15

Hematology

15

Hematology

15

Hematology

15

Hematology

15.1 Hematopoiesis

Lothar Thomas

Circulating blood cells play a multifactorial role in the maintenance of organ function. They react to stimuli and adapt their number and function to the requirements of the organism. The lifespan of the individual blood cells in the circulation is shown in Tab. 15.1-1 – Life span and daily turnover of blood cells in the circulation. Changes in the number and function of blood cells can result from diseases of the hematopoietic system or hematopoiesis is activated or compromised due to disorder of an organ or a systemic disease.

15.1.1 Clonal hematopoiesis of indeterminate potential (CHIP)

CHIP is a hematologic precursor lesion that is defined by the presence of somatic mutations in peripheral blood cells but without evidence for the presence of leukemia or another hematologic neoplasm.

15.1.1.1 Blood count abnormalities in CHIP

The majority of individuals with CHIP show a normal leukocyte morphology. While a slight elevation in the red cell size has been described in some studies. An increased red cell distribution width (RDW) is the most consistent blood count feature found in CHIP. Next generation sequencing based detection of clonal hematopoiesis has become a diagnostic standard in the hematologic workup of patients with suspected myeloid neoplasms. In contrast testing for CHIP is currently not recommended in the general population. In addition to the hematologic progression, CHIP has been associated with a plethora of non-hematologic diseases of which cardiovascular disorders are currently best understood and clinically established /34/.

15.1.2 Red blood cells

Red blood cells are comprised of erythrocytes (99%) and reticulocytes (1%). They transport O2 from the lungs to the tissues and CO2 from the tissues to the lungs. The amount of O2 that can be carried per unit volume of blood is determined by the concentration of hemoglobin (Hb) in blood. About 1.34 to 1.39 mL O2 can be bound per gram of Hb. For osmotic reasons Hb is packed into red cells. They perform their function exclusively in the circulation. The human organism contains, on the average, a red blood cell mass of 2–3 liters, and the daily total circulating red blood cell mass is 3,000 liters. Every day some 2.0 × 1011 senescent erythrocytes (approximately 1% of all red blood cells) are removed from the circulation in adults and retained in the spleen. Spleen macrophages degrade the cells, and the components, especially iron, are reused by the bone marrow for the production of new red cells. Every erythrocyte enters the circulation as reticulocyte. Within 1–2 hours of acute onset of hypoxia circulating erythropoietin levels begin to increase and reticulocyte counts can be doubled one to two days following the hypoxia /1/.

Red blood cell membrane

The red blood cell (RBC) membrane is composed of a lipid bilayer in which approximately 20 major proteins and at least 850 minor ones are embedded /2/. The lipid bilayer acts as a barrier for the retention of cations and anions within the red cells, while it allows water molecules to pass through freely. The maintenance of high intracellular K+ and low intracellular Na+ compared with the corresponding ion concentrations in the plasma involves a passive outward movement of K+, which is pumped back by the action of an ATP dependent Na+/K+ pump in exchange for Na+. A further component of the RBC membrane is the cytoskeleton. This protein network laminates the inner surface of the membrane and contains the proteins spectrin and actin which are connected to each other in two protein complexes; ankrin and protein 4.1 complex. The red cell membrane is attached to the intracellular cytoskeleton by protein-protein and lipid-protein interactions that confer the erythrocyte shape, stability and deformability. During the life span the RBC is forced to cross the pores of splenic sinusoids thounds of times and has an ongoing relationship with the spleen that contributes to remodeling during the first week of its life. In addition the spleen plays the primary role in the removal of aged erythrocytes.

Dynamics of red cells

RBCs undergo a rapid reduction in volume and hemoglobin (Hb) in the few days after release from the bone marrow. This rapid phase is followed by a much longer period of slower reduction during which the volume and Hb are co regulate. The correlation between volume and Hb content increases as the cells mature from an initial correlation coefficient of approximately 0.40 in the reticulocyte population to approximately 0.85 in the full population /3/.

Erythroblast adaption to iron

During RBC maturation abundant synthesis of Hb is required. Since iron is essential for Hb synthesis, erythropoiesis consumes a major part of the body iron. The transcription factor Bach 1 is involved in the responses of erythroblasts to iron status. In Iron deficiency Bach 1 depresses the genes of globin synthesis in erythroblasts to balance the levels of heme and globin /4/.

The erythroid regulator erythroferrone facilitates iron acquisition e.g., in hemorrhage-induced anemia. Erythroferrone is produced by erythroid precursors in the marrow and the spleen and mediates hepcidin suppression during increased erythropoietic activity stimulated by erythropoietin. Thus erythroferrone increases iron availability by suppressing hepcidin /5/.

15.1.3 Thrombocytes (platelets)

Approximately 2 × 1010 thrombocytes per liter of blood are released from the bone marrow daily. The plasma membrane of the platelets contains receptors such as the glycoprotein GPIIb/IIIa receptor and the von Willebrand receptor (GPIb/V/IX). Thrombocytes repair vascular injuries and prevent excessive bleeding.

15.1.4 Leukocytes

The function of leukocytes is carried out extra vascularly. Therefore the blood merely serves as a transport way which the leukocyte uses to move from one place to the other /1/.

Polymorphonuclear neutrophil granulocytes (PMN)

The PMN are mature neutrophil granulocytes and have a segmented nucleus with the segments separated by a filamentous strand. The PMN pool in blood is divided in two compartments of approximately equal size: the circulating pool and cells which adhere to the walls of the small vessels (marginal pool). Upon blood sampling cells of the circulating pool, but not the marginal pool, are determined reliably with the leukocyte count. There is, however, a constant exchange of cells between the two pools. In the tissues the PMN are capable of phagocytosis.

Eosinophils

Eosinophils have the same morphologic character and maturation sequence as neutrophils. They are filled with orange-colored acidophilic granules that contain basic proteins, eosinophilic peroxidase and other substances. IL-5 mobilizes eosinophils, induces chemotaxis and enhances the production of reactive oxygen radicals. Following a short circulation time in the blood, they participate in inflammatory tissue reactions, stimulated by the immune system, and they are also involved in defense mechanisms against helminth infections.

Basophils

Basophils have the same morphologic character and maturation sequence as neutrophils. Basophils lack mobility and phagocytosis. Their granules are filled with angiotonic substances involved in blood vessel contraction, such as histamine and serotonin.

Lymphocytes

Lymphocytes vary in size from slightly larger than erythrocytes to as large or larger than monocytes. Lymphocytes are broadly classified in T cells, B cells and NK cells and are concerned with innate and adaptive immunity (see Section 21.1.4 – Innate immune response). Their sub populations are determined flowcytometrically.

Monocytes

For monocytes, as for PMNs, the blood serves as a street from the bone marrow to the tissues. In blood the monocytes are distributed into a circulating and a marginal pool. Following stimulation, which usually takes place in the tissues, they are transformed into metabolically active macrophages. Their primary functions are the phagocytosis of aging erythrocytes and of microorganisms and the release of inflammatory cytokines for the regulation of the cellular and humoral immune defense.

Cell distribution in the bone marrow

The hematopoietic cells in the bone marrow are comprised of up to 60% granulocytes (mainly neutrophils), 20% erythropoietic precursors, and 15% lymphocytes, plasma cells, monocytes and megakaryocytes.

15.1.5 Hematopoietic system

The hematopoietic system provides the blood with cells, maintains the steady state level of circulating blood cells, and responds to acute challenges. There is a constant turnover of hematopoietic cells as they reach their “use-by date” and are removed from the system /6/. Over a period of 7 years, an adult forms a quantity of blood cells that corresponds to his body weight.

The hematopoietic system is organized in a hierarchical manner (Fig. 15.1-1 – Hierarchy of the hematopoietic system with myelopoiesis and lymphopoiesis). Pluripotent stem cells exist primarily in the bone marrow of adults and generate the numerous lineages found in blood. The differentiation of stem cells into blood cell lineages is regulated via a group of hematopoietic growth factors and cytokines. Activation of receptors on the surface of hematopoietic cells by growth factors leads to signalling events which enable each cell to differentiate.

Among all of the hematopoietic cells, stem cells have the greatest capacity for self-renewal. Committed progenitor cells are limited to one or two cell lineages and have a low capacity for replication.

Growth factors are required for the survival and proliferation of hematopoietic cells at all stages of development /7/.

Effective erythropoiesis is the result of the interaction of hematopoietic cells and their supporting stroma. More than 95% of hematopoiesis takes place in the bone marrow; this is the only location where erythropoiesis, granulopoiesis, lymphopoiesis, monopoiesis and megakaryopoiesis proceed simultaneously. Under certain conditions, hematopoiesis can also occur in the spleen.

15.1.5.1 Hematopoietic micro environment

Bone marrow stroma provides the micro environment for the hematopoietic cells and influences their proliferation and differentiation. Components of the micro environment are /8/:

  • Fibroblasts, macrophages, adipocytes and accessory cells such as T lymphocytes and monocytes
  • Extracellular matrix (e.g., collagen, laminin, fibronectin, and proteoglycans).

The cells of the micro environment affect hematopoiesis, in a positive and negative manner, by the following mechanisms:

  • Direct cell-cell contact; thus, pluripotent stem cells require contact with stromal cells
  • Secretion of proteins which serve to maintain the structure of the extracellular matrix
  • The formation of soluble and cell-associated cytokines. Colony stimulating factors (CSF), interleukins (IL-1, IL-3, IL-6, IL-12), as well as inhibitors such as tumor necrosis factor-α, transforming growth factor-β and interferon-γ are produced.

15.1.5.2 Hematopoietic stem cell

The maintenance of blood cell populations is achieved by the proliferation and differentiation of precursor cells located primarily in the bone marrow. The precursor cells are derived from a common hematopoietic stem cell population that is established during embryogenesis and functions for the lifetime of the organism. In addition to its differentiation potential the stem cell has the capacity to produce daughter cells with the same or very similar proliferative and developmental potential as the parental cell /9/. Stem cells are defined as self-renewing cells, capable of forming all blood cell lineages for at least 4 months following transplantation in a recipient mouse /10/. Stem cell division proceeds asymmetrically. One daughter cell retains the pluripotency of the mother cell, while the other is limited (committed) to develop into a cell of a hematopoietic lineage. In adults, stem cells are in a resting stage or of minimal activity. These stages are induced by the pleiotropic hormone transforming growth factor-β (TGF-β) /11/.

The hematopoietic progenitor cells are named according to the colony type (colony-forming unit, CFU) that they form in vitro culture /12/. The pluripotent stem cell is called CFU-blast (Fig. 15.1-1 – Hierarchy of the hematopoietic system with myelopoiesis and lymphopoiesis). The CFU-blast can form cells of all hematopoietic cell lineages and comprises 0.05% of the bone marrow cells. The CFU-blast expresses CD34+, but no lineage specific antigen and is, consequently, defined as CD34+DRlin. The expression of the HLA antigen DR is one of the earliest differentiation markers and leads from the CFU-blast to the committed progenitor cells. From these, the CFU-GEMM can differentiate into one of the following cell lineages: G, granulocytes; E, erythrocytes; M, monocytes; and M, megakaryocytes /13/.

The differentiation of the CFU-blast into a committed progenitor cell (lineage-bound progenitor cells) is subject to control by growth factors. The most important of these is the stem cell factor (SCF), which is produced by the stromal cells. The SCF is responsible in particular for the long term survival of non-dividing, pluripotent stem cells.

Pluripotent stem cells are also present in the peripheral blood; the CD34+ cells account for 0.15% of the mononuclear blood cells. Their fraction is increased to 0.6% during a course of chemotherapy, and they are harvested for stem cell transfer by means of apheresis.

15.1.5.3 Hematopoietic growth factors

Hematopoietic growth factors are required /7/:

  • For the survival and proliferation of hematopoietic factors at all stages of cell development. Factors that affect multi potential cells are the steel factor, the Fms-like tyrosine kinase 3 (FLT3) ligand, the granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-2, IL-3 and IL-7.
  • For the development of progenitor cells, that are committed to one or two cell lineages. Lineage-specific hematopoietic growth factors are erythropoietin and thrombopoietin.

Stem cell factor

The stem cell factor (SCF) is synthesized by the bone marrow stroma. SCF binds to c-kit, a tyrosine kinase receptor localized on stem cells and committed progenitor cells. SCF generates or activates these cells to become colony stimulating factors (CSF). In cell culture, SCF manifests effects that are synergistic with those of the CSF /14/.

Lineage-specific hematopoietic growth factors

The lineage-specific hematopoietic growth factors are synthesized in the kidneys (erythropoietin), endothelial cells, fibroblasts, macrophages (GCSF), bone marrow stromal cells, and the liver (thrombopoietin). The effects of the lineage-specific growth factors are mediated by receptors of the cytokine receptor super family. The receptors are transmembrane proteins with one or two extracellular binding domains and one intracellular domain, which activates kinases of the Janus kinase (JAK) family. JAKs mediate signal transduction to the cell nucleus via multiple intermediate steps /7/.

FMS-like tyrosine kinase 3 gene

The FMS-like tyrosine kinase 3 (FLT3) is a transmembrane ligand-activated receptor tyrosine kinase that is normally expressed by hematopoietic stem cells and early myeloid and lymphoid progenitor cells, and is involved in the proliferation, differentiation and apoptosis of hematopoietic cells through various signaling pathways. The FTL3 gene is mutated in 25-30% of patients with acute myeloid leukemia (AML) because of the poor prognosis associated with FTL3- internal tandem duplication mutated AML, allogeneic hematopoietic stem-cell transplantation is commonly performed in first complete remission /32/.

15.1.5.4 Embryonic and fetal hematopoiesis

The blood cells are formed in the 3rd–6th week of gestation in the yolk sac, the para aortic splanchnopleura, and the ventral region of the embryo, which is responsible for the aorta-gonad-mesonephros (AGM) development. Soon after their initial development in yolk-sac blood islands, primitive erythr­oblasts enter the newly formed vasculature. The colonization of the rudimentary liver, which is the principle location of embryonic and fetal blood formation until week 22 of gestation, ensues from these tissues very early on. The colonization of the bone marrow and the spleen takes place at this time. During the second half of pregnancy, the bone marrow becomes the major site of blood cell formation and it retains this function life long. The spleen remains a lymph organ.

The first erythropoietic blood cells are primitive erythro­blasts in the yolk sac on days 16–19 post-conception. The hemoglobin in these cells contains embryonic globin. The cells also maintain their nucleus for a certain period of time in the circulation. The differentiation of primitive erythro­blasts occurs in the circulation through the accumulation of increasing quantities of hemoglobin. From the 8th week of development onwards definitive red blood cells, formed by the liver, gradually replace the primitive erythro­blasts. The change is believed to be based upon a dose effect of the EKLF gene /15/. The definitive red blood cells are smaller, they no longer have a nucleus, and the formation of globin is limited to fetal or adult hemoglobin. The synthesis of erythropoietic cells is dependent upon erythropoietin, which is a mitogen as well as a survival factor for erythroid progenitor cells /16/. During the fetal period, the hemoglobin concentration and the hematocrit increase, while the mean corpuscular volume (MCV) decreases.

Hematopoietic stem cells continue to self-renew and differentiate into mature blood cell lineages throughout their lifetime. Until a sufficient number of hematopoietic stem cells are produced in the fetal liver to supply the adult-type (definitive) erythrocytes, embryo/fetus-specific erythrocytes, called primitive erythrocytes, which are produced in the extra-embryonic yolk sac tissue, play a pivotal role in transporting oxygen to embryonic and fetal tissue. A committed hematopoietic cell population serves as a common precursor for primitive erythrocytes and lympho-myeloid lineages. Primitive erythrocytes are large in size and enter into circulation in their nucleated form. The erliest hematopoietic progenitors are found in the CD41+CD45 cell stage /16/.

It has long been thought that hematopoiesis occurs in two phases (primitive and definitive erythropoiesis) during development, but some findings include that there are intermediate cell populations /16/.

Definitive hematopoiesis produces all lympho-myeloid lineages and adult-type erythropoiesis. The leukocytes are initially formed from para aortic splanchnopleure and aorta-gonad-mesonephros cells. The stem cells that have migrated from there are responsible for the formation of B and T lymphocytes, granulocytes and natural killer (NK) cells. These cells are detectable from the 15th week of gestation onwards, and the mean leukocyte count is 0.8 × 109/L. From the 21st week of gestation, the leucocyte count is above 1 × 109/L.

15.1.6 Erythropoiesis

Erythropoiesis occurs in three stages: the primitive, the fetal definitive and the adult definitive stages. After the primitive stage, which takes place in the yolk sac, definitive erythropoiesis moves to the fetal liver and the spleen but is finally restricted to the bone marrow, as adult definitve red blood cells, for the rest of the live. After birth, the location of erythropoiesis gradually switches to spongy flat bones, such as ilium, sternum, ribs and cranium, the sites which adults rely on mostly for steady-state erythropoiesis /17/.Extramedullary erythropoiesis results /17/:

  • In the development of erythroblastic islands in other organs and tissues, in particular the spleen and liver
  • In the abundance of erythroid precursor cells co-expressing CD71+.

Continuous red cell production is required for the maintenance of the erythrocyte and hemoglobin concentration. Following a period of about 10 days for maturation and differentiation in the bone marrow some 2 × 1011 reticulocytes are released into the blood stream, daily. During maturation of the red cell, a marked accumulation of hemoglobin, making up over 95% of the total red blood cell protein, occurs. The differentiation of erythropoiesis begins at the stage of the pluripotent hematological stem cell. All successive cells have lost the capability of renewing erythropoiesis; only cell division and differentiation remains possible. The development of erythropoietic cells in the bone marrow takes place in the proliferation pool at first and thereafter in the maturation pool (Fig. 15.1-2 – Erythropoiesis develops a proliferation pool and a maturation pool).

Proliferation pool

In this phase of erythropoiesis, the stem cell acquires the potential of surface receptor expression to acquire signals for proliferation and differentiation. The most immature form of the committed erythroid progenitors is the burst-forming unit erythroid (BFU-E). The BFU-E requires 14 days or more for the formation of a cluster of mature erythroblasts. The BFU-E stage is followed by the colony forming unit erythroid (CFU-E). Both progenitors constitute some 0.3% of the nucleated erythropoietic bone marrow cells. A time period of 7 days is required for the formation of a cluster of 8–64 mature erythroblasts from the CFU-E.

Maturation pool

CFU-E cells enter this pool and mature to reticulocytes which are released in the circulation and become erythrocytes (Fig. 15.1-2 – Erythropoiesis develops a proliferation pool and a maturation pool). The maturation phase begins with the first progenitor cell that is cytologically distinguishable in the bone marrow, the pronormoblast. It has basophilic cytoplasm, a nucleus with, as a rule, six nucleoli and a diameter of 15–20 μm. As the stage of maturation progresses, the cell diameter and density of the erythropoietin receptors decrease and cellular hemoglobin content increases. Following rejection of the nucleus by orthochromatic erythroblasts the reticulocyte, which remains with the RNA residues, is released into circulation, where it matures within 24–48 hours to an erythrocyte.

Erythropoietin (EPO) is an important regulatory hormone of erythropoiesis. It prevents apoptosis of CFU-E cells and induces their clonal expansion. The CFU-E has the highest EPO receptor density of the erythroid progenitor cells. Under stimulation with EPO, CFU-E proceeds through division stages up to orthochromatic erythroblasts which are no longer capable of division.

The normal red blood cell achieves a mean cellular hemoglobin concentration (MCHC) of up to 360 g/L. A further increase of 10–20 g/L can take place if the cell loses water or membrane, which occurs only relatively seldom. The hemoglobin concentration of the maturing red blood cell regulates the number of cell divisions that the cell is to undergo. The pronormoblast normally undergoes four divisions, resulting in a total of 16 erythrocytes. With every division the volume of the cells declines.

For heme formation see Section 7.1 – Iron metabolism and disorders and Ref. /18/.

Changes in erythrocyte volume and hemoglobin concentration can result from:

  • Iron deficiency or disorders of globin synthesis (thalassemia). The hemoglobin content of the erythrocyte is reduced. Normally, after four cell divisions in the maturation pool, the erythroblast is saturated with hemoglobin and the cell nucleus receives a signal to refrain from further cell division. This is not the case in iron deficiency and an additional cell division occurs with a volume reduction of the red cell.
  • Vitamin B12 or folic acid deficiency. In this case the cellular hemoglobin content, which signals the erythroblast nucleus to cease cell division and to implement the expulsion of the nucleus, occurs earlier, resulting in fewer divisions and a larger erythrocyte volume.
  • Hereditary disorders which result in microcytic normochromic anemia (MCHC normal). Examples are the spherocyte and the keratocyte. Hereditary spherocytosis is a disease in which the cell membrane of mature erythrocytes is reduced. Cellular hemoglobin and cell volume are normal, but the cell diameter is decreased. Due to the reduced diameter, the hematocrit is lower than expected, the calculated MCHC is elevated and the erythrocyte is seemingly hyperchromic. Keratocytes that develop in vivo due to the effect of oxidative medication, have a reduction of the cell membrane and of hemoglobin. Erythrocyte hemoglobin precipitates with the development of Heinz bodies, a proportion of the erythrocyte contains less hemoglobin and looks pale in the blood smear.
  • Senescent erythrocytes are removed from the blood stream through phagocytosis by the reticuloendothelial system of the spleen. Senescent erythrocytes show volume deformation and decrease and increase in cell density. Cell membrane changes lead to a loss of carbohydrates on the cell surface.

Based on the volume (MCV) and the mean hemoglobin concentration (MCHC) of erythrocytes anemias are classified as:

  • Normocytic and normochromic
  • Macrocytic and hypochromic
  • Microcytic and hypochromic
  • Microcytic and normochromic

The classification of anemia can never be:

  • Normocytic and hyperchromic
  • Macrocytic and hyperchromic.

Regulation of erythroid differentiation

Red cell production increases 5 to 7-fold after blood loss or hemolysis, but does not overshoot because it is tightly regulated /19/. The regulation involves proliferation, differentiation and survival of erythroid progenitor and precursor cells. Although the cells of the proliferation pool act in concert each of these processes can be regulated independently of another. Following blood loss or hemolysis, stem cell factor (SCF/kit-ligand) and glucocorticoids increase proliferation in the BFU-E to colony-forming unit-erythroid (CFU-E) stages. However, SCF and glucocorticoids have no influence on the survival and differentiation of these erythroid progenitors. EPO, in turn, supports the maturation of erythroid progenitor cells from CFU-E to basophil erythroblasts, but it influences neither their maturation nor their differentiation. Proliferation, differentiation and survival of erythroid progenitors are controlled through activation and repression of specific genetic programs. An important transcriptional regulator in erythropoiesis is LIM domain-only protein 2 (LMO2). This protein is regulated at both a post-transcriptional and post-translational level in erythroid progenitors and contributes to the precision with which erythrocyte production is controlled.

Erythroid precursor cells co-expressing CD71+ and the erythroid lineage marker CD235a (glycophorin A) are cells of extramedullary erythropoiesis (EE). These cells are defined as CD71+ erythroid cells (CEC). CEC are mainly erythroid precursors expressing high levels of CD71, including reticulocytes but excluding erythrocytes. EE may be considered a normal physiological process during pregnancy and in developing newborns. Regarding of the underlying mechanism, EE results in the development of erythroblastic islands in other organs/tissues, in particular the spleen and liver. EE results in an abundance of erythroid precursors or CEC in the periphery. Erythroid precursors have immunosuppressive or immunomodulatory properties and their expression can, therefore, have an impact on the effector functions of various different immune cells /17/.

15.1.7 Granulopoiesis

The phagocytic cell lineages of granulocytes and monocytes have a bipodent committed progenitor the colony forming unit granulocyte-monocyte (CFU-GM). Colony stimulating factors (CSFs) such as the granulocyte colony stimulating factor (G-CSF) and the granulocyte macrophage-CSF (GM-CSF) stimulate the proliferation, differentiation and maturation of neutrophil and eosinophil granulocytes and monocytes. The importance of GM-CSF in granulopoiesis is similar to that of erythropoietin in erythropoiesis.

Neutrophile production and storage

Following the maturation from the stem cell, there is a continuous maturation from the earliest recognizable precursor the myeloblast to the polymorphonuclear neutrophil. In the bone marrow neutrophils and their precursors mature in two pools:

  • The mitotic or production pool that is divided into myeloblasts, promyelocytes, and myelocytes. Four to five cell divisions occur from myeloblast to myelocyte stages, leading to a 32-fold increase in the number of cells. Transition time from myeloblast to myelocyte is 3–9 days; in proliferative stress the rate of granulopoiesis can be elevated by a factor of 20.
  • The post-mitotic maturation and storage pool which is divided in metamyelocytes, bands and segmented neutrophils. In this pool mitosis no longer occurs, but a steady process of maturation and storage of bands and segmented neutrophils is taking place. Upon demand these cells are released into the circulation. Under non-pathological conditions, only post mitotic cells are released. Normally the cells spend about 10 days in the storage and maturation pool. The pool contains about 15–20 times as many granulocytes as are in the blood.

Formation of granules

Granules start to form at the stage of neutrophil maturation marked by transition from myeloblast to promyelocyte. From here on, formation of granule protein continues even up to the stage of segmented cells. Granules are believed to be formed by aggregation of immature transport vesicles that bud off from the trans-Golgi network (TGN). In TGN sorting takes place between constitutively secreted proteins and proteins that are routed into the regulated secretory pathway (i.e., go to granules) /20/.

Stimulation and activation of granulocytes

The increase in granulocytes in the circulation can be triggered by the following mechanisms /121/:

  • Demarcation; administration of epinephrine or severe exercise will decrease the proportion of marginated granulocytes and therefore induce neutrophilia. Since demarcation only represents redistribution of existing intravascular pools of granulocytes, it does not increase numbers of cells available to control infectious organisms.
  • Leukocyte egress; increased numbers of maturating band-form neutrophils in the circulation results from the release of a post-mitotic storage pool rather than an increased rate of production of theses cells. Under non-pathological circumstances, only post-mitotic cells are released. Responses to endotoxin and infection occur within minutes to hours.
  • Shift to the left; when an increased ratio of bands to segments is measured in the blood, with or without an increase in more mature neutrophils such as metamyelocytes, it is indicative of accelerated marrow release, usually accompanied by a reduction in size of the storage pool. The increased rate of granulopoieses as in severe inflammation results in a significant increase in post-mitotic cells only after 2–3 days.
  • Neutrophil count remains high; this may be the case in chronic infection. The neutrophil count is in a new steady state of balanced but increased input and output which is maintained by accelerating production.
  • Expression of the Fc-receptor (CD64). In resting neutrophils the level of CD64 expression is relative low (about 1000 molecules/cell), following activation it can increase up to 5 to 10-fold /22/.

15.1.8 Monopoiesis

In bone marrow, the principal cytologically detectable cell is the monoblast. Immature cells migrate into tissues and body cavities where they mature into so-called wandering macrophages (pulmonary-alveolar macrophages, macrophages lining sinuses, Kupffer cells in liver, Langerhans’ cells in epidemis) /23/. Monocytes circulate in the blood with a half life time of 1–3 days, and then migrate into the tissue. Monocyte populations in the blood are CD14+CD16 and CD14+CD16+.

Cluster of differentiation 64 (CD64): The membrane gylycoprotein CD64 (FC-gamma receptor 1) is found only on macrophages. CD64 binds monomeric antibodies of IgG type with high affinity. Treatment of polymorphonuclear granulocytes with interferone (IFN) gamma can induce CD64 expression on these cells. Some authors /35/ recommend monitoring of neutrophil CD64 as a prognostic marker in septic shock. In these patients CD64 is a valuable marker indicating the course of septic shock. In the initial 48 hours after admission decrease of CD64 concentration can aid in predicting the prognosis of septic patients.

15.1.9 Megakaryopoesis

Megakaryopoiesis is a complex, stepwise process that takes place in the bone marrow. Committed progenitor cells, the colony forming unit megakaryocyte (CFU-MK), undergo a number of lineage commitment decisions lead to the production of polyploid megakaryocytes. As of a certain stage CFU-MK cease to proliferate, and endomitosis begins /24/. At this stage they are megaloblasts or immature megakaryocytes. The process of endomitosis involves DNA replication without cell division, and a polyploid cell with a single frequently lobed nucleus develops. In the process of DNA replication the nucleus does not divide and cytoplasm remains intact /25/. Replication usually occurs three times so that the mature megakaryocyte has a ploidy of 16, which, however, is variable and can be as high as 256.

Maturing megakaryocytes acquire the competence of forming thrombocytes. This occurs through /24/:

  • Up-modulation of a vast array of cytoskeletal, membrane and granule regulatory proteins
  • Acquisition of stores of ribosomes, α-granules and dense granules
  • Differentiation into cells that are large, polyploid, and have a cytoplasm filled with a complex system of interconnected cytoplasmic membranes (demarcation membrane system; DMS). The demarcations marks preformed platelet regions, also termed pro platelets.
  • In the bone marrow sinusoids, pro platelets are separated from the cytoplasm by shear forces. One megakaryocyte can release 1,000–5,000 platelets. The pro platelet formation is a terminal process, and once has been completed the residual megakaryocyte nucleus in engulfed by macrophages within the bone marrow.

Thrombopoietin

Megakaryopoiesis and formation of thrombocytes is regulated by thrombopoietin (TPO), but other cytokines such as IL-6 and IL-11 also play a role /26/. The latter make it possible for the megakaryocyte to adapt to regions that permit maturation and thrombocyte formation. The effect of TPO on megakaryocytes and thrombocytes is mediated by the c-Mpl (CD110) receptor; however, only a low number is present on the thrombocytes. TPO is synthesized in constant amounts by the liver and its concentration is regulated to a certain extent by binding to its thrombocyte receptor and thus by the number of thrombocytes as well. If the blood thrombocyte count is high, increased amounts of TPO bind to c-Mpl and only small quantities bind to megakaryocytes; the result is down-regulation of megakaryopoiesis. If, on the other hand, the thrombocyte count is low, then plasma TPO concentration will be high and megakaryopoiesis is stimulated.

Von Willebrand factor receptor

The receptor GPIb/V/IX is an important regulator of pro platelet formation. In immune thrombocytopenia, autoantibodies develop and inhibit the release of pro platelets.

Bernard-Soulier syndrome: in this hereditary autosomal recessive macro thrombocytopenia, defined platelet regions that are marked by the demarcation membrane system are larger than normal /31/.

15.1.10 Splenectomy

Thromboembolism and pulmonary hypertension constitute frequently occurring complications (Odds ratio 4.08) in patients with transfusion-dependent thalassemia and can be associated with splenectomy. Thalassemias are congenital autosomal recessive hemoglobinopathies. The patients are unable to produce enough hemoglobin to survive without red blood cell transfusions. The two primary types of the disease, alpha- and beta-thalassemia, are distinguished by decreased or absent synthesis of either alpha-globin chains or beta-globin chains of the hemoglobin molecule. Nowadays thalassemias are differentiated as follows /36/:

  • Beta thalassemia minor: asymptomatic microcytic anemia.
  • Thalassemia syndromes: they are classified depending on clinical severity and transfusion requirement. The patients need maintaining a pre-transfusion level of hemoglobin of 95–105 g/L which suppresses erythropoiesis. Despite advances in therapeutic care, splenectomy is a treatment option in some patients. Splenectomy is related to major long-term complications (thromboembolic, pulmonary hypertension, and infections). The spleen normally removes damaged red blood cells (RBCs). In the absence of spleen negatively charged RBCs and hyperactive thrombocytes contribute to the hypercoagulable state of thalassemia, as these cells stay in blood circulation and activate thrombin production.

15.1.11 Hematopoietic equilibrium

In normal hematopoiesis there is an equilibrium between the cell mass for each cell lineage in the blood, and the tissues, and their formation in bone marrow. There are three important variations that disturb this equilibrium:

  • Hypo proliferation of the marrow or of one or more cell lineages. In this case either the marrow is incapable of reacting adequately to the increased stimulatory effect of growth factors (intrinsic marrow disorder), or substances that are important for the formation and maturation of cells are lacking (e.g., iron deficiency).
  • Ineffectiveness of one or more cell lineages. Hematopoiesis is hypo proliferative due to the inhibitory effect of inflammatory cytokines. This is the case in anemia of chronic disease.
  • Bone marrow failure, defined as the inability of hematopoiesis to meet the physiologic demands for the production of a sufficient number of functional blood cells is classified based on presumed etiology in inherited, secondary or idiopathic. An approach to evaluating patients presenting for the presence of inherited bone marrow failure syndromes is shown in Ref. /30/.
  • Hyper proliferation of the marrow or of one or more cell lineages. This results in an intensified hematopoietic response to the demand. The compensatory capacity of the marrow is often inadequate to fulfill the requirements and cytopenia results. An example is thrombocytopenia in disseminated intravascular coagulation.

Primary disorders of hematopoiesis

A disease of one or more hematopoietic cell lineages is present (e.g., in leukemia, thalassemia, or pancytopenia). The differential diagnosis of pancytopenia is shown in Table 15.1-3 – Differential diagnosis of pancytopenia.

Secondary disorders of hematopoiesis

Hematopoiesis is compromised because of a local organ disease (splenomegaly) or a systemic disease (inflammation). The consequence is diminished or flawed blood cell formation or a reactive hematopoietic response with augmented synthesis and overproduction of precursors. Disorders may be caused by:

  • Important substances that are deficient (iron, folic acid, vitamin B12, vitamin B6)
  • A reactive response of the marrow (granulocytosis, granulocytopenia, thrombocytosis, thrombocytopenia, anemia) in systemic disease of the organism
  • A reactive response according to hypoxia (polycythemia at high altitudes of above 3,000 meters).

Secondary disorders of hematopoiesis are often complex and require special hematological and biochemical tests in addition to a blood count for clarification.

15.1.12 Investigation of hematopoiesis

For the investigation of hematopoiesis, basic tests are distinguished from functional tests. Basic tests are descriptive measures of the condition (e.g., Hb value or leukocyte count). Functional tests describe hematopoietic function in response to a compromising situation (e.g., reticulocytosis in blood loss or a left shift of granulopoiesis in sepsis).

15.1.13 Basic hematologic tests

Basic hematological tests for the detection of hematopoietic disorders are performed using hematology analyzers:

  • Complete blood count
  • Partial or complete leukocyte differentiation
  • Reticulocyte count. Some analyzers also measure, in addition, reticulocyte indices like reticulocyte Hb content (CHr, RetHe), reticulocyte RNA content and the proportion of hypochromic red cells (%HYPO).
  • Blood smear. This test is required as a supplementary investigation in symptomatic patients and if the hematology analyzer provides a signal indicating the presence of atypical cells. Important hematological tests are shown in Tab. 15.1-2 – Hematological investigations for assessment of the hematopoieses.
  • Flow cytometric measurement of blood cell surface markers using specific antibodies (immunophenotyping).

15.1.14 Dermatoses associated with hematological malignancy

Cutaneous manifestations of hematologic malignancy supports the concept of molecular defects associating with specific clinical features /37/. Neutrophilic or eosinophilic dermatoses are classified according to their clinico-pathological picture into superficial/epidermal, dermal, and deep forms, with a forth category that encompasses mixed as well as syndromic neutrophilic dermatoses.

Neutrophilic and eosinophilic dermatoses associated with hematological malignancy represent a heterogenous group of skin conditions, which may either parallel the course of the underlying hematological disorder or be independent from it.

Proposed pathophysiology of dermatosis of hematological malignancy is that leukemic cells may promote systemic inflammation, resulting in the clinico-pathological picture of Sweet syndrome, a rare neutrophilic dermatosis.

References

1. Nelson DA. The biology of myelopoiesis. Clin Lab Med 1990; 10: 649–59.

2. Andolfo I, Russo R, Gambale A, Iolascon A. New insights on hereditary erythrocyte membrane defects. Haematologica 2016; 101: 1284–94.

3. Higgins JM, Mahadevan L. Phyiological and pathological population dynamics of circulating human red blood cells. PNAS 2010; 107: 20587–92.

4. Kobayashi M, Kato H, Hada H, Itoh-Nakadai A, Fujiwara T, Muto A, et al. Iron-heme-Bach1 axis is involved in erythroblast adaption to iron deficiency. Haematologica 2017; 102: 454–65.

5. Kautz E, Jung G, Du X, Gabayan V, Chapman J, Nasoff M, et al. Erythroferrone contributes to hepcidin suppression and iron overload in a mouse model of β-thalassemia. Blood 2015; 126: 2031–7.

6. Valent P, Büsche G, Theurl I, Uras IZ, Germing U, Stauder R, et al. Normal and pathological erythropoiesis in adults: from gene regulation to targeted treatment. Haematologica 2018; 103 (10) 1593–1603

7. Kaushansky K. Lineage-specific hepatopoietic growth factors. N Engl J Med 2006; 2006; 354: 2034–45.

8. Mayani H, Guibert LJ, Janowska-Wieczorek A. Biology of the hemopoietic microenvironment. Eur J Haematol 1992; 49; 225–33.

9. Graham GJ, Wright EG. Hemapoietic stem cells: their heterogeneity and regulation. Int J Exp Path 1997; 78: 197–218.

10. Orlic D, Bodine D. What defines a pluripotent hematopoietic stem cell (PHSC): will the real PHSC please stand up? Blood 1994; 84: 3991–4.

11. Fortunel NO, Hatzfeld A, Hatzfeld JA. Transforming growth factor-β: pleiotropic role in the regulation of hematopoiesis. Blood 2000; 96: 2022–36.

12. Huss R. Applications of hematopoietic stem cells and gene transfer. Infusionsther Transfusionsmed 1996; 23: 147–60.

13. Migliaccio AR, Vannucchi AM, Migliaccio G. Molecular control of erythroid differentiation. Int J Hematol 1996; 64: 1–29.

14. Ashman LK. The biology of stem cell factor and its receptor c-kit. IJBCB 1999; 31: 1037–51.

15. Isen J, Fraser ST, He Z, Zhang H, Baron MH. Dose-dependent regulation of primitive erythroid maturation and identity by the transcription factor Eklf. Blood 2010; 116: 3972–80.

16. Yamane Toshiyuki. Cellular basis of embryonic hematopoiesis and its implications in prenatal erythropoiesis. Int J Mol Sci 2020; 21: 9346. doi: 10.3390/ijms21249346.

17. Elahi S, Mashhouri S. Immunogical consequences of extramedullary erythropoiesis:immunoregulatory functions of CD71+ erythroid cells. Haematologica 2020; 105 (6): 1478–83.

18. Chiabrando D, Mercurio S, Tolosano E. Heme and erythropoieses: more than a structural role. Haematologica 2014; 99: 973–83.

19. Brandt SJ, Koury MJ. Regulation of LMO2 mRNA and protein expression in erythroid differentiation. Haematologica 2009; 94: 447–8.

20. Borregard N, Cowland JB. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 1997; 89: 3503–21.

21. Jagels MA, Hugly TE. Mechanisms and mediators of neutrophilic leukocytosis. Immunopharmacology 1994; 28: 1–18.

22. Hoffmann JJML. Neutrophil CD64: a diagnostic mar- ker for infection and sepsis. Clin Chem Lab Med 2009; 47: 903–16.

23. Ziegler-Heitbrock HWL. Definition of human blood monocytes. J Leukoc Biol 2000; 67: 603–6.

24. Geddis AE. The regulation of proplatelet production. Haematologica 2009; 94: 756–9.

25. Cazzola M. Molecular basis of thrombocytosis. Haematologica 2008; 93: 646–8.

26. Kaushansky K. Thrombopoietin: understanding and manipulating platelet production. Annu Rev Med 1997; 48: 1–11.

27. Gulati GL, Hyun BH. The automated CBC. Hematol Oncol Clin North Am 1994; 8: 593–603.

28. Krause JR. The automated white blood cell differential. Hematol Oncol Clin North Am 1994; 8: 605–16.

29. Israels LG, Israels ED. Mechanisms in hematology. Winnipeg 1996; The University of Manitoba.

30. West AH, Churpek JE. Old and new tools in the clinical diagnosis of inherited bone marrow failure syndromes. Hematology 2017; 1: 79–87.

31. Veninga A, De Simone I, Heemskerk JWM, ten Cate H, van der Meijden PEJ. Clonal hematopoietic mutations linked to platelet traits and the risk of thrombosis or bleeding. Haematologica 2020; 105 (8): 2020–31.

32. Bazarbachi A, Bug G, Baron F, Brissot E, Ciceri F, Dalle IA, Döhner H, et al. Clinical practice recommendation on hematopoietic stem cell transplantation for acute myeloid leukemia patients with FTL3-internal tandem duplication: a position statement for the Acute Leukemia Working Party of the European Society for Blood and Marrow transplantation. Haematologica 2020; 105 (6): 1507–16.

33. Leaf RK, Dhami RS,Chun NS,Ta R. Case 11-2022: An 80-year-old woman with pancytopenia. N Engl J Med 2022; 386 (15): 1453–61.

34. Hoermann G. Clonal hematopoiesis of indeterminate potential: clinical relevance of an incidental finding in liquid profiling. J Lab Med 2022; 44 (4): 301–310.

35. Pham HM, Nguyen DLM, Duong MC, Phan XT, Tran LT, Tan LT, et al. Neutrophil CD64 – a prognostic marker of sepsis in intensive care unit: a prospective cohort studx. Front Med 2023. doi: 103389/fmed.2023.1251221.

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15.2 Erythrocytes (cell count and indices)

Lothar Thomas

The red blood cell (RBC) count is a basic examination for the evaluation of erythropoietic disorders. RBCs are investigated in further detail by measuring the hemoglobin concentration, the mean cell volume (MCV)of the erythrocyte and the red cell distribution width (RDW).

Based on the measured RBC count, the MCV, and the hemoglobin concentration, hematology analyzers calculate the following parameters:

  • Hematocrit, also referred to as packed cell volume
  • Mean cell hemoglobin (MCH)
  • Mean cell hemoglobin concentration (MCHC).

MCV, MCH, and MCHC are referred to as RBC indices and serve to describe RBC changes and to differentiate erythropoietic disorders. A sensitive marker is the determination of the proportion of hypochromic red blood cells (%HYPO).

15.2.1 Erythrocyte count

The main significance of the RBC count is based on the measurement of erythrocyte indices such as the hematocrit, MCV and MCHC.

15.2.1.1 Indication

In combination with erythrocyte indices:

  • Differentiation of anemia
  • Diagnosis of polycythemia and polyglobulia.

15.2.1.2 Method of determination

Current major analytic methods are variations of the flow cytometry using electrical resistance or impedance measurements. The methods incorporate laser technology (flow cytometry) or cytochemical techniques or both /1/.

15.2.1.2.1 The automated complete blood count (CBC)

Principle: analysis of the CBC begins with the automatic aspiration of a specific amount of well-mixed blood specimen. The specimen is automatically aliquoted, diluted with appropriate diluents with or without added red cell lysing agent, and channeled to specific counting chambers, baths or flow cells for cell counting and sizing and to a spectrophotometric cuvette for hemoglobin determination /2/.

Light scattering technology

A specific amount of diluted blood sample (cell suspension) is hydrodynamically focused in a flow cell, which is illuminated by a narrow beam of light. As a cell passes through the illuminated area of the flow cell, it scatters light. The light scattered by each cell is detected by a photo detector and converted to an electrical pulse, whose amplitude is proportional to the cell volume. The number of pulses generated is proportional to the number of cells passing through the illuminated area. The measurement of scattered light at two angles (2.5° to 3.5° and 5 to 15°) in the forward direction permits the simultaneous determination of the red cell and platelet count, the size and Hb content of red cells and the differentiation of leukocytes /2/.

The red blood cell count (RBC) and the platelet (PLT) count are obtained from the number of pulses generated within a predetermined size range (PLT 0–20 fL, RBC 30–180 fL).

The MCV and CHCM (comparable to MCHC) are derived from histograms of the red blood cell volume and the Hb concentration (Fig. 15.2-1 – Erythrogram).

The HCT, MCH and MCHC are calculated by formulas.

The white blood cell count (WBC) is obtained from another cell suspension in which red blood cells have been lysed and leukocytes are fixed and stained for enzymes like myeloperoxidase. This stained cell suspension is hydrodynamically focus in another flow cell, which is illuminated by a tungsten-halogen light source. As the cells pass through the illuminated area, the light scatter as well as the light absorption are measured. The data obtained are displayed in a cytogram along with leukocyte count and differentiation. See Fig. 15.2-2 – Histogram of red cell distribution width.

Impedance technology

A specific amount of diluted blood sample flows through a small aperture located between two sensing electrodes. As each cell passes through the aperture, a momentary increase in impedance is recorded in the form of an electrical pulse. The amplitude of the pulse is proportional to the cell volume. The number of pulses generated is proportional to the number of cells passing the aperture /2/.

The RBC and the platelet count, although performed on the same cell suspension, are determined separately by analyzing the number of pulses of cell size > 36 fL (red cells) and 2–20 fL (platelets).

The HCT, MCH and MCHC are calculated by formulas.

For hemoglobin determination red cells are lysed in a specific lysing bath and measured by the spectrophotometric cyanmethemoglobin method.

The leukocyte count and differential are based on the principle of cell counting and sizing by detection and measurement of changes in electrical resistance as cells pass through an aperture between two electrodes suspended in a conductive diluent. The different cell types are distinguished electronically by the pulses they generate. Using a specific reagent that results in differential shrinkage of leukocytes, these cell types may be differentiated and expressed on a histogram /1/.

15.2.1.3 Specimen

EDTA blood: 1 mL

Capillary blood (heparinized) from finger tip or heel.

15.2.1.4 Reference interval

Refer to references /234/.and Tab. 15.2-1 – Erythrocyte reference intervals.

15.2.1.5 Clinical significance

The RBC count as a single parameter is of little diagnostic value. An evaluation of the red cell mass of the body (i.e., the differentiation between erythrocytopenia, erythrocytosis or a normal RBC mass) can only be achieved in combination with the hematocrit. The reason for the minimal significance of the RBC count is that changes in the plasma volume are reflected by the RBC count (e.g., as observed during pregnancy or in the case of disorders of water and electrolyte balance). For instance, in strenuous physical activity, depending on the intensity and the duration of the exercise, the RBC count, the hematocrit, and the hemoglobin concentration rise by 10–30% as compared to the baseline values.

The red cell mass of healthy individuals determined with radioactively labeled autologous erythrocytes is 30–36 mL/kg body weight and the plasma volume, determined with radioactively labeled albumin, 33–39 mL/kg body weight /6/. The data are not valid in cachectic or obese individuals.

In routine diagnostic investigation, the erythrocyte number is useful essentially for validating the credibility of the Hb value and the hematocrit. Thus, according to a rule the following relation is valid in cases with a normal blood count and in normocytic normochromic anemia:

Erythrocyte count (106/μL) × 3 = Hb value (g/dL) × 3 = hematocrit (%)

The rule assumes that the erythrocyte hemoglobin content changes linearly with erythrocyte volume, but this is only the case in normal erythropoiesis. Discrepancies are the rule with pathological changes such as:

  • Iron deficiency anemia. Hypochromic erythrocytes are deformed, consequently the cell volume and the hematocrit are underestimated in impedance measurement.
  • Beta thalassemia. These patients generally have mild anemia, elevated red cell count, normal or slightly reduced hematocrit, and decreased MCV.
  • Macrocytic anemia caused by folic acid or vitamin B12 deficiency, alcoholism and chronic liver disease
  • Hereditary spherocytosis with elevated hemoglobin content in relation to the MCV of the erythrocyte
  • Interference with hemoglobin determination due to hyperlipidemia
  • Cold agglutinins. The erythrocyte number is underestimated because of red cell aggregation.
  • Marked hemolysis of the sample. The erythrocyte number is inappropriately low in comparison to the hemoglobin concentration.
15.2.1.5.1 Anemia

Acute anemia (e.g., due to acute hemorrhage) is hardly recognizable by a decrease in the RBC count or the hematocrit during the first 24 h because RBC and plasma are lost in an equal ratio. A decline in the RBC count does not occur until fluid shifts occur to correct the blood volume deficit created by the blood loss.

In chronic anemia, the blood volume is usually normal (i.e., decline in red cell mass and increase in the plasma volume). The RBC count and the hematocrit are usually reduced. However, in the case of pronounced microcytosis (e.g., severe iron deficiency anemia or thalassemia) the RBC count may be within the reference interval because of increased compensatory red cell production. The number of RBC can be also elevated in hereditary spherocytosis. See Section 15.4 – Hematocrit.

15.2.1.5.2 Cancer-associated anemia

Use of erythropoiesis-stimulating agents (ESAs) to manage anemia rises hemoglobin levels and reduces the need for red blood cell transfusions. ESA therapy is suspected of causing thrombosis The ESMO and the ASCO/ASH clinical practice guidelines recommend /2728/:

  • ESMO: ESA therapy is recommended in patients with symptomatic anemia who receive chemotherapy and present with an Hb level < 10 g/dL
  • ASCO/ASH: ESAs may be offered to patients with chemotherapy-associated anemia whose cancer treatment is not curative in intent and whose hemoglobin has declined below 10 g/dL.
15.2.1.5.3 Polycythemia

Conditions with an elevated red blood cell count are termed polycythemia or erythrocytosis. Absolute polycythemia, which is based on an expansion of erythropoiesis in the bone marrow, is distinguished from relative polycythemia, which is contingent upon a reduction in plasma volume. Polycythemias can be congenital or acquired. In secondary polycythemia, (erythrocytosis) increased erythrocyte formation results from augmented erythropoietin synthesis in hypoxic patients. Congenital polycythemia is rare and in newborns it is associated with a hematocrit of above 0.65 and hyper viscosity syndrome. Polycythemia, which is not to be mistaken for polycythemia vera, is present if the red blood cell mass is greater 25% of the expected value. In addition to the erythrocyte number, the hemoglobin level and hematocrit are elevated. For detailed information on polycythemia see Section 15.4 – Hematocrit. Mild polycythemia, which results from an increase in red blood cell mass and a reduction in plasma volume are observed in smokers.

15.2.1.6 Comments and problems

Anticoagulant

EDTA is recommended at 1.5–1.8 mg/mL of blood /7/. Among the EDTA salts, K3EDTA is the least suitable since it causes:

  • The strongest shrinkage of the RBCs as the EDTA concentration rises
  • The biggest time dependent MCV decline during the time interval between blood collection and automated measurement of the complete blood count.

K2EDTA appears to cause only mild shrinkage while Na2EDTA, in comparison, leads to mild swelling /7/. A rise in the EDTA concentration generally results in a decline in the MCV.

Sampling

If the blood is collected from an individual in a sitting position after a change from an upright position that lasted for at least 15 min., the RBC count is 5–10% higher than after at least 15 min. in a supine position /7/. A venous occlusion time of more than 2 min. results in a rise of the cell count by an average of 10%; this corresponds to a 1–2-fold increase above the maximally tolerable imprecision. Blood collection immediately after strenuous physical activity is associated with an increase of the RBC count by about 10%. All of these changes are due to hemoconcentration. In comparison to venous blood collection in skin puncture specimens in children the red blood cell count is increased by 6% and in adults by 1,8% /8/.

Interfering factors

Cold agglutinins: cold agglutinins of a high titer lead to the aggregation of erythrocytes if the sample is stored at room temperature. Measuring the automated complete blood count, the RBC count is falsely low and the MCV is too high. Consequently, the calculated hematocrit is too low and the MCH as well as the MCHC are too high /9/.

White blood cells: these cells are counted as part of the red cell count. The proportion is negligible as long as the WBC count is normal. If the WBC count is high as in chronic myeloid or lymphoid leukemia, the WBC count must be subtracted from the RBC count.

Platelets: large platelets (e.g., essential thrombo-cythemia) are also counted as part of the red cell count.

Intraindividual variation

Within day variation 4%, day-to-day variation 5.8%, month-to-month variation 5.0% /10/.

Stability

At room temperature (20 °C) and at 4–8 °C 3 days, at 37 °C 36 h; after that continuous decrease /11/.

15.2.2 MCV, MCH, MCHC, RDW, %HYPO

The evaluation of RBC is accomplished by additional measuring or calculating the following indices /6/:

  • MCV (mean cell volume) of red cells. The MCV is expressed in femtoliters (fL = 10–15 L), and is either directly measured by the hematology analyzer or calculated as follows:
MCV (fl) = Hematocrit (fraction) × 10 3 Number of RBC (10 12 /L)
  • MCH (mean cellular hemoglobin) of the RBC. MCH is expressed as pg/RBC and is calculated by the hematology analyzer according to the following equation:
MCH (pg) = Hemoglobin (g/L) Number of RBC (10 12 /L)
  • MCHC (mean cellular hemoglobin concentration). MCHC is expressed as red cell hemoglobin (g/dL or g/L) and is calculated as follows:
MCHC (g/L) = Hemoglobin concentration (g/l) Hematocrit (fraction)
  • RDW (red cell distribution width). The distribution of the MCV in a sample is shown in the form of a graph (Fig. 15.2-1 – Erythrogram). Some hematology analyzers also generate an erythrogram (the RBC hemoglobin content is plotted against their volume) (Fig. 15.2-2 – Histogram of red cell distribution width (RDW)).
  • %HYPO, the proportion of hypochromic RBCs with a hemoglobin concentration of less than 280 g/L, expressed in % of the total RBCs.

Based on the MCV, anemia is classified as normocytic, microcytic or macrocytic; and based on the MCH , as normochromic and hypochromic. Hyperchromic anemia does not exist.

15.2.2.1 Indication

Differentiation and monitoring of anemia.

Early diagnosis of anemia (%HYPO).

15.2.2.2 Method of determination

MCV (see also Section 15.2.1.2 – Method of determination)

Impedance technology: when the red blood cell flows through the aperture of the sensing electrodes, a sudden increase in impedance is recorded in the form of an electrical pulse. The amplitude of the pulse is proportional to the cell volume.

Light scattering technology /2/: a specific amount of diluted blood sample is hydrodynamically focused in a flow cell that is illuminated by a laser or mercury-halogen light. The light that is scattered by every red blood cell in the flow cell is converted to an electrical pulse whose amplitude is proportional to the cell volume. The measurement of scattered light at two angles in the forward direction permits simultaneous determination of the size and hemoglobin content of the red cell.

Proportion of hypochromic red cells (%HYPO)

Determination can only be performed by hematology analyzers that transform the red blood cell to a sphere. The hematology analyzer is capable of separately measuring red cell corpuscular volume, corpuscular hemoglobin concentration (CHCM) together with the MCV. The proportion of red blood cells with a hemoglobin concentration of less than 280 g/L is recorded and expressed as %HYPO. The %HYPO play a prominent role in the assessment of iron deficient erythropoiesis. In addition the analyzer determines the fraction of microcytic (% MICRO) and macrocytic (% MACRO) red blood cells /12/.

15.2.2.3 Specimen

EDTA blood: 1 mL

15.2.2.4 Reference interval

See references /2351314/ and Tab. 15.2-2 – Reference intervals of erythrocyte indices.

15.2.2.5 Clinical significance

The erythrocyte indices MCV, MCH, MCHC, RDW are important tools for /15/:

  • Classification of anemia
  • Detection of latent anemia
  • Etiological clarification of anemia.

The MCV should be estimated along with the RDW. Thus, in microcytic anemia, an elevated RDW is indicative of iron deficiency anemia, while a normal RDW suggests thalassemia.

15.2.2.5.1 MCV

Determination of MCV supports the diagnostically important classification of anemia into normocytic, microcytic and macrocytic forms. It must be noted that the MCV is an averaged value, and that small populations of microcytic or macrocytic erythrocytes cannot be recognized.

MCV depends on the hemoglobin content and hydration of the erythrocyte (i.e., plasma osmolality). Markedly hypochromic erythrocytes are subject to more severe deformation than those with normal Hb content and, in consequence, the MCV is underestimated.

Normal MCV

The MCV is normal if:

  • The majority of the RBCs have a cell size within the reference interval; this is associated with an RDW that does not exceed 15 fL
  • Both large and small RBCs are present; this is associated with a RDW that is > 15 fL. Such a situation occurs, for example, in immune hemolytic anemias (IHA) or in a microangiopathic hemolytic anemia. In IHA the simultaneous presence of microspherocytes which are smaller than normal RBCs and reticulocytes as well as polychromatic RBCs which are larger than normal RBCs may cause a mean of the MCV to be within the reference interval. In an increased RDW, a peripheral blood smear should be examined which, in this situation, will demonstrate anisocytosis, polychromasia, and microcytosis. In disseminated intravascular coagulation, fragmentocytes that are counted as small RBCs and large polychromatic RBCs may be present but the MCV may still be within the reference interval.

Decreased MCV

Microcytosis may be caused in iron, copper and vitamin B6 deficiency as well as in hereditary conditions. The most common cause is iron deficiency. Because of iron deficiency, the erythropoiesis undergoes one or more cell divisions as normal and with each cell division the RBC size becomes smaller. The RDW is > 15 fL, the peripheral blood smear shows microcytosis and anisocytosis. An increase in the RDW is an early sign of iron deficiency anemia.

Hereditary sideroblastic anemia is a rare microcytic anemia, commonly with a markedly decreased MCV of below 60 fL. It is often misdiagnosed as thalassemia, particularly thalassemia inter media.

Increased MCV

Macrocytosis occurs due to the following causes:

  • In regenerative anemia (e.g. supplementation of deficiency-induced anemias)
  • Smoking, liver cirrhosis
  • Alcoholism; the mean increase is about 5 fL i.e., 5–10% above the mean value of healthy controls. A cutoff level ≤ 96 fL is considered to be a threshold of non chronic alcoholism. MCV normalization is to be anticipated 3–4 months after abstention from alcohol; a cutoff of ≤ 94 fL is taken as the threshold for the assessment of alcohol abstinence. Due to its long half-life time, MCV is unsuitable as a clinical control marker for alcohol withdrawal /16/.
  • Chronic liver disease. Some 20% of patients with non-alcoholic liver disease have macrocytosis that cannot be accounted for by folic acid or vitamin B12 deficiency or reticulocytosis due to bleeding.
  • Vitamin B12 or folate deficiency. These deficiencies impair DNA synthesis which reduces the rate of nuclear replication and mitosis, resulting in fewer cellular divisions during RBC development. The decrease in divisions causes RBCs to be larger than normal. The rate of hemoglobin synthesis is not impaired. A normal MCV does not rule out a vitamin B12 or folate deficiency /17/. A major reason is that some 20% of vitamin deficient patients are also iron-deficient.
  • Reticulocytosis. Depending on the regenerative erythropoietic response, the volume of reticulocytes is 3–10% greater than that of erythrocytes. Since the MCV measurement includes reticulocytes, a rise in the MCV of erythrocytes is to be expected in reticulocytosis of about 15% or more.
  • Myelodysplastic syndrome, hereditary stomatocytosis.
15.2.2.5.2 MCH

Generally, the erythrocyte volume is provided with 95% of the maximum possible content of Hb and, consequently, MCH correlates with MCV in most types of anemia. Accordingly, microcytosis corresponds to hypochromia, normocytosis to normochromia.

Normal MCH

Normal MCH is typical of healthy individuals but is often also observed in acute hemolytic anemia and in anemia of chronic disease (e.g., infection, inflammation, chronic liver disease, and malignant tumors).

Low MCH

Low MCH indicates a decrease in the hemoglobin content of the red cells and is, for example, typical of iron, copper and vitamin B6 deficiency.

High MCH

Conditions with a high MCH also cause an increase in the MCV. This is mainly the case in macrocytic anemias, (e.g., due to folate and vitamin B12 deficiency) as well as in regenerative anemias (e.g., as observed during the supplementation of iron deficiency anemia).

15.2.2.5.3 MCHC

The MCHC is an estimate of the hemoglobin concentration of the red blood cell. Because of the comparable behavior of cell size and hemoglobin content of the individual red cell, the MCHC remains constant in many hematopoietic diseases. Hemogram abnormality related to red blood cell count, Hb concentration, MVC or hematocrit leads to abnormal calculated red blood cell indices, especially MCHC.

Normal MCHC

In many types of anemia the MCHC is within the reference interval.

Decreased MCHC

A decreased MCHC may occur in deficiency-induced anemias (e.g., iron, copper and vitamin B6 deficiency). If falsely low hemoglobin concentrations or a falsely elevated hematocrit are measured, the MCHC is also decreased.

Increased MCHC

An increased MCHC is found in the presence of cold agglutinins at a high titer and in hereditary spherocytosis.

RDW

The RDW allows evaluation of whether the RBC of a patient are isocytic or anisocytic. Very high RDW values are measured in acute hemolytic anemias and are a sign of an underlying reticulocytosis. Tab. 15.2-3 – Classification of anemia based on MCV and RDW shows the classification of anemias, dependent on the RDW and the MCV.

According to the results of the Third National Health and Nutrition Examination Survey 1988–1994 (NHANES III), RDW is an indicator of mortality risk in middle-aged individuals and in elderly. The mortality risk in individuals without carcinoma or cardiovascular disease, but with an RDW of over 14.05%, was twice as high as in those with an RDW of below 12.6% /19/.

15.2.2.5.4 %HYPO

Determination of the proportion of hypochromic red cells is a more sensitive marker than erythrocyte indices for detection of hypochromic red cells and thus iron-restricted erythropoiesis. A decrease in erythrocyte hemoglobin content of up to 10% does not lead to a significant change in MCV, MCH or MCHC. The determination of %HYPO has a detection limit of 2% with acceptable precision. Incipient erythrocyte iron deficiency is, therefore, recognized earlier than by the determination of erythrocyte indices. This is also the case with monitoring of iron supplementation. A response is indicated with the decline in %HYPO after 2–3 weeks /20/.

In individuals with low ferritin levels, the iron stores are empty. In this situation, a normal %HYPO indicates that iron-restricted erythropoiesis has not, as yet, occurred, and that the iron supply for erythropoiesis is still at a level that enables normal hemoglobin synthesis (latent iron deficiency, iron deficiency without anemia).

Patients with chronic kidney disease should have a balanced iron metabolism, particularly under treatment with erythopoiesis stimulating agents (ESA). The European Best Practice Guidelines for the Management of Anemia in Patients with Chronic Renal Failure recommend a minimal ferritin concentration ≥ 100 μg/L (better 200–500 μg/L), %HYPO below 10 (better below 2.5) or, instead of %HYPO, transferrin saturation above 20% (better 30–40%) /21/.

15.2.2.5.5 %MICRO/%HYPO

This ratio is a screening test for β-thalassemia if the microcytic red blood cell fraction is above 18%. A ratio above 0.90 with greater than 18% microcytic erythrocytes is indicator of β-thalassemia /22/. The diagnostic sensitivity of the ratio is acceptable and its specificity is moderate.

15.2.2.5.6 %MACRO

If reticulocytosis is not present, the macrocytic red blood cell fraction (%MACRO) is an earlier indicator than MCV with regard to the following:

  • Detection and assessment of the progression of vitamin B12 or folate deficiency anemia
  • Assessment of alcohol abstinence by monitoring of red blood cell volume.

The classification of anemia based on MCV, MCH and MCHC is shown in Tab. 15.2-4 – Classification of anemia based on MCV, MCH and MCHC.

15.2.2.6 Comments and problems

MCV

If the lower threshold of the hematology analyzer is set too high, the MCV is too high because small RBCs are no longer measured. If the upper threshold is too high, white blood cells are measured as well.

In the presence of several RBC populations, the MCV represents only the arithmetic mean. Therefore, without knowledge of the RDW, microcytosis in particular may be overlooked. The MCV measured by hematology analyzers yields lower results than the manual method because in the procedure for determining packed cell volume by the micro hematocrit method, the hematocrit is measured too high because of the inclusion of a minimal amount of plasma.

MCH

In the case of pronounced hypertriglyceridemia and in leukocytosis of > 50 × 109/L, the hemoglobin concentration and thus also the MCH are determined at too high a level because of light absorption and light scattering.

MCHC

Since the inter individual variation of MCHC is small, this red cell index is well suited for plausibility control of hemogram. It is also suited for controlling the analytical reliability of the hematology analyzer used (e.g., by comparison of the variability of the daily mean from day to day, and for verifying the adjustment of the threshold values).

Spurious increased MCHC induces an analytical alarm. Either it is just an artefact, or it refers to a true pathological sample. If it is a problem of the analytical reliability of the analyzer it needs prompt corrective action to ensure delivery of right results to the clinician e.g., red blood cell count (RBC), Hb, and MCV. Problems causing an increase of MCHC are /25/:

  • decreased RBC (cold agglutination)
  • RBC disease
  • lipemic, hemolytic, and icteric plasma.
  • leukocytosis
  • spherocytosis
  • electrolyte disorders.

Hyperglycemia

Due to the swelling of RBCs, blood glucose levels above 600 mg/dL (33.3 mmol/L) cause a rise in the MCV and the hematocrit as well as a decline in the MCHC /23/.

Cold agglutinins

See section 15.2.1.6. Cold agglutinins are found to be increased in /24/:

  • Infection with Mycoplasma pneumoniae. In this case high-titer IgM class cold agglutinins are detectable
  • The Epstein-Barr virus (EBV) infection (infectious mononucleosis). This refers to class IgM or IgG anti-i cold agglutinins.
  • Malignant, lymphoproliferative B-cell diseases such as chronic lymphocytic leukemia or other malignant lymphomas. In these cases monoclonal immunoglobulins, particularly of the IgM class, are responsible for the cold agglutination of red blood cells.

Warming of blood samples to 37 °C prior to measurement cancels the cold agglutination. Cold agglutination can be verified with a blood smear. Agglutination of the erythrocytes is confirmed at 250-fold magnification.

RDW

Intraindividual variation is within 24 hours 1.7%, day-to-day 5.9% and month-to-month 5.3% /10/.

Stability /11/

  • Erythrocyte count: with room temperature (RT) and at 4 °C 72 h
  • MCV: at 4 °C 3 days, at RT 12 h, at 37 °C 8 h
  • MCH: at 4 °C and RT 3 days; at 37 °C 24 h
  • MCHC: at 4 °C 3 days; at RT 7 h
  • RDW: at 4 °C 3 days; at RT 7 h.

Reticulocyte count

In reticulocyte count 8.6% of cases exhibited interference. The main interferents of spuriously high reticulocyte count were caused by parasites, such as malaria, as well as autofluorescence due to drugs. The main interferents of spuriously low reticulocyte count were caused by red blood cell fragments. The presence of numerous red blood cell fragments in blood samples can cause spuriously low red blood cell count and high platelet count, thus affecting the reticulocyte count /26/.

References

1. Krause JR. The automated white blood cell differential. Hematol Oncol Clin North Am 1994; 8: 593–603.

2. Gulati GL, Hyun BH. The automated CBC. Hematol Oncol Clin North Am 1994; 8: 605–616.

3. Nebe T, Bentzien F, Bruegel M, Fiedler GM, Gutensohn K, Heimpel H, et al. Multizentrische Ermittlung von Referenzbereichen für Parameter des maschinellen Blutbildes. J Lab Med 2011; 35: 3–28.

4. Saarinen UM, Siimes MA. Developmental changes in red blood cell counts and indices of infants after exclusion of iron deficiency by laboratory criteria and continuous iron supplementation. J Pediatr 1978; 92: 412–6.

5. Taylor MRH, Holland CV, Spencer R, Jackson JF, O’Connors GI, Donnell JRO. Haematological reference ranges for school children. Clin Lab Haem 1997; 19: 1–15.

6. Geaghan SM. Hematologic values and appearances in the healthy fetus, neonate and child. Clin Lab Med 1999; 19: 1–37.

7. NCCLS. Procedure for determining packed cell volume by the microhematocrit method – second edition; approved standard. NCCLS Document H7–A2, Vol 13, No 9. Villanova: NCCLS, 1993.

8. Schalk E, Scheinpflug K, Mohren M. Kapilläre Blutbildanalysen in der klinischen Praxis: eine sichere, zuverlässige und valide Methode. J Lab Med 2009; 33: 303–9.

9. Wisser H. Störungen der Messgrößen des kleinen Blutbildes durch Antikörper. GIT Labor-Medizin 1995; 83–5.

10. Costongs GMPJ, Janson PCW, Bas BM, et al. Short-term and long-term intra-individual variations and critical differences of haematological laboratory parameters. J Clin Chem Clin Biochem 1985; 23: 69–76.

11. Empfehlungen der Arbeitsgruppe Präanalytik der DGKC. Stabilität der Messgröße in der Probenmatrix. DG Klinische Chemie Mitteilungen 1996; 27: 74–8.

12. Buttarello M, Bulian P, Venudo A, et al. Laboratory evaluation of the Miles H3 automated reticulocyte counter. A comparative study with manual reference method and Sysmex R-1000. Arch Pathol Lab Med 1995; 119: 1141–8.

13. Thomas C, Thomas L. Biochemical markers and hematologic indices in the diagnosis of functional iron deficiency. Clin Chem 2002; 48: 1066–76.

14. Segel GB, Oski FA. Hematology of the newborn. In: Williams WJ, Beutler E, Erslev AJ, Lichtman MA (eds). Hematology. New York 1990; McGraw Hill: 476.

15. Tyler RD, Cowell RL. Classification and diagnosis of anemia. Comp Haematol Int 1996; 6: 1–16.

16. Peach HG, Bath NE, Farish S. Predictive value of MCV for hazardous drinking in the community. Clin Lab Haem 1997; 19: 85–7.

17. Oosterhuis WP, Niessen RWLM, Bossuyt PMM, Sanders GTB, Sturk A. Diagnostik value of the mean corpuscular volume in the detection of vitamin B12 deficiency. Scand J Clin Lab Invest 2000; 60: 9–18.

18. Anagnou J. Mikrozytose, Hypochromie und Erythrozytenindizes im Wandel. Dtsch Med Wschr 1985; 110: 657–8.

19. Patel KV, Ferrucci L, Ershler WB, Longo DL, Guralnik JM. Red blood cell distribution width and the risk of death in middle-aged and older adults. Arch Intern Med 2009; 169: 515–23.

20. McDougal IC. What is the most appropriate strategy to monitor functional iron deficiency in the dialysed patient on rHuEPO therapy? Merits of percentage hypochromic red cells as a marker of functional iron deficiency. Nephrol Dial Transplant 1998; 13: 847–9.

21. European Best Practice Guidelines for the Management of Anemia in Patients with Chronic Renal Failure. Target guideline 6: assessing and optimizing iron stores. Nephrol Dial Transplant 1999; 14 (Suppl 5): 14–5.

22. d’Onofrio G, Zini G, Ricerca BM, Mancini S, Mango G. Automated measurement of red blood cell microcytosis and hypochromia in iron deficiency and β thalassemia trait. Arch Pathol Lab Med 1992; 116: 84–9.

23. van Duijnhoven HLP, Treskes M. Marked interference of hyperglycemia in measurements of mean (red) cell volume by Technicon H analyzers. Clin Chem 1996, 42: 76–80.

24. Strobel SL, Panke TW, Bills GL. Cold erythrocyte agglutination and infectious mononucleosis. Laboratory Medicine 1993; 24: 219–21.

25. Haddad YB, Faure C, Boubaya M, Arpin M, Cointe S, Frankel D, et al. Increased mean corpuscular hemoglobin concentration: artefact or pathological condition? Int Jnl Lab Hem 2017; 39. 32–41.

26. Jiang H, Wang J, Wang K, Gu J, Chen J, Wang Z. Interferents of automated reticulocyte analysis integrated with relevant clinical cases. Clin Lab 2019: 65; 1251–9.

27. Bohlius J, Bohlke K, Castelli R, Djulbegovic B, Lustberg MB, Martino M, et al. Management of cancer-associated anemia with erythropoiesis-stimulating agents: AsCO/ASH clinical practice guideline update. Blood Advancea 2019; 3 (8): 1197–1210.

28. Aapro M, Beguin Y, Bokemeyer C, Jordan K, Herrstedt J., et al Management of anaemia and iron deficiency in patients with cancer: ESMO clinical practice guidelines. doi: 10.1093/annonc/mdx/7658.

15.3 Hemoglobin concentration

Lothar Thomas

The hemoglobin (Hb) concentration is a function of the number of red blood cells their MCH and the proportion of blood plasma. With a constant plasma volume, there is a direct relationship between blood Hb level and red blood cell mass. A decrease in an organisms’ Hb content is termed anemia. Anemia is present if the red blood cell mass of the body is reduced (normal 21–27 mL/kg BW in women and 24–32 mL/kg BW in men) /1/. Since the red blood cell mass can only be measured with radioactive methods the determination of the Hb value in a defined blood volume is a simple alternative. This is because the Hb concentration includes the product of the erythrocyte number and the Hb content of the red cell. Anemia is diagnosed from the Hb level if the blood volume is normal. This is the case in anemia that has been present for longer than 48 hours. The reason is that in this case, the decrease in red blood cell mass is compensated for by an increase of plasma volume. The blood volume is 90 mL/kg BW in newborns and in older children and in adults it is 80 mL/kg BW.

15.3.1 Indication

Diagnosis, and monitoring in anemia, erythrocytosis, and polycythemia.

15.3.2 Method of determination

Hemiglobin cyanide method /2/

Principle: in solution, Fe2+ of Hb is oxidized by potassium hexacyanoferrate [K4Fe(CN)6] to Fe3+. Hemiglobin (Hi) is formed and generates HiCN with cyanide ions (CN), which are provided in the solution by potassium cyanide (Tab. 15.3-1 – Formation of hemiglobin cyanide from hemoglobin). HiCN has an absorption maximum at 540 nm, and the absorption of HiCN is proportional to Hb concentration. Hematology analyzers are calibrated with secondary HiCN standards that contain 500–800 mg/L HiCN. For HiCN methods employing a 250-fold dilution of blood samples, this provides an equivalent Hb concentration of 125–200 g/L. The HiCN method is the reference method. See also Section 15.2.1.2 – Method of determination.

15.3.3 Specimen

EDTA blood (disodium or dipotassium EDTA): 1 mL

Capillary blood (heparinized capillaries): 0.02–0.05 mL.

15.3.4 Reference interval

See references /3456/ and Tab. 15.3-2 – Hemoglobin reference intervals.

15.3.5 Clinical significance

The Hb concentration combined with the hematocrit and the RBC count is an important criterion for the diagnosis and differentiation of anemia, erythrocytosis, and polycythemia. Frequent causes of anemia are shown in Fig. 15.3-1 – Frequent causes of anemia.

15.3.5.1 Diagnosis of anemia

The term anemia describes a decline of the Hb level, due to /78/:

  • An absolute reduction in the erythrocyte number (e.g., in anemia of chronic disease)
  • A decrease in erythrocyte Hb content. This can be the case with a normal, slightly reduced or even increased cell count (e.g., in iron deficiency anemia, heterozygous β-thalassemia).
  • An increase in plasma volume with a relative reduction of the erythrocyte number with normal or even increased red blood cell mass (e.g., during the last trimester of pregnancy). In this case, the condition is referred to as pseudoanemia.

The diagnosis of anemia is an important aspect of the practice of hematology. To decide whether a patient is anemic compared on the basis of the population distribution of Hb values is problematic /9/. Proposed lower thresholds are shown in Tab. 15.3-3 – Proposed low hemoglobin thresholds for adults.

In children, the threshold values for anemia can only be related as a function of age. The proposed thresholds of normal of the Centers for Disease Control (CDC) in the USA /10/ are shown in Tab. 15.3-4 – Proposed low hemoglobin thresholds for children .

For certain groups of individuals such as smokers, or as a function of living conditions (living at high altitudes), adjustments in the Hb level are required. Refer to:

15.3.5.2 Extent of anemia

Depending on the Hb level, the extent of anemia is classified as /11/:

  • Mild; from the lower reference interval value to 100 g/L
  • Moderate 100–80 g/L
  • Severe 80–65 g/L
  • Life-threatening; below 65 g/L.

15.3.5.3 Clinical symptoms of anemia

Clinical symptoms are cold and pale skin, fatigue, palpitation, low endurance level, depression, disturbance of cognitive function, and a general reduction in quality of life. In severe anemia, if the Hb value is reduced by about 50%, cardiac decompensation with congestive heart failure may occur, since coronary blood flow will have reached its maximum. This situation is particularly critical in patients with pre-existing coronary heart disease. Mild proteinuria can also occur in severe anemia, due to a decrease in renal blood flow /12/.

The severity of clinical symptoms is dependent upon the extent of anemia and other factors /12/:

  • The physiological status of the patient. Healthy young individuals have fewer and milder symptoms than elderly individuals.
  • Co morbidity. Thus, in individuals with multiple co morbidities or in bed-ridden patients, even only slight decreases in the Hb concentration lead to symptoms such as tiredness, falling upon standing up, claudication or angina pectoris-like symptoms.
  • The pace of occurrence of anemia. Anemias that develop slowly are often first noticed in apparently healthy individuals in situations of physical or mental stress.

15.3.5.4 Prevalence of anemia

In Europe and the USA, approximately 1% of adult males and 3–5% of adult females are anemic; in Africa, the numbers are 27% and 48%, and in southeast Asia they are 40% and 57%. The most common causes are deficient nutrition and malnutrition, as well as hookworm infestation /13/. Approximately half of all anemia is contingent upon iron deficiency; worldwide, some 500 million individuals are believed to suffer from iron deficiency anemia, while the number of individuals with iron deficiency but without anemia is 3 times as high.

15.3.5.5 Tolerance to anemia

Healthy young individuals with normovolemia

These individuals show no indication of a critical change in O2 supply down to a Hb value of 50 g/L. From ≤ 60 g/L, however, ECG changes and disturbances of cognitive function may occur. Hb values of 45–50 g/L are an absolute indication for substitution with banked blood.

Patients with cardiovascular disease (CVD)

Patients with stable CVD tolerate Hb values of 70–80 g/L without hypoxic damage. Values below 70 g/L increase morbidity and mortality.

Surgical patients /14/

A preoperative Hb value ≤ 100 g/L is associated with increased peri operative mortality. This is not the case with a peri operative decline in Hb to ≤ 100 g/L in patients without CVD. The mortality risk is greatest if, in CVD patients, the intraoperative fall is ≥ 40 g/L. With regard to all patients, a peri operative fall in Hb down to 70 g/L is associated with increased morbidity but not with increased mortality. Nonetheless, each reduction of 10 g/L below 70 g/L increases the mortality risk 1.5-fold.

Patients without CVD and a preoperative Hb value of 60–90 g/L and only minimal pre-operative blood loss have an odds ratio of 1.4 for mortality, in comparison with those with pre-operative Hb values > 120 g/L (see also Tab. 15.4-2 – Hematocrit reference intervals.

Intensive care patients

Ventilated intensive care patients with poly trauma and sepsis do not seem to benefit from transfusion to Hb values > 90 g/L. Only in the event of massive blood loss or diffuse bleeding diathesis does an Hb value > 100 g/L seem to contribute to the stabilization of blood coagulation.

15.3.5.6 Classification of anemia

Anemia can be classified as follows:

  • According to pathogenesis. However, this classification is problematic, since in many forms of anemia multiple mechanisms contribute to pathogenesis.
  • Based on erythrocyte morphology into microcytic, normocytic and macrocytic forms; or based on red blood cell Hb concentration into hypochromic and normochromic forms. This classification has gained acceptance in the differential diagnosis of anemia.
  • According to erythropoietic regenerativity as hypo-, normo- and hyper-regenerative anemia
  • According to the form of progression into acute and chronic
  • Into congenital and acquired anemia.

Classification of anemia according to erythropoiesis regenerativity is shown in Tab. 15.3-7 – Classification of anemia according to erythropoietic activity of the bone marrow.

Acute anemia

Acute anemia (e.g., due to hemorrhage) can only be recognized with the hematocrit, the erythrocyte count and the Hb concentration following 24 hours, because no sufficient compensatory increase in plasma volume occurs during the initial hours.

Chronic anemia

In patients with chronic anemia the blood volume is normal, since plasma volume has increased according to the decrease in red blood cell mass. As a result there is a decrease in the erythrocyte count and a reduction of the hematocrit and Hb value.

Relative anemia

Relative anemia is a condition with normal red blood cell mass, but in which the total blood volume is increased due to an increased plasma volume. The cause is a regulatory change in the water and electrolyte balance (e.g., during pregnancy). In contrast to chronic anemia, in which serum total protein is usually found to be normal, total protein in relative anemia (pseudoanemia) is low or low-normal, with the exception of Waldenströms’ macroglobulinemia.

Aplastic anemia

Aplastic anemia refers to the failure of marrow to form blood, and hematopoietic failure is the end-organ effect of diverse pathophysiological mechanisms. Bone marrow cellularity is decreased, the blood count indicates pancytopenia /73/.

15.3.5.7 Differentiation of anemias

Microcytic hypochromic anemia is the most common form of anemia. It is a very heterogenous group of diseases that may be either acquired or inherited. Microcytic hypochromia is often diagnosed in children, adolescents, women before menopause and in pregnant women (mostly due to iron deficiency) /64/.

15.3.5.7.1 Microcytic hypochromic anemias

Microcytic hypochromic anemia can result from /64/:

  • Iron deficiency
  • A defect in globin genes (hemoglobinopathies or thalassemias)
  • A defect in heme synthesis
  • A defect in iron availability or in iron acquisition by the erythroid precursors.

The microcytic anemias can be sideroblastic, a trait which reflects the implications of different gene abnormalities.

The inherited microcytosis due to defects in heme synthesis are /64/:

The inherited microcytosis due to iron metabolism deficiency are /64/:

  • Iron deficiency anemia (refer to Tab. 7.1-2)
  • Ferroportin disease
  • Hereditary atransferrinemia (refer to Tab. 7.1-9)
  • Hereditary aceruloplasminemia (refer to Tab. 7.1-9).

For anemia differentiation the following Tables are recommended:

15.3.5.7.2 Macrocytic anemias

The interaction between folate and vitamin B12 is responsible for the megaloblastic anemia seen in both vitamin deficiencies. Dyssynchrony between the maturation of cytoplasm and that of nulei leads to macrocytosis, immature nuclei, and hyper segmentation in granulocytes in the peripheral blood. The dysplastic and hyper cellular bone marrow can be mistaken for signs of acute leukemia. The ineffective erythropoiesis results in intramedullary hemolysis and release of lactate dehydrogenase. Clinical and Laboratory findings of megaloblastic anemia are shown in:

15.3.5.7.3 Which test to choose?

Important laboratory tests for the differentiation and evaluation of the pathogenesis of anemias are:

  • Hematocrit as a measure for assessing the red cell blood volume fraction
  • Erythrocyte indices MCV, MCH and blood smear findings, for the identification of iron deficiency, vitamin B12 or folic acid deficiency or changes in erythrocyte shape
  • Reticulocyte count or reticulocyte index (RI) as a measure of the effectiveness of erythropoiesis; based on the RI, anemias are classified into normo-, hypo- and hyper regenerative
  • Reticulocyte indices such as the reticulocyte Hb content (CHr, Ret-He) for early diagnosis of iron-restricted erythropoiesis
  • Ferritin as an indicator of iron stores
  • Zinc protoporphyrin for diagnosis of iron-restricted erythropoiesis
  • Transferrin saturation (TSAT) or soluble transferrin receptor (sTfR) for estimation of functional iron
  • Soluble transferrin receptor (sTfR) in relation to hematocrit for the detection of intrinsic hypo proliferative erythropoiesis. See Section 7.4 – Soluble transferrin receptor.
  • Erythropoietin (EPO) concentration relative to the hematocrit value, for assessing the adequate stimulation of erythropoiesis (see Fig. 15.10-1 – Expected range of EPO concentration as a function of the hematocrit value)
  • EPO and assessment of observed/predicted (O/P) ratio; see also Section 7.4 – Soluble transferrin receptor. An O/P ratio of below 0.8 is indicative of inadequately low EPO stimulation and a value of above 1.2 suggests hyper proliferative erythropoiesis due to augmented EPO stimulation.
  • C-reactive protein for the detection of anemia of inflammation and infection
  • Haptoglobin as indicator of hemolytic anemia.

In most cases of anemia erythropoiesis is compromised due to:

  • Nutritional deficiencies (e.g., iron, folate, vitamin B12)
  • Chronic disease (e.g., systemic inflammation, solid malignant tumor, leukemia, lymphoma)
  • Organic disease (e.g., kidney, liver, thyroid or small intestine).

A bone marrow investigation is usually not necessary:

  • In cases of compromised erythropoiesis (except lymphoma and leukemia)
  • In all cases of microcytic anemia (except for suspected sideroblastic anemia).

15.3.5.8 Red blood cell function in anemia

The symptoms and severity of anemia depend on various factors, including the degree of anemia, the rapidity of onset, and the age and the physiologic status of the patient /12/. At rest, the amount of O2 required by the whole body ranges between 200 to 300 mL per minute. In health, the amount of O2 delivered to the whole body exceeds resting O2 requirements by a factor of 2–4-fold. In cardiac output of 5 liters per minute and oxygen saturation of 99% the delivery will be 1032 mL per minute. An isolated decrease in Hb to 100 g/L will result in an O2 delivery of 688 mL per minute and a Hb decrease to 50 g/L to an delivery of 342 mL per minute /15/.

There are several mechanisms by which the body tries to counterbalance the effects of anemia /12/:

  • Increased cardiac output with increase in the oxygen transport capacity, which is very effective, but metabolically expensive
  • Increase of the respiratory rate
  • Oxygen binding by hemoglobin
  • Reduction of pH in capillary blood and tissues, which leads to better dissociation of O2 from Hb and to vasodilation. The plasma bicarbonate concentration is reduced due to hyperventilation and the compensatory renal bicarbonate excretion.
  • Increased erythropoietin EPO secretion (see Tab. 15.10-1 – Reference intervals for erythropoietin); within 1–2 hours of acute onset of hypoxia (normobaric or hypobaric) circulating EPO levels begin to increase and erythropoiesis is stimulated. However, the erythropoietic activity is diminished dramatically when the arterial O2 saturation decreases below 60%.
  • The blood is shunted from presumably non vital donor organs to oxygen-sensitive recipient organs.

15.3.5.9 Increase in the O2 transport capacity

Stimulated erythropoiesis increases the number of red blood cells and the amount of Hb in the circulation. Any increase in the Hb level brings a proportional increase in O2 per volume of blood transported. The contribution of enhanced heart rate to the increase in cardiac output and the increase in the O2 transport capacity is variable. Hemodynamic compensation occurs solely via the increased stroke volume down to a fall in the Hb level to 75 g/L. If, additionally, extreme conditions are present, this compensation is limited by myocardial tissue. Under conditions of more intense performance requirements or a further decline in Hb, the shift in the oxyhemoglobin curve to the right is important.

With utilization-related compensation the oxygen reserves are exploited in an extreme manner. This utilization is as high as 90% in most organs, corresponding to a decline in blood O2 saturation from normally 150 mL/L to 10 mL/L.

The shift of the oxyhemoglobin curve to the right does not occur with acute loss of blood but only following a number of days due to enhanced formation of 2,3-DPG and its effect on Hb (Fig. 15.3-3 – Structure and function of hemoglobin). Due to an increased erythrocyte 2,3-DPG formation, there occurs a reduction in O2 affinity for Hb and, in consequence, a shift to the right of the Hb-O2 affinity curve, with increased O2 release in the tissues (see Fig. 15.4-4 – Effect of 2,3-diphosphoglycerate (2,3-DPG) on O2 saturation curve). In this way, half of the oxygen deficit caused by moderate anemia is compensated for.

15.3.5.10 Oxygen binding by hemoglobin

The O2 transport by Hb depends on the concentration of Hb and the Hb-O2 affinity, which determines the ease of O2 loading onto or unloading from the Hb molecule /16/. The dependency of the oxygen saturation of Hb on the PO2 in the blood is described by the Hb-O2 affinity curve (Fig. 15.3-2 – O2 affinity curve). The curve is characterized by the P50, the PO2 at which Hb is saturated by 50% with O2.

The physiological significance of a variable Hb-O2 affinity lies in the appropriate adjustment of the HbO2-binding in order to optimize both arterial O2 loading and peripheral O2 release. A shift to the left of the Hb-O2 affinity curve facilitates arterial oxygen loading a shift to the right indicates a low Hb-O2 affinity, which favours the oxygen release from Hb to the tissues.

The Hb-O2 affinity curve is shifted /16/:

  • To the right due to acidosis, a high CO2, high erythrocyte 2,3-DPG and an elevated body temperature
  • To the left due to alkalosis, hypocapnia, a decreased 2,3-DPG, and a low temperature.

Effect of pH /16/

The effect of pH on the Hb-O2 affinity is due to pH-dependent pK changes of ionizable amino acid residues of the Hb molecule. Acidosis stabilizes the deoxy form of the Hb molecule and decreases the Hb-O2 affinity (Bohr effect).

Effect of CO2 /16/

The main effect of CO2 on the Hb-O2 affinity is due to its effect on cell pH. Another effect is that an increase in CO2 at constant pH shifts the Hb-O2 affinity curve towards the right through the reversible formation of carbamates from CO2 and N-terminal residues of alpha- and beta chains of the Hb molecule.

15.3.5.11 Oxygen delivery in anemia

The erythrocytes are endowed with a content of 2,3-bisphosphoglycerate (2,3 BPG), that is nearly 103 times as high as the content in other body cells /1683/. The 2,3-BPG is synthesized in a side path of red cell glycolysis. Alkalosis stimulates glycolysis and increases 2,3-BPG in erythrocytes, whereas acidosis reduces the erythrocyte content of 2,3-BPG. The enzyme responsible for 2,3-BPG synthesis is 2,3-DPG mutase and for breakdown is 2,3-BPG phosphatase.

A reduction of free 2,3-BPG by binding to Hb upon deoxygenation increases its rate of synthesis and elevates to total 2,3-BPG content in the erythrocyte. Generally, organic phosphates bind preferentially to deoxy-Hb with an affinity 100 times higher than to that to oxy-Hb. The binding stabilizes the deoxy form of Hb.

2,3 BPG binds specifically to deoxy-Hb and lowers the fractional oxygen saturation, and the oxygen-binding curve is shifted to the right. The function of 2,3-BPG in the binding and release of O2 to the Hb molecule is shown in Fig. 15.4-4 – Effect of 2,3-diphosphoglycerate (2,3-BPG) on O2 saturation.

All patients with anemia have increased content of 2,3-BPG that enhances oxygen delivery (P02). In a person with anemia at any given oxygen tension the oxygen saturation is reduced. At a venous PO2 of 40 torr the oxygen saturation is about 80% and 20% of oxygen in the blood is unloaded. In cases of anemia elevate concentrations of 2,3 PBG in erythrocytes lower oxygen affinity of the blood allows a much higher fraction of oxygen to be unloaded.

Example /83/: In an individual with a Hb concentration of 150 g/L the oxygen-carrying capacity of the blood is 20 mL per 100 mL of blood. In the arteries and the capillary bed about 20% of the oxygen (about 4 mL of blood) will be unloaded per 100 mL of blood. In anemic patients with a Hb concentration of 75 g/L the oxygen binding capacity is halved. In this patient 20% of the oxygen (about 2 mL) would be unloaded per 100 mL of blood. However, the patient’s erythrocytes have increased content of 2,3 BPG and lower affinity for oxygen, for this reason about 3 mL is released. By this way the deficit in red cells is compensated.

15.3.5.12 Findings in hypoxic anemia

The following findings are indicative of hypoxic anemia: tachycardia, hypotension, O2 extraction > 50%, mixed venous O2 saturation < 50%, central venous O2 saturation < 60%, mixed venous PO2 < 32 mm Hg and lactic acidosis (lactate > 2 mmol/L).

15.3.5.13 Polycythemia and polyglobulia

In Caucasians suspicion of polycythemia or polyglobulia is unlikely if in women the Hb value is < 165 g/L or the hematocrit is < 0.50 and in men the Hb value is < 180 g/L or the hematocrit is < 0.55 /1/. Refer to Chapter 15.4 – Hematocrit.

15.3.6 Comments and problems

Anticoagulation

For venous blood 1.5–2.2 mg EDTA (dipotassium or di sodium salt) per mL, the final EDTA concentration is 3.7 or 5.4 μmol/L /2/.

Method of determination

The use of quartz cuvettes for the spectrophotometric measurement is an interfering factor with respect to the hemiglobin cyanide method. The use of reagent blanks solves the problem. A further problem is the turbidity of samples. Ultrafiltration reduces turbidity so that the ratio of absorption required for the reference method A540/504 is ≥ 1.59 /17/.

Lipemia and leukocytes

Turbid blood leads to a rise in Hb of up to 30 g/L because the HiCN solution becomes turbid /2/. Leukocyte values > 100 × 109/L can have the same effect as lipemia /2/.

Thrombocytes

Values > 700 × 109/L cause the HiCN solution to become turbid and lead to erroneously high Hb values /2/.

References

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2. NCCLS. Reference and selected procedures for the quantitative determination of hemoglobin in blood – second edition; approved standard. NCCLS Document H15–A2, Vol 14 No 6. Villanova: NCCLS, 1994.

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18. Hillman RS, Finch CA, eds. Red cell manual. Philadelphia 1996; Davis Publ.

19. Israels LG, Israels ED (eds). Mechanisms in hematology. Winnipeg 1997; The University of Manitoba: Section 8.

20. Detti L, Mari G. Noninvasive diagnosis of fetal anemia. Clin Obstet Gynecol 2003; 46: 923–30.

21. Takala T, Mäkelä E, Suominen P, Matomäki J, Lapinleimu H, Lehtonen L, et al. Blood cell and iron status analytes of preterm and full-term infants from 20 weeks onwards during the first year of life. Clin Chem Lab Med 2010; 48: 1295–1301.

22. Stauder R, Thein SL. Anemia in the elderly: clinical implications and new therapeutic concepts.Haematologica 2014; 99: 1127–30.

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24. Riva E, Tettamanti M, Mosconi P, Apolone G, Gandini F, Nobili A, et al. Association of mild anemia with hospitalization and mortality in the elderly: the Health and Anemia population-based study. Haematologica 2009; 94: 22–8.

25. Patel KV. Variability and heritability of hemoglobin concentration: an opportunity to improve understanding of anemia in older adults. Haematologica 2008; 93: 1281–3.

26. Letzky EA. Erythropoiesis in pregnancy. J Perinat 1995; 23: 39–45.

27. Richter C, Huch A, Huch R. Erythropoiesis in postpartum period. J Perinat 1995; 23: 51–9.

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29. Vincent JL, Sakr Y, Creteur J. Anemia in the intensive care unit. Can J Anesth 2003; 50: S53–S55).

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31. Munoz M, Acheson AG, Bisbe E, Butcher A, Gomez-Ramirez S, Khalafallah AA, et al. An international consensus statement on the management of postoperative anaemia after major surgical procedures. Anaesthesia 2018. doi: 10.1111/anae.14358.

32. European Best Practice Guidelines for the Management of Anemia in Patients with Chronic Renal Failure. Nephrol Dial Transplant 1999; 14, Suppl 5: 14–15.

33. Macdougall IC, White C, Anker SD, Bhandari S, Farrington K, Katra PA, et al. Intravenous iron in patients undergoing maintenance hemodialysis. N Engl J Med 2019; 380, 5: 447–58.

34. Thomas MC, MacIsaac J, Tsalamandris C, Molyneaux L, Goubina I, Fulcher G, et al. The burden of anemia in type 2 diabetes and the role of nephropathy: a cross-sectional audit. Nephrol Dial Transplant 2004; 19: 1792–7.

35. Massey CN. Microcytic anemia. Med Clin North Am 1992; 76: 549–66.

36. Iannou GN, Spector J, Scott K, Rockey DC. Prospective evaluation of a clinical guideline for the diagnosis and management of iron deficiency anemia. Am J Med 2002; 113: 281–7.

37. Kulozik AE. β-thalassaemia: molecular pathogenesis and clinical variability. Eur J Pediatr 1992; 151: 78–84.

38. d’Onofrio G, Zini G, Ricerca BM, Mancini S, Mango G. Automated measurement of red blood cell microcytosis and hypochromia in iron deficiency and β thalassemia trait. Arch Pathol Lab Med 1992; 116: 84–9.

39. Eber S, Lux SE. Hereditary spherocytosis – defects in proteins that connect the membrane skeleton to the lipid bilayer. Semin Hematol 2004; 41: 118–41.

40. Bolton-Maggs PHB, Stevens RF Dodd NJ, Lamont G, Tittensor P, King MJ. Guidelines for the diagnosis and management of hereditary sperocytosis. Br J Haematol 2004; 126: 455–74.

41. Mariani M, Barcelli W, Vercellati C, Marcello AP, Fermo E, Pedotti P, et al. Clinical and hematologic features of 300 patients affected by hereditary spherocytosis grouped according to the type of membrane protein defect. Haematologica 2008; 93: 1310–7.

42. Harigae H, Furuyama K. Hereditary sideroblastic anemia: pathophysiology and gene mutations. Int J Hematol 2010; 92: 425–31.

43. Fishbane S, Cohen DJ, Coyne DW, Djamali A, Sigh AK, Wish JB. Posttransplant anemia: the role of sirolimus. Kidney Int 2009; 76: 376–82.

44. Mix TCH, Kazmi W, Khan S, et al. Anemia: a continuing problem following kidney transplantation. Am J Transplant 2003; 3: 1426–33.

45. Taylor RD, Cowell RL. Classification and diagnosis of anemia. Comp Haematol Int 1996; 6: 1–6.

46. Tabbara IA. Hemolytic anemias. Med Clin North Am 1992; 76: 649–68.

47. Ataga KI. Hypercoagulability and thrombotic complications in hemolytic anemias. Haematologica 2009; 94: 1481–3.

48. Firth PG, Head A. Sickle cell disease and anesthesia. Anesthesiology 2004; 101: 766–85.

49. Dorn-Beineke A, Frietsch T. Sickle cell disease – pathophysiolpgy, clinical and diagnostic implications. Clin Chem Lab Med 2002; 40: 1075–84.

50. Ware RE, Rees RC, Sarnaik SA, Iyer RV, Alvarez OA, Casella JF, et al. Renal function in infants with sickle cell anemia: baseline data from the Baby Hug Trial. J Pediatr 2010; 156: 66–70.

51. Dobie G. Sickle cell disease and thromboembolism: New insights on the pathophysiology, diagnosis, and treatment. Clin Lab 2023; 69: 1335–48.

52. Lee JE, Jang JH, Kim JS, Yoon SS, Lee JH, Kim YK, et al. Clinical signs and symptoms associated with increased risk for thrombosis in patients with paroxysmal nocturnal hemoglobinuria from a Korean Registry. Int J Hematol 2013; 97: 749–57.

53. Scheiring J, Rosales A, Zimmerhackl LB. Today’s understanding of the haemolytic uremic syndrome. Eur J Pediatr 2010; 169: 7–13.

54. Robson KJH, Weatherall DJ. Malarial anemia and STAT6. Haematologica 2009; 94: 157–9.

55. Weiss G. Iron metabolism in the anemia of chronic disease. Biochem Biophys Acta 2009; 48: 57–63.

56. Thomas C, Thomas L. Anemia of chronic disease: pathophysiology and laboratory diagnosis. Lab Hematol 2005; 11: 14–23.

57. Aapro M, Österborg A, Gascon P, Ludwig H, Beguin Y. Prevalence and management of cancer-related anaemia, iron deficiency and the specific role of i.v. iron. Ann Oncol 2012; 23: 1954–62.

58. Ludwig H, van Belle S, Barrett-Lee P, Birgegard G, Bokemeyer C, Gascon P, et al. The European Cancer anemia survey (ECAS): a large, multinational, prospective survey defining the prevalence, incidence, and treatment of anemia in cancer patients. Eur J Cancer 2004; 40: 2293–306.

59. Caro JJ, Salas M, Ward A, Goss G. Anemia as an independent prognostic factor for survival in patients with cancer: a systematic quantitative review. Cancer 2001; 91: 2214–21.

60. Bron D, Meuleman N, Mascaux C. Biological basis of anemia. Seminars Oncol 2001; 28 , Suppl 8: 1–6.

61. Aapro M, Brguin Y, Bokemeyer C, Dicato M, Gascon P, Glaspy J, et al. Management of anemia and iron deficiency in patients with cancer: ESMO Clinical Practice Guidelines. Ann Oncol 2018. doi: 10.1093/annonc/mdx758.

62. Sleijfer S, Lugtenburg PJ. Aplastic anemia: a review. Netherl J Med 2003; 61: 157–63.

63. Coyle TE. Hematologic complications of human immunodeficiency virus infection and the acquired immunodeficiency syndrome. Med Clin North Am 1997; 81: 449–70.

64. Iolascon A, Esposito M, Rosso R. Clinical aspects and pathogenesis of congenital dyserythropoietic anemias: from morphology to molecular approach. Haematologica 2012; 97: 1786–94.

65. Heimpel H. Congenital dyserythropoietic anemias: epidemiology, clinical significance, and progress in understanding their pathogenesis. Ann Hematol 2004; 83: 613–21.

66. Maitra P, Caughey M, Robinson L, Desal PC, Jones S, Nourale M, et al. Risk factors for mortality in adult patients with sickle cell disease: a meta-analysis of studie in North America and EUrope. Haematologica 2017; 102: 626–36.

67. Stabler SP. Vitamin B12 deficiency. N Engl J Med 2013; 368: 149–60.

68. Chadebech P, Loustau V, Janvier D, Languille L, Ripa J, Tamagne M, et al. Clinical severity in adult warm autoimmune hemolytic anemia and its relationship to antibody specificity. Haematologica 2018; 103: e35.

69. Inamoto Y, Lee SJ. Late effects of blood and marrow transplantation. Haematologica 2017; 102: 614–25.

70. Dierickx D, Habermann TM. Post-transplantation lymphoproliferative disorders in adults. N Engl J Med 2018; 378. 549–62.

71. De Falco L, Sanchez M, Silvestri L, Kannengieser C, Muckenthaler MU, Iolascon A, et al. Iron refractory iron deficiency anemia. Haematologica 2013; 98: 845–53.

72. Millot S, Delaby C, Moulouel B, Lefebvre T, Pilard N, Ducrot N, et al. Hemolytic anemia repressed hepcidin level without hepatocyte iron overload: lesson from Günther disease model. Haematologica 2017; 102: 260–70.

73. Young NS. Aplastic anemia. N Engl J Med 2018; 379 (17): 1643–55.

74. Petrelli F, Ghidini M, Ghidini A, Sgroi G, Vavassori I, Petro D, et al. Red cell transfusions and the survival in patients with cancer undergoing curative surgery: a systematic review and meta-analysis. Surgery Today 2021. doi: 10.1007/s00595-020-02192-3.

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76. Pawlak R, Berger J, Hines I. Iron status of vegetarian adults: a review of literature. Analytic Review 2018; 6: 486–97.

77. Ambroszkiewicz J, Klemarczyk W, Mazur J, Gajewska J, Rowicka G, Strucinska M, Chelchowska M. Serum hepcidin and soluble transferrin receptor in the assessment of iron metabolism in children on a vegetarian diet. Biol Trace Elem Res 2017; 180: 182–90.

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81. Notaro R, Luzzatto L. Breakthrough hemolysis in pNH with proximal or terminal complement inhibition. N Engl J Med 2022; 387 (2): 160–6.

82. Block J, Rashkova C, Castanon I, Zoghi S, Platon J, Ardy RC, et al. A systemic inflammation and normocytic anemia in DOCK11 deficiency. N Engl J Med 2023; 389 (6): 527–39.

83. Bunn HF. Oxygen delivery in the treatment of anemia. N Engl J Med 2022; 387 (25): 2362–5.

15.4 Hematocrit (HCT)

Lothar Thomas

The HCT, or packed cell volume (PCV) is a measure of the ratio of the volume occupied by the red cells to the volume of whole blood in a sample of capillary or venous blood. The ratio is measured after appropriate centrifugation and is expressed as a fraction (e.g., 0.42 and not 42%). The HCT is used, together with the red cell count, in calculating the MCV and, together with hemoglobin content, in calculating the MCHC /1/.

15.4.1 Indication

  • Detection of anemia or polycythemia
  • Estimating changes in hemodilution and hemoconcentration
  • Reference for assessing erythropoietin formation in relation to the extent of anemia; see section 15.10.

15.4.2 Method of determination

Micro hematocrit method

Disposable type I, class B borosilicate glass capillary tubes, 75 mm in length, internal diameter 1.15 mm, are recommended /1/. Wall thickness should be 0.20 mm. Use of a special micro hematocrit centrifuge with rotor radius greater than 8 cm, reaching maximal speed within 30 seconds, capable of sustaining a relative centrifugal force (RCF) of (10,000 to 15,000) × g at the periphery for 5 min., without the rotor exceeding a temperature of 45 °C. Calculation of RCF: see Tab. 15.4-1 – Equations for HCT calculation.

Hematology analyzer

Principle: see Section 15.2.1.2 – Method of determination. In the most simple case, HCT is calculated as shown in the equation as product of RBC × MCV. The RBC is the red blood count. Some hematology analyzers determine the sum of electrical pulses and divide them by the number of pulses. Calculation is performed according to the equation in Tab. 15.4-1 – Equations for HCT calculation. The hematology analyzer results are adapted to the micro hematocrit method.

15.4.3 Specimen

  • Whole blood (di sodium EDTA, tripotassium EDTA or heparin as anticoagulant): 1 mL
  • Capillary blood (heparinized capillaries): 0.05 mL

15.4.4 Reference interval

Refer to references /12, 3, 4, 5, 6, 78/ and Tab. 15.4-2 – Hematocrit reference intervals.

15.4.5 Clinical significance

The HCT determination is a simple method for the detection of anemia, and erythrocytosis. It is useful, in addition, for identifying the extent of changes with hemodilution and hemoconcentration. The HCT is dependent upon:

  • The red blood cell mass of the organism (17–32 mL/kg body weight in women and 20–36 mL/kg body weight in men) /9/
  • The mean cell volume of erythrocytes
  • The plasma volume, the reference interval is 30–45 mL/kg body weight.

15.4.5.1 Decrease of HCT

Besides a decrease of the Hb level, a decrease in HCT is a diagnostic criterion of anemia. Exceptions are patients:

  • With hyper hydration (e.g., postoperative patients with minimal blood loss but overcompensated volume substitution). In such cases there is an increased plasma volume in the presence of still normal red blood cell mass, called pseudoanemia.
  • With acute bleeding, in which the erythrocyte loss has not yet been compensated for by an increase in plasma volume, anemia is already present pathophysiologically, but is not yet indicated by a reduction of HCT or the Hb level.

In healthy normovolemic adults, HCT can decline to 0.15–0.20 before abnormal regional myocardial blood flow distribution, indicated by a rise in cardiac lactate formation, develops. In the fetus and the newborn, anemia can be very severe before disturbances of cardiac function occur /10/.

An elevated incidence of postoperative cardiac complications is found in patients with peripheral vascular disease in whom HCT is below 0.29 /11/. Cardiac disease is the most frequent cause of death in hemodialysis patients. An increase in HCT from below 0.30 to the range of 0.30–0.38 reduces the incidence of myocardial infarction by 30% within a time period of 30 months /12/.

In patients with congestive heart failure and HCT ≤ 0.24, mortality and re-hospitalization risks are increased by 51% and 17%, respectively, in comparison to patients with HCT of 0.40–0.44. Although anemia is an independent risk factor, a patient’s co morbidities also play an important role /13/.

HCT plays a major role in primary hemostasis by influencing blood viscosity and platelet adhesion. HCT values below 0.30 lead to a reduction in blood viscosity and platelet adhesion and the bleeding time may be prolonged /14/. It is that during continuous venovenous hemofiltration (CVVH) higher HCT values are accompanied by an increased hemostasis activation during CVVH. This is not the case in CVVH patients with HCT values between 0.30 and 0.35 /15/.

Diseases and conditions with decreased HCT are shown in Tab. 15.4-3 – Diseases and conditions with decreased HCT.

In patients with acute blood loss the number of blood donations is dependent on the actual HCT and the desired HCT in patients with anemia. A calculation is shown in Tab. 15.4-4 – Calculation of desired packed red cells in dependence of HCT.

15.4.5.2 Increase of HCT

HCT elevations of over 0.48 in women and over 0.51 in men are due to:

  • An absolute increase in the red cell mass, associated with erythrocytosis and polycythemia
  • A plasma volume decrease (e.g., associated with exsiccosis).

Clinically, there is an association between a high-normal or high HCT and increased blood viscosity, blood vessel disease, metabolic disease and thrombosis. According to:

  • The Framingham Study /16/, Caucasians with a HCT over 0.50 had a 2–6 fold higher relative risk of stroke over the study period of 87 months than individuals with lower values
  • The Puerto Heart Program /17/, individuals from the urban population with a HCT over 0.49 had a 2-fold higher risk of cardiovascular disease within 8 years than individuals with a HCT below 0.42
  • A British study /18/ the risk of non-insulin dependent diabetes mellitus increased significantly with increasing HCT levels. There was more than a fourfold elevation in relative risk of diabetes among men with a HCT of ≥ 0.48 relative to those with a HCT below 0.42, adjusted for age and body mass index.

15.4.5.3 Erythrocytosis

Erythrocytosis is defined as an absolute increase in red blood cell mass and can be associated with an elevated HCT and hemoglobin level (Fig. 15.4-1 – Classification of erythrocytosis). An HCT > 0.51 in a male and > 0.48 in a female individual is above the normal threshold and thus elevated. Initial classification of erythrocytosis is on the basis of whether it is a primary process (also known as familial and congenital polycythemia) or a secondary process where the red cell production is driven by some other process /19/. Some clinicians use the term polycythemia interchangeably with erythrocytosis, the two are not synonymous. Polycythemia refers to an increased number of any hematopoietic cell in blood, be it erythrocytes, thrombocytes or leukocytes. A complicating matter is the term polycythemia vera, a type of chronic myeloid leukemia that only affects the erythroid lineage.

15.4.5.3.1 Primary erythrocytosis

Primary erythrocytosis (also known as primary familial and congenital polycythemia, PFCP) is a pathology of erythroid progenitors, which display hypersensitivity to erythro­poietin /1920/. PFCP is characterized by an isolated primary polycythemia in which an increased red cell mass is associated with subnormal erythropoietin levels.

Refer to:

A genetic abnormalites in concern to the EPO level are:

15.4.5.3.2 Secondary erythrocytosis

Secondary erythrocytosis results from a mechanism other than intrinsic to the bone marrow and is driven by increased production of erythropoietin (EPO)(predominantly reactive) /1920/. The responsiveness of erythroid progenitors to circulating EPO is usually normal. Erythrocytosis is associated with inappropriately normal or raised EPO levels indicating a defect in the control of EPO synthesis by the oxygen-sensing pathway. The increased EPO secretion may represent either a physiologic response to tissue hypoxia, or a deregulation of the oxygen-dependent EPO synthesis. Refer to Tab. 15.4-5 – Diseases and conditions with elevated HCT.

Etiologically, there are a number of different ways where a secondary erythrocytosis can result /25/:

  • Central hypoxia driven process (e.g., reduced oxygen supply will lead to stimulation of EPO production and erythrocytosis)
  • Local renal hypoxia driven process (e.g., a local hypoxia in the kidney leads to increased EPO production and erythrocytosis)
  • Pathological EPO production (e.g., meningioma, cerebellar hemangioblastoma hepatocellular carcinoma, parathyroid adenomas)
  • Exogenous EPO administration (EPO doping).
15.4.5.3.3 Idiopathic erythrocytosis

The cause of erythrocytosis cannot be elucidated. These patients can be differentiated on the basis of their EPO levels /19/:

  • One third have levels below the normal range and are likely display hypersensitivity to EPO
  • Two thirds have inappropriately normal EPO levels for elevated HCT or increased EPO concentrations. This group are likely to have defects in the oxygen sensing pathway.

15.4.5.4 Decreased plasma volume

Decreased plasma volume (hemoconcentration) in the presence of normal red blood cell mass leads to relative erythrocytosis. The decrease in plasma volume is due to disturbances of the water and electrolyte balance. These can be caused by /27/:

  • Insufficient intake of fluids (e.g., small children, elderly individuals, seriously ill individuals)
  • Insensible loss of water (e.g., perspiration)
  • Diarrhea and vomiting
  • Polyuria (e.g., due to disorders of the ADH thirst mechanism, abuse of diuretics, diabetes mellitus)
  • Ingestion of medicines for the improvement of cardiac function
  • Smoking; smokers may have relative, absolute or combined erythrocytosis
  • Abuse of alcohol, tea, caffeine-containing and cola-containing drinks.

15.4.6 Comments and problems

Blood collection

Venous occlusion for too long a period (more than 2 min.) causes a significant rise in HCT /22/.

Anticoagulant

If EDTA concentrations are used that are higher than recommended (see Section 15.3 – Hemoglobin concentration), a falsely low HCT occurs because of a decline in the MCV /2/.

Sample

Arterial blood has an approximately 2% higher HCT than venous blood.

Method of determination

Because of trapped plasma, the values of the PCV are approximately 2% higher than the HCT determined by hematology analyzers. The differences are higher yet if abnormal RBCs are present (e.g., sickle cells, thalassemia, iron deficiency, spherocytes, macrocytes) /2/.

In the presence of a high reticulocyte or WBC count, HCT determinations using hematology analyzers result in the calculation of falsely elevated values, because the higher cell volumes of reticulocytes and white blood cells enter into the calculation of the HCT. Falsely low values are determined in the case of in vitro hemolysis, autoagglutination, and microcytosis.

Point of care analyzers that are used for bedside HCT determinations, measure the HCT by means of the conductivity of undiluted blood. In patients with an increased plasma osmolality, the HCT determination gives lower values /23/.

Stability

Up to 24 h at room temperature or 4–8 °C, if the HCT is determined by an automated hematology analyzer. Centrifugation within 6 h for the micro hematocrit method /1/.

Leukemia

The white leukocyte pellet must be ignored when the results of the capillary method are interpreted /1/.

Intraindividual variation

Within-one-day variation 4.6%, day-to-day variation 4.1%, month-to-month variation 3.4% /24/.

References

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12. Besarab A, Bolton K, Brown JK, et al. The effects of normal as compared with low hematocrit values in patients with cardiac disease who are receiving hemodialysis and Epoetin. N Engl J Med 1999; 339: 584–90.

13. Kosiborod M, Curtis JP, Wang Y, Smith GL, Masoudi FA, Foody JM, et al. Anemia and outcomes in patients with heart failure. Arch Intern Med 2005; 165: 2237–44.

14. Boneu F, Fernandez F. The role of the hematocrit in bleeding. Transfus Med Rev 1987; 1: 182.

15. Stefanidis I, Heintz B, Frank D, Mertens PR, Kierdorf HP. Influence of hematocrit on hemostasis in continuous venovenous hemofiltration during acute renal failure. Kidney Int 1999; 56 Suppl 72: 51–5.

16. Heyman A, Karp HR, Heyden S, et al. Cerebrovascular disease in the biracial population of Evans county, Georgia. Arch Intern Med 1971; 128: 949–51.

17. Soriie PD, Garcia-Palmieri MR, Lstas R, Havlik RJ. Hematocrit and the risk of coronary heart disease: the Puerto Rico Heart Program. Am Heart J 1981; 101: 456–61.

18. Wannamethee SG, Perry IY, Shaper AG. Hematocrit and risk of NIDDM. Diabetes 1996; 45: 576–9.

19. McMullin MF. The classification and diagnosis of erythrocytosis. Int Jnl Lab Hem 2008; 30: 447–59.

20. Cao M, Olsen RJ, Zu Y. Polycythemia vera. Arch Pathol Lab Med 2006; 130: 1126–32.

21. Landaw SA. Polycythemia vera and other polycythemic states. Clin Lab Med 1990; 10: 857–71.

22. Junge B, Hoffmeister H, Feddersen HM, Röcker L. Standardisierung der Blutentnahme. Dtsch Med Wschr 1978; 103: 260–5.

23. Stott AW, Hortin GL, Wilhite TR, Miller SB, Smith CH, Landt M. Analytical artifacts in hematocrit measurements by whole-blood chemistry analyzers. Clin Chem 1995; 41: 306–11.

24. Costongs GMPJ, Janson PCW, Bas BM, et al. Short-term and long-term intraindividual variations and critical differences of haematological laboratory parameters. J Clin Chem Clin Biochem 1985; 23: 69–76.

25. Balloch AJ, Cauchi MN. Reference ranges for haematology parameters in pregnancy derived from patient populations. Clin Lab Haemat 1993; 15: 7–14.

26. Selby GB, Eichner ER. Hematocrit and performance: the effect of endurance training on blood volume: Sem Hematol 1994; 31: 122–7.

27. Vincent JL, Sakr Y, Creteur J. Anemia in the intensive care unit. Can J Anesth 2003; 50: S53–S55).

28. Sachs V. Hämotherapie – ein Leitfaden. Ärztl Lab 1984; 30: 204–20.

29. Musallam KM, Tamim HM, Richards T, Spahn DR, Rosendaal FR, Habbal A, et al. Preoperative anemia and postoperative outcomes in non-cardiac surgery: a retrospective cohort study. Lancet 2011; 378: 1396–1407.

30. Bernard AC, Davenport DL, Chang PK, Vaughan TB, Zwischenberger JB. Intraoperative transfusion of 1 U to 2 U packed red blood cells is associated with increased 30-day mortality, surgical site-infection, pneumonia, and sepsis in general surgery patients. J Am Coll Surg 2009; 208: 931–7.

31. Cao M, Olsen RJ, Zu Y. Polycythemia vera. Arch Pathol Lab Med 2006; 130: 1126–32.

32. Spivak JL. Myeloproliferative neoplasms. N Engl J Med 2017; 376: 2168–81.

33. Gordeuk VR, Key NS, Prchal JT. Re-evaluation of hematocrit as a determinant of thrombotic risk in erythrocytosis Haematologica 2019; 104 (4): 653–8.

34. Pasquier F, Marty C, Balligand T, Verdier F, Grosjean S, Gryshkova V, et al. New pathogenic mechanisms induced by germline erythropoietin receptor mutations in primary erythrocytosis. Haematologica 201(, 103. 675–86.

35. Drew JH, Guaran RL, Grauer S, et al. Cord whole blood viscosity: measurement, definition, incidence and clinical features. J Pediatr Child Health 1991; 27: 363–5.

36. Gaston RS, Julian BA, Curtis JJ. Posttransplant erythrocytosis: an enigma revisited. Am J Kidney Dis 1994; 24: 1–11.

37. Fallert-Müller A. Lexikon der Biochemie. Heidelberg 1999; Spektrum Akademischer Verlag: 430.

38. Camps C, Petousi N, Bento C, Cario H, Copley RR, McMullin MF, et al. Gene panel sequencing improves the diagnostic work-up of patients with idiopathic erythrocytosis and identifies new mutations. Haematologica 2016, 101: 1306–18.

39. Drevon L, Maslah N. Anemia and hemodilution: analysis and a single center cohort based on 2,858 red cell mass measurements. Haematologica 2020; 106 (4): 1166–71.

15.5 Dyshemoglobins and cell free hemoglobin

Lothar Thomas

Increased concentrations of dyshemoglobins such as methemoglobin (hemiglobin), carboxyhemoglobin and sulfhemoglobin limit the oxygen-carrying capacity of arterial blood. These hemoglobin (Hb) fractions are not detected by blood gas analysis when measuring blood oxygen status and therefore can cause misinterpretation when the functional oxygen saturation is used for patient evaluation /1/.

15.5.1 Methemoglobin (metHb), hemiglobin (Hi)

Methemoglobin is an oxidized form of Hb. Many oxidant chemicals and drugs are capable of inducing methemoglobinemia and can also cause hemolysis.

15.5.1.1 Indication

Cyanosis and suspicion in:

  • Toxic methemoglobinemia
  • Hereditary methemoglobinemia
  • Decrease in arterial oxygen saturation which, from the clinical point of view, primarily cannot be accounted for.

15.5.1.2 Method of determination

Principle: metHb has a spectral absorbance curve with a characteristic maximum at 630 nm, in weak acid solution. For increase of the analytical specificity a measurement of absorption difference is made. MetHb reacts with cyanide ions provided by KCN to form hemiglobin cyanide which has no absorption at 630 nm /2/.

15.5.1.3 Specimen

EDTA blood, heparinized blood: 1 mL

For the determination, hemolysate is used (1 part of whole blood + 5 parts of distilled water).

15.5.1.4 Reference interval

MetHb: 0.2–1.0% /3/

15.5.1.5 Clinical significance

In vivo, ferrous (Fe2+) Hb is continuously oxidized to ferric (Fe3+) Hb and hence metHb is produced. The reduction of metHb to Hb is catalyzed by the NADH-dependent methemoglobin reductase. To a small extent, a nonenzymatic reduction of metHb is also possible by ascorbic acid or reduced glutathione. The presence of methemoglobinemia reduces the oxygen-binding capacity, since Fe3+ is no longer able to reversibly bind oxygen. Methemoglobinemia is said to be present if the proportion of oxidized iron in hemoglobin exceeds 1%.

MetHb inducers act by oxidizing Fe2+ to Fe3+ hemoglobin, resulting in the formation of metHb. Methemoglobinemia should be considered if a patient’s arterial oxygen saturation measured with pulse oximetry, is as low as about 85%.

15.5.1.5.1 Hereditary methemoglobinemia

The term describes an autosomal recessive inherited condition in which there is a deficiency in NADH-dependent methemoglobin reductase; its activity is below 20%. The metHb proportion in blood is 8–40%; finding of a chocolate brown blood color. Heterozygous metHb reductase deficiency is not associated with methemoglobinemia /4/.

Neonates and individuals with congenital NADH-dependent metHb reductase activity or glucose-6-phosphate dehydrogenase deficiency have an impaired ability to regenerate normal Hb and are therefore more likely to accumulate metHb after oxidant exposure. Thus, the administration of drugs or nitrate-containing water, which is converted to nitrite in the gastrointestinal tract, can cause methemoglobinemia. The clinical manifestations of congenital methemoglobinemia are cyanosis and neurological disturbances.

15.5.1.5.2 Toxic methemoglobinemia

Activity of NADH-dependent met-Hb reductase is normal in toxic methemoglobinemia. Methemoglobinemia occurs /35/:

  • In occupational risk groups especially chemical and munitions workers
  • Dependent upon metHb inducers (drugs, chemicals) which directly convert Hb to metHb or indirectly promote conversion via the formation of oxygen radicals in the circulation. An important environmental source of methemoglobinemia in infants is nitrate-contaminated well water.

Refer to:

Treatment involves administration of oxygen and infusion of methylene blue for the reduction of Fe3+ to Fe2+. 1 mg of methylene blue/kg body weight is administered in a 10 minute infusion. Mild methemoglobinemia (below 20%) will resolve spontaneously without intervention.

15.5.1.6 Comments and problems

Stability

In intact RBCs, metHb is stable only for about 5 h because it is rapidly converted back to Hb. However, if the blood sample is diluted with 5 parts of distilled water and the erythrocytes are hemolyzed, stability is guaranteed for at least 45 h. Fluoride should not be used, as its interaction with metHb leads to erroneously low total Hb and metHb values /1/.

Interference factors

Hypertriglyceridemia and hyperbilirubinemia interfere with the metHb determination. Using multi-wavelength photometry, interference by triglycerides or by bilirubin does not occur until the concentration reaches levels of about 1000 mg/dL (11.4 mmol/L) or about 10 mg/dL (171 μmol/L), respectively /6/.

15.5.2 Carboxyhemoglobin (COHb)

Carbon monoxide (C0) is a colorless, odorless, tasteless and nonirritating gas produced by incomplete combustion of any carbon-containing material. Sources of exposures include smoke inhalations in fires, automobile exhaust fumes, faulty or poorly ventilated charcoal, kerosene or gas stoves, cigarette smoke and methyl chloride /5/. Silent killer is the most common nickname for CO.

15.5.2.1 Indication

Non-specific signs such as flu-like symptoms, headache, syncope, seizures occurring for the first time, and a history of carbon monoxide exposure.

15.5.2.2 Method of determination

CO measurement methods /9/:

  • CO monitor: the volume of CO is measured in the end tidal breath in parts per million (ppm) and is correlated to blood Hb concentration
  • COHb measurement: Hb is measured spectrophoto-metrically at multiple wavelengths. Different correction fractures are used in order to eliminate interferences (e.g., due to elevated bilirubin or high triglyceride levels). A triple wavelength measurement is performed (e.g., at 415, 380 and 450 nm /17/ or at 578, 562 and 598 nm)
  • total blood carbon monoxide (TBCO): The measurement of TBCO allows the measurement of total amount of CO present in the blood sample at the time of sampling and includes the CO bound to Hb and the amount of free CO present in the blood sample. A disadvantage of TBCO measurement is that it is analyzed using GC-MS/MS only
  • HPLC combined with absorption spectrophotometry: the method is implemented in two steps. At first, protein-bound and free Hb are separated by HPLC. The free Hb fraction is diluted with acetic acid, H2O2 and tetramethylbenzidine. A blue dye is formed and measured spectrophotometrically at 600 nm.

15.5.2.3 Specimen

Whole blood (EDTA, oxalate, heparin): 5 mL

The sample tube needs to be filled in such a manner that, if at all possible, only a small volume of air is above the blood.

15.5.2.4 Reference interval

COHb /8/

Non-smokers

≤ 3,0%

Smokers

≤ 10,0%

Recommended values

15.5.2.5 Clinical significance

Environmental CO exposure is typically below 0.001% (10 ppm), but may be higher in urban areas. The quantity of CO absorbed by the body is dependent upon the respiratory minute volume, duration of exposure and CO and O2 concentrations in the environment. After cooking with a gas stove indoor CO concentration reach 100 ppm. A cigarette smoker is exposed to 400–500 ppm of CO and automobile exhaust may contain 10,000 ppm CO. A 4-hour exposure to 70 ppm results in a COHb content of 10%, and following an exposure of the same duration to 350 ppm CO may lead COHb level of 40% /9/. In the USA, the Occupational Safety and Health Administration permissible CO exposure in workers is 50 ppm averaged over an 8-hour work day /9/.

The most frequent sources of CO poisoning include exposures to smoke inhalations in fires, automobile exhaust fumes, faulty or poorly ventilated charcoal, kerosene or gas stoves and cigarette smoke. In Germany, the maximal allowable CO concentration is set at 30 ppm (33 mg/m3/8/.

The pathological effect of CO is due to a decrease in O2 transport capacity of the blood and a reduction in tissue O2 extraction and a direct CO toxicity at the cellular level. At low levels endogenously CO functions as a neurotransmitter (see Section 19.2 – Oxidative stress) and modulates inflammation, apoptosis, and cell proliferation /10/. CO affinity for Hb is more than 240 times that of O2 and shifts the oxyhemoglobin dissociation curve to the left (see Fig. 15.4-4 – Effect of 2,3-diphosphoglycerate on O2 saturation curve). CO is preferentially bound in the lungs and causes a blockade of O2 diffusion in the lungs and muscles, since CO dissociates less rapidly from Hb than O2. The presence of COHb always signifies diminished O2 supply to the tissues, which is not detected by measuring the Hb value and which is very much more marked than an equivalent reduction in Hb /10/.

Clinical symptoms are shown in Tab. 15.5-3 – Clinical symptoms in dependence of the COHb concentration.

15.5.2.5.1 COHb in healthy individuals

Serum COHb levels in nonsmokers would be expected to have levels of less than 1–3% from endogenous production and background environmental exposure. Increases in non-smokers to some 3% are mainly associated with passive smoking, rather than environmental pollution. CO content in tobacco smoke is 4% and most smokers have, depending on the number of cigarettes they smoke daily, a blood COHb content of 3–8%. Certain professional groups (e.g., non-smoking fire fighters) have COHb values that are 1–2% higher than those in non-smokers. The elimination half-life time of COHb is approximately 4 hours with the breathing of room air and 1 hour with the breathing of 100% O2 /9/.

15.5.2.5.2 Constant mild CO elevations

At a constant CO fraction of 25 ppm, the blood COHb content will be 3.5%, at 50 ppm it increases to 6–8% /9/. Even short-lasting low CO elevations in the inhaled, air can lead to COHb values of 2–6% and can trigger angina pectoris-like symptoms and arrhythmia in patients with atherosclerosis, under stress /11/.

15.5.2.5.3 Pregnancy and smoking

CO crosses the placenta readily and small quantities of COHb in maternal blood have a considerable effect on the fetal O2 supply. Fetal Hb has high O2 affinity (P50, 19.4 mm Hg) in comparison with adult Hb (P50, 26.3 mm Hg at sea level), and this favors O2 uptake from the hypoxic maternal uterine blood. At the nadir of the HbO2 dissociation curve, fetal O2 saturation is only 75–80%. Therefore the fetus is vulnerable even to small fluctuations in O2 saturation /10/. If a pregnant woman smokes, for example, one pack of cigarettes daily, the COHb fraction may be 6% or more. In consequence, the maternal P50 is decreased from 26 to 23 mmHg and the O2 partial pressure in the uterine blood declines from 38 to 32 mmHg, which leads to a reduction of diffusion gradients toward the placenta. As a result, the umbilical cord blood O2 partial pressure drops from 28 to 22 mmHg, fetal arterial O2 saturation decreases from 75% to 58% and the fetus goes into a state of O2 hypoxia /12/.

15.5.2.5.4 CO poisoning

The clinical symptoms of acute CO poisoning are headache, nausea, confusion, stupor and coma. In mild poisoning with CO values up to 25%, the patient represents the physician with flue-like symptoms /9/.

The symptoms in chronic CO poisoning are headache, nausea light-headedness, cerebellar dysfunction and mood disorders. Nonetheless there is, in addition, considerable impairment of intellectual function, with lapses of concentration and cognitive disturbances. Even 3 years after the patient is removed from the environment, more than 40% of patients still manifest neurological problems. CO-associated symptomatology is often not taken into consideration so that the corresponding examinations are not performed.

The clinical severity of CO poisoning often correlates poorly with the blood COHb level and is, rather, determined by the duration of poisoning. Therefore, a patient who attains a high COHb level after a brief, high-level exposure may not manifest any clinical symptomatology, whereas a patient who attains the same COHb level after a prolonged lower-level exposure may be significantly symptomatic /9/.

Five severity grades of CO poisoning are established and, accordingly, a COHb fraction of over 50% is considered to be potentially fatal (Tab. 15.5-3 – Clinical symptoms in dependence of the COHb concentration). The cherry red appearance of blood and tissues is an unreliable sign and is only found with the most severe CO poisonings.

In forensic medicine, a COHb content exceeding 50% is indicative of CO poisoning as the primary cause of death. Values of 10–50% indicate that smoke was inhaled and that CO may be a factor related to the cause of death, but that the victim was still alive when the fire began.

Pathophysiologically, the poor correlation between CO poisoning and clinical effects is explained on the basis of the combination of

  • Hypoxia due to formation of COHb and
  • a direct toxic effect of soluble CO in blood at the cellular level.

The effects of CO are not confined to the period immediately after exposure /9/. Persistent or delayed neurological symptoms with loss of memory, confusion, ataxia, seizures, urinary and bowel incontinence, disorientation and psychiatric symptoms develop after a latency period of 2–40 days /9/.

15.5.2.5.5 Increased endogenous CO accumulation

Endogenous CO accumulates during degradation of Hb and myoglobin as well as of enzymes containing heme structures, such as peroxidase, catalase or cytochrome C. The endogenous formation of CO is responsible for physiological concentrations of COHb in the blood. Elevated endogenously dependent COHb concentrations occur in conditions of severe hemolysis or myolysis. In COPD patients, COHb values are higher in stage IV than in stages II or III /13/. Values of COHb as high as 12% are measured in neonatal jaundice.

In sickle cell anemia, endogenous CO leads to a worsening of the situation:

15.5.2.6 Comments and problems

Sample

Blood sampling should be performed with hermetic test tubes with screw caps. For laboratory storage, there should be as little free airspace as possible on top of the anticoagulated blood /15/.

Method of determination

  • COHb measurement: Despite being a direct biomarker of CO exposure often low COHb concentrations are measured in patients with CO exposure suspicion or with symptoms that are normally associated to higher COHb concentrations.
  • CO can be measured in breath through a CO monitor which measures the volume of CO in the end tidal breath in parts per million (ppm) and is correlated to blood COHb values.
  • Total blood carbon monoxide (TBCO). The poisoning diagnosis is only confined to the effects caused by direct CO toxicity e.g., increased oxidative stress, impaired mitochondrial function, inflammation of the brain.
  • Gas chromatography: this methode is the gold standard and capable of detecting low concentrations below 2.5% COHb. For this reason, tests in which hemolysis-dependent COHb accumulation is to be measured should only be performed using gas chromatography.

CO spectrometric results correlate with those of gas chromatography as of COHb values of ≥ 2.5%/9/, but good comparability is only ensured with values of over 5% /14/. Gas chromatography has a limit of detection of less than 0.1% COHb /15/.

Interference factors

Hypertriglyceridemia leads to erroneously high values with spectrophotometric determinations, because a constant absorption factor is added. Methemoglobinemia also causes falsely high COHb values with the use of this method /615/.

Stability

Whole blood should be stored refrigerated or deep frozen and exposure of samples to strong light must be avoided. The total sample CO content can remain constant over weeks to years at 3 °C or deep frozen /15/.

15.5.3 Free hemoglobin

Cell free hemoglobin (plasma free heme, plasmaprotein bound heme and cell free hemoglobin) are mediators of tissue injury in hemolytic disease. Cell free hemoglobin is a potent scavenger of nitric oxide (NO), activates inflammation and undergoes redox cycling reactions that cause oxidative stress. Cell free heme also stimulates oxidative stress, activates TLR4, and the inflammasome leading to exacerbated inflammation-mediated tissue injury. TLR4 is a transmembrane protein, member of the toll-like receptor family, which belongs to the pattern recognition receptor family. Cell free hemoglobin and total Hb will co-exist and need to be distinguished from each other when quantifying /16/.

15.5.3.1 Indication

Hemolytic disease

  • Extracorporeal membrane oxygenation support
  • Cardiopulmonary bypass
  • Transfusion toxicity
  • Dialysis
  • Sickle cell disease

15.5.3.2 Method of determination

Spectrometric method

Principle: Hb is measured at multiple wavelengths and different correction factures are used in order to eliminate interferences (e.g., due to elevated bilirubin or high triglyceride levels). A triple wavelength measurement, is performed (e.g., at 415, 380 and 450 nm /17/ or at 578, 562 and 598 nm).

Immunonephelometric quantification

Rabbit antiserum raised against human hemoglobin A with no cross reactivity against myoglobin is used /18/.

HPLC combined with absorption spectrophotometry

Principle: the method is implemented in two steps. At first, protein-bound and free Hb are separated by HPLC. The free Hb fraction is diluted with acetic acid, H2O2 and tetramethylbenzidine. A blue dye is formed and measured spectrophotometrically at 600 nm.

Inspection of plasma

Hemolysis is visible as free Hb at concentrations ≥ 300 mg/L /19/.

15.5.3.3 Specimen

Heparin plasma, citrate plasma: 1 mL

15.5.3.4 Reference interval

Free hemoglobin:

Plasma < 100 μg/L /20/

15.5.3.5 Clinical significance

Normal catabolism of hemoglobin (Hb) takes place through extravascular hemolysis. Tissue macrophages engulf senescent erythrocytes or take up Hb of ruptured erythocytes. Within macrophages degradation of Hb and heme, catalyzed by hemoxygenase, leads to the formation of amino acids, iron, CO and biliverdin, then converted to bilirubin /20/.

In intravascular hemolysis free Hb dissociates in αβ-dimers or follows spontaneous oxidative reactions. The clearance of αβ-dimers or oxidative products takes place after they are complexed by the plasmaproteins haptoglobin and hemopexin. Haptoglobin reacts with αβ-dimers and neutralizes its reactive ferric part whereas hemopexin binds free heme. The complexed haptoglobin (Hp) and hemopexin (Hx) are taken up by hepatocytes and splenocytes (receptor CD163 for Hb-Hp complexes and CD91 for heme-Hx complexes).

Part of the dissociated αβ-dimers (32 kD) cross the wall of the vessels and reach the extravascular spaces. Hb dimers freely circulate in the blood stream /20/:

  • In overwhelming scavenging capacity of haptoglobin
  • In patients with haptoglobin phenotype Hp 1-1. Phenotype Hp 2-2 individuals with complexes of free Hb and Hp 2-2 exhibit higher functional affinity for the scavenger receptor CD163 on macrophages as other phenotypes. There is a marked relationship between free Hb concentration in plasma and phenotypes of haptoglobin. Individuals with Hp 1-1 have the highest concentration free Hb, followed by phenotype Hp 2-1. The concentration of free Hb of phenotype Hp 2-2 is 61% than in those with the Hp 1-1 phenotype. In Hp 1-1 and Hp 2-1 individuals the concentration of free Hb is also positively correlated with the Haptoglobin concentration /21/.

Diseases and pathophysiologic aspects of consequences of free hemoglobin are shown in Tab. 15.5-4 – Hemolysis and plasma free hemoglobin.

In addition to the normal degradation of erythrocytes, daily hemolysis of 1% of the red blood cell mass, representing some 3 g of Hb, leads to complete disappearance of haptoglobin from plasma and to detection of free Hb /21/.

A rise in free Hb is a more sensitive indicator of intravascular hemolysis than an increase in LD. Thus, with LD activity of 165 U/L, only hemolysis that leads to a free Hb concentration of 800 μg/L causes a rise in LD of 58% and thus a value that is above the upper reference interval value /22/.

Severe hemolysis always manifests increased LD and if the rate of hemolysis is > 5%, representing a daily release of Hb of ≥ 15 g, non-conjugated bilirubin is also increased.

In vivo and in vitro hemolysis can be distinguished by determination of potassium and haptoglobin. A hemolytic sample with elevated potassium and normal haptoglobin value raises suspicion of the presence of in vitro hemolysis.

Refer to Tab. 15.5-4 – Hemolysis and plasma free hemoglobin.

15.5.3.6 Comments and problems

Anticoagulant

EDTA must not be used as anticoagulant. Free Hb is some 20-fold higher in EDTA plasma than in heparin plasma. Serum should not be investigated, since Hb is released from the erythrocytes during coagulation. In absence of intravascular hemolysis, the serum concentration of free Hb can be 50–100 mg/L /18/.

Method of determination

COHb measurement: Despite being a direct biomarker of CO exposure often low COHb levels are measured in patients with CO exposure suspicion or with symptoms that are normally associated to higher COHb levels. These discrepancies can be due to previously administered oxygen that reduced the COHb levels prior to hospitak admission.When using COHb as biomarker the effects caused by direct CO toxicity (e.g., increased oxidative stress, impaired mitochondrial function inflammation in brain tissue) are not taken into account.

Carbon monoxide pulse oximetry can not be a screening tool for CO poisoning in the emergency department due to its low sensitivity. This method of determination can have a role in the prehospital setting as a tool to quickly identify CO poisoned patients /29/.

While bilirubin and hyperlipidemia strongly interfere with the spectrophotometric methods according to reference /17/, they hardly do so /23/ with the method described in reference /24/. Only hyperlipidemia visible to the naked eye causes interferences in the immunonephelometric method /23/.

Reference range /20/

Reference ranges are dependent on the method of determination. Old data from healthy individuals found free Hb concentrations below 50 mg/L. New studies agree to consider as pathologic cut-off value of 100 mg/L.

References

1. Lim SF, Tan IK. Quantitative determination of methemoglobin and carboxyhemoglobin by co-oximetry, and effect of anticoagulants. Ann Clin Biochem 1999; 36: 774–76.

2. Taulier A, Levillain P, Lemonnier A. Determining methemoglobin in blood by zero crossing, point first, derivative spectrophotometry. Clin Chem 1987; 33: 1767–70.

3. Beutler E. Methemoglobinemia and sulfhemoglobinemia. In: Williams WJ, Beutler E, Erslev AJ, Lichtman MA. Hematology, 4th ed. New York: McGraw-Hill, 1990: 743.

4. Wolak E, Byerly FL, Mason T, Cairns BA. Methemo- globinemia in critically ill burned patients. Am J Crit Care 2005; 14: 104–8.

5. Olson KR (ed). Poisoning & Drug Overdose. Norwalk 1990, Appleton and Lange.

6. Carl B, Kaehler H, Schlegel R. Einfache photometrische Bestimmung von Carboxyhämoglobin und Methämoglobin durch Mehrwellenlängenmessung. Lab Med 1990; 14: 299–305.

7. Brown CM, Levy SA, Susann PW. Methemoglobinemia: life-threatening complication of endoscopy premedication. Am J Gastroent 1994; 89: 1108–9.

8. Deutsche Forschungsgemeinschaft. Photometrische Bestimmung von Carboxy-Hämoglobin (COHb) im Blut. Mitteilung VIII der Senatskommission für klinisch-toxikologische Analytik. Weinheim: VCH, 1988: 1–104.

9. Oliverio S. Current challenges in carbon monoxide poisoning diagnosis from an analytical perspective. Frontiers in Medicine 2023. doi: 10.3389/fmed.2023.1304294.

10. Hsia CCW. Respiratory function of hemoglobin. N Engl J Med 1998; 338: 239–47.

11. Allred EN, Bleeker ER, Chaitman BR, et al. Short term effects of carbon monoxide exposure on the exercise performance of subjects with coronary artery disease. N Engl J Med 1989; 321: 1426–32.

12. Eichhorn L, Thudium M, Jüttner B. The diagnosis and treatment of carbon monoxide poisoning. Dtsch Arztebl Int 2018; 115: 863–70.

13. Yasuda H, Yamaya M, Nakayama K, Ebihara S, Sasaki T, Okinaga S, et al. Increased arterial carboxyhemoglobin concentrations in chronic obsructive pulmonary disease. Am J Respir Crit Care Med 2005; 171: 1246–51.

14. Sears DA, Udden MM, Thomas LJ. Carboxyhemoglobin levels in patients with sickle cell anemia: relation- ship to hemolytic and vasoocclusive severity. Am J Med Sci 2001; 322: 345–8.

15. Widdop B. Analysis of carbon monoxide. Ann Clin Biochem 2002; 39: 378–91.

16. Oh J-Y, Hamm J, Xu X, Genschmer K, Zhong M, Lebensburger J, et al. Absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matrices. Redox biology 2016; 9: 167–77.

17. Harboe M. A method for determination of hemoglobin in plasma by near-ultraviolet spectrophotometry. Scand J Clin Lab Invest 1959; 11: 66–70.

18. Lammers M, Gressner AM. Immunonephelometric quantification of free hemoglobin. J Clin Chem Clin Biochem 1987; 25: 363–7.

19. Guder W, Fonseca-Wollheim F. Heil W, et al. Die hämolytische, ikterische und lipämische Probe. Empfehlungen zur Erkennung und Vermeidung klinisch relevanter Störungen. DG Klinische Chemie-Mitteilungen 1999; 30: 167–77.

20. Dufour N, Radjou A, Thuong M. Hemolysis and plasma free hemoglobin during extracorporeal membrane oxygenation support: from clinical implications to laboratory details. ASAIO Journal 2020; 66: 239–46.

21. Na Na, Ouyang J, Taes YEC, Delanghe J. Serum free hemoglobin concentrations in healthy individuals are related to haptoglobin type. Clin Chem 2005; 51 (9): 1754–5.

22. Podlasek SJ, McPherson RA. New lactate dehydrogenase-IgM complexes. Laboratory Medicine 1989; 20: 617–9.

23. Kahn SE, Watkins BF, Bermes EW. An evaluation of a spectrophotometric scanning technique for measurement of plasma hemoglobin. Ann Clin Lab Sci 1981; 11: 126–31.

24. Bednar R, Bayer PM. Freies Hämoglobin im Plasma. Vergleich zweier spektralphotometrischer Methoden. Bilirubin als Störfaktor. Lab Med 1994; 18: 196–9.

25. Rother RP, Bell L, Hillmen P, Gladwin MT. The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease. JAMA 2005; 293: 1653–62.

26. Chambers SD, Ceccio SL, Annich GA, Bartlett RH. Extreme negative pressure does not cause erythrocyte damage in flowing blood. ASAIO J. 1999; 45: 431–5.

27. Adamzik M, Hamburger T, Petrat F, Peters J, de Groot H, Hartmann M. Free hemoglobin concentration in severe sepsis: methods of measurement and predictin of outcome. Critical Care 2012; 16: R125 431–5.

28. Gladwin MT, Kanias T, Kim-Shapiro DB. Hemolysis and cell-free hemoglobin drive an intrinsic mechanism for huma disease. J Clin Invest 2012; 122 (4): 1205–8.

29. Ramponi G. The diagnostic accuracy of carbon monoxide pulse oximetry in adults with suspected acute carbon monoxide poisoning: a systematic review. Front Med 2023. doi: 10.3389/fmed.2023.1250845.

15.6 Reticulocyte count and reticulocyte indices

Lothar Thomas

The reticulocyte analysis of peripheral blood samples is an indicator of the erythropoietic activity of the bone marrow. Automated reticulocyte analysis involves the measurement of the reticulocyte count and most often of reticulocyte indices such as

  • The cell volume (MCVr)
  • The cellular concentration (CHCMr) and content of hemoglobin (CHr, RetHe)
  • The reticulocyte maturity pattern.

15.6.1 The reticulocyte

The reticulocyte is an immature red blood cell (RBC) from which the nucleus has been extruded. Reticulocytes are transitional RBC between nucleated RBC and the mature RBC. A reticulocyte is a RBC which, when stained with a supravital stain, contains precipitable ribonucleic acid (RNA). The RNA content is still high enough to be identified using optical fluorescent methods in the flow cytometer or panoptically under the microscope. To be identified with microscopic techniques as a reticulocyte, the cell must contain two or more blue-colored RNA particles that are visible without requiring fine focus adjustment on the individual cell to confirm their presence The granules should be distant from the cell margin to avoid confusion with Heinz bodies /1/.

The following groups are distinguished morphologically /2/:

  • Group 0; normoblasts and megaloblasts, both contain a nucleus and a dense perinuclear reticulum
  • Group 1; reticulocytes with a reticulum in the form of dense clumps
  • Group 2; reticulocytes with an annular reticulum
  • Group 3; reticulocytes with a diffusely scattered reticulum
  • Group 4; reticulocytes with a reticulum in the form of scattered granules and fragments. At this point the final maturation process stage has been reached. The reticulocyte gradually loses its granules and fragments and becomes a mature erythrocyte.

In the blood of healthy individuals, only a small fraction of reticulocytes belongs to groups 1 and 2, approximately 30% to group 3 and over 60% to group 4 /3/. In hyper-regenerative erythropoiesis, there occurs an increase of group 1 and 2 reticulocytes and the blood smear shows polychromasia of the erythrocytes /4/.

15.6.1.1 Reticulocyte maturation

During the normal maturation of RBC in the bone marrow, there is a gradual condensation of the nuclear chromatin with a reduction of both nuclear and cellular size. When the nucleus becomes pyknotic, it is extruded. In parallel, the synthesis of Hb increases. The heme portion of Hb is synthesized in the mitochondria, and the globin chains in the polyribosomes. In erythroblasts the globin chains in RNA-containing ribosomes as well as mitochondrial heme are formed. Coupling of both components takes place in the mitochondria /5/.

The RNA containing poly ribosomes can remain in the non nucleated RBCs for up to 4 days. During this period of time there is a continuous decrease in poly ribosomes and in Hb formation. About 25% of Hb is synthesized in the reticulocyte or the equivalently polychromatophilic anucleate stage of erythroid development. These cells have a greater volume than mature erythrocytes. Since Hb synthesis is not yet complete, the macrocytic erythrocytes will stain poly chromatically with Wrights-Giesma stain. The appearance of polychromatophilic macrocytes in the blood reflects the premature release of reticulocytes from the bone marrow and indicates enhanced erythropoiesis /4/.

The primary reticulocyte maturation (3 days) occurs in the bone marrow. The reticulocyte leaves the bone marrow and completes its final maturity in the peripheral blood within one day. With the loss of protein-synthesizing poly ribosomes there is cessation of Hb synthesis and transformation from reticulocyte to mature erythrocyte /5/.

Following pyknosis of the nucleus, reticulocytes can be stained with supravital dyes such as brilliant cresyl blue or new methylene blue. The polyribosomal RNA is stained. RNA can also be displayed with fluorescent dyes such as thiazole orange, acridine orange or pyronin Y /6/.

15.6.1.2 Disorders of reticulocyte maturation

In acute blood loss with a decline in the Hb level to below 80 g/L, the bone marrow is stressed with resulting hyper regenerative erythropoiesis. This leads to the following changes in maturation:

  • Reticulocytes no longer reside for 3 days but rather for only 1.5 days in the bone marrow and their final maturation is achieved in the blood. They reside in the circulation 1.7–3 for instead of 0.8–1.2 days /7/. The result is a rise in the reticulocyte count, with a shift of the reticulocytes towards maturation groups 1–3.
  • Stress reticulocytes appear in the peripheral circulation. These are reticulocytes with a large volume and high RNA content (macro reticulocytes). Erythrocytes that are formed from these stress reticulocytes have a shortened life span /8/.

15.6.2 Reticulocyte count and derived indices

The absolute reticulocyte count, the reticulocyte index and the reticulocyte production index are indicators of erythropoietic activity and permit conclusions regarding the effectiveness of erythropoiesis.

15.6.2.1 Indication

  • Assessment of bone marrow erythropoietic activity following a diagnosis of anemia
  • Distinguishing hemolytic or posthemorrhagic anemia (hyper regenerative) from anemia of chronic disease (hypo regenerative)
  • Monitoring therapy (megaloblastic anemia, iron deficiency anemia)
  • Checking early regeneration after marrow or stem cell transplant
  • Monitoring therapy with erythropoiesis stimulating agents (ESA).

15.6.2.2 Method of determination

The following can be determined:

  • Reticulocyte count
  • Reticulocyte index (hematocrit correction)
  • Reticulocyte production index (shift correction).
15.6.2.2.1 Reticulocyte count

Microscopic counting

Staining of unfixed cells with vital stains produces a net-like precipitate, the granulofilamentous material, in the immature red blood cell. This is achieved by mixing whole blood with a vital stain (brilliant cresyl blue or new methylene blue) in a 1 : 1 ratio, followed by the preparation of blood smears on several slides. After air-drying, a microscopic examination is performed. Among 1000 red blood cells, all cells that contain a bluish, thread-like or granular precipitate are counted /9/.

Automated reticulocyte counting

The automated systems have in common the rapid analysis of whole blood suspensions in flow-through systems, with the red cell population interrogated on a cell-by-cell basis by laser light. The reticulocytes are stained in a separate procedure. Some analyzers utilize a detection principle based upon light absorption or scatter caused by the reagent-RNA precipitate within the reticulocyte. Others employ fluorescent reticulocyte reagents /10/.

Fluorescence-activated cytometry: a fluorescent dye (e.g. thiazole orange, acridine orange, pyronin Y) binds to the RNA of the reticulocyte. The extent of the fluorescence emission that is produced by the excitation of the bound dye is directly proportional to the RNA content of the reticulocyte and inversely proportional to its maturity stage /6/.

Flow cytometry: the detection of reticulocytes is based on the principle that precipitation and staining, due to the use of fixation and staining reagents, causes light to be absorbed or scattered. The stains employed include, for example, methylene blue or oxazine /10/.

The reticulocyte count is either expressed as a percentage (number of reticulocytes/100 red blood cells) or as absolute cell count (109/L) /9/.

15.6.2.2.2 Reticulocyte index; RI (hematocrit correction)

The reticulocyte count can be increased in relation to erythrocyte count either because more reticulocytes are in the circulation, or there are fewer erythrocytes /9/. Therefore, the observed reticulocyte count may be corrected to a normal hematocrit of 0.45 (45%) according to the equation 1 in Tab. 15.6-1 – Hematocrit correction and shift correction.

15.6.2.2.3 Reticulocyte production index; RPI (shift correction)

Counts corrected for hematocrit (HCT) are not perfect indices of production, because the reticulocyte count can also be altered by premature release of cells from the marrow (shift) /9/. If shift cells (polychromatophilic macrocytes) are detected in the Wright-stained smear, an empirical correction must be applied for RBC maturation. The maturation time of the reticulocyte is taken as:

  • 1 day at HCT of 0.45 (45%)
  • 1.5 days at a HCT of 0.35 (35%)
  • 2 days at a HCT of 0.25 (25%)
  • 3 days at a HCT of 0.15 (15%)

The calculation of the RPI is shown in Tab. 15.6-1 – Hematocrit correction and shift correction. If the patient has a corrected reticulocyte count (RI) of 10% and a HCT of 25% the RPI is 10/2 = 5. In this case a shift (RPI) > 3 is considered to represent an adequate erythropoietic response while an RPI < 2 is inadequate. The expected lower limits of the RPI for an adequate erythropoietic response in dependence of the HCT are shown in Tab. 15.6-2 – Expected lower RPI limits and reticulocyte count as function of HCT.

15.6.2.3 Specimen

EDTA blood: 1 mL

15.6.2.4 Reference interval

Refer to Tab. 15.6-3 – Reticulocyte reference intervals.

15.6.2.5 Clinical significance

The reticulocyte count is an important indicator of bone marrow erythropoietic activity and of a significant reduction of erythrocytes.

15.6.2.5.1 Absolute reticulocyte count

The reticulocyte count per volume is a measure of the bone marrow’s effectivity in forming mature RBC. This holds true for normo regenerative erythropoiesis (steady state), as well as for hyper- and hypo regenerative erythropoiesis /15/.

15.6.2.5.2 Relative reticulocyte count

Expression of reticulocytes as percentage of red cells is a measure of the erythrocyte life span. It allows for an estimation of the shortening of the erythrocyte life span in chronic anemia in the steady state. The higher the relative reticulocyte count (%), the shorter the life span. Often, inexperienced physicians do not know that an apparently elevated percentage reticulocyte count may not be elevated when correction is made for a low erythrocyte count /15/.

15.6.2.5.3 Reticulocyte index (RI)

In the steady state, after HCT correction an increased percentage of reticulocytes can be an indicator of a shortened erythrocyte life span. The higher the RI, the shorter the life span.

15.6.2.5.4 Reticulocyte production index (RPI)

An elevated reticulocyte count can reflect /15/:

  • A hyper regenerative erythropoiesis because of a shortening of the erythrocyte life span
  • A premature release of red cells from the marrow.

The RPI reflects the increase or reduction in erythropoiesis. Under ESA stimulation, an increase in reactivity of up to 8-fold is possible. Hypo reactivity is present if an increase in reactivity is below 2. Erythropoietin-stimulated reactivity increases as the HCT falls. In hyper regenerative erythropoiesis, the reticulocyte maturation time in bone marrow is shortened, proportional to the decline in HCT and the residence time in peripheral blood is prolonged. The HCT dependent prolonged residence time in blood is corrected with calculation of the RPI /1/. Without correction of the prolonged residence time, with increasing anemia erythrocyte production will be overestimated due to the absolute reticulocyte count and the erythrocyte life span will be underestimated due to the percentage of reticulocyte count. The expected RPI in dependence of HCT and reticulocyte count that can be achieved in intact marrow function are shown in Tab. 15.6-2 – Expected lower RPI limits and reticulocyte count as function of HCT /16/. With an HCT of 0.35, an RPI ≥ 2 is indicative of regenerative, an RPI ≥ 3 points to hyper regenerative erythropoiesis. An RPI < 2 denotes hypo regenerative erythropoiesis (e.g. anemia associated with chronic disease due to inflammation, infection, malignant disease or intrinsic hypoplasia of erythropoiesis).

15.6.2.5.5 Reticulocytosis

With a HCT below 0.30 it is necessary to determine the RPI since, otherwise, reticulocytosis will be diagnosed too frequently /15/.

Reticulocyte determination is particularly relevant in normocytic anemia. In macrocytic anemia, reticulocytosis is indicative of untreated folate or vitamin B12 deficiency /17/. In microcytic anemia, the reticulocyte count should only be determined if ferritin level or the transferrin saturation do not convey an unequivocal statement. Refer to:

15.6.2.5.6 Reticulocytopenia

Reticulocytopenia is a sign of hypo regenerative erythropoiesis and occurs in:

  • Deficiency anemia (e.g. iron, copper or vitamin B6, vitamin B12 and folate deficiency)
  • Anemia associated with chronic disease (infection, chronic inflammation), malignancy, chronic liver disease) caused by inflammatory cytokine-induced hypo proliferation of erythropoiesis /18/
  • Chronic renal insufficiency. Erythropoietic proliferation is diminished due to inappropriately low erythropoietin secretion /19/.
  • Myelodysplastic syndrome (MDS). Anemia associated with MDS is hypo proliferative, with peripheral reticulocytopenia in spite of hyper cellular marrow and up to 90% ineffective erythropoiesis /20/. Concentration of soluble transferrin receptor is often elevated.
  • Congenital dyserythropoietic anemia. Three types are distinguished. They manifest moderate anemia with Hb values around 90 g/L and a low to normal reticulocyte count /21/.

15.6.2.6 Comments and problems

Method of determination

Microscopic method: the microscopic analysis of the blood smear is imprecise, with intra- and inter laboratory coefficients of 25% and 25–50%, respectively, if the reticulocyte count is determined on the basis of 1000 red cells /9/. Howell-Jolly bodies, Heinz bodies and malaria parasites are also stained with the smear /9/.

Hematology analyzer: counting accuracy is higher since some 10,000 cells are counted. The inter laboratory coefficient of variation for reticulocytosis > 2.5% is 24%, being approximately half as large as with microscopic counting with the same reticulocyte count /26/.

Potential sources of error associated with flow cytometry are caused by Howell-Jolly bodies, nucleated red blood cells, sickle cells, giant thrombocytes, cold agglutinins, parasites (malaria, babesiosis), and platelet clumps. Cytometric methods distinguish systematically from one another based on the use of different dyes.

Stability

Strong depending on the staining technique and the method of determination. At 20 °C, decline after 24 h, at 4– 8 °C possible for up to 72 h and longer /27/.

15.6.3 Retikulocyte maturity index and immature reticulocyte fraction

The Reticulocyte maturity index (RMI) and Immature Reticulocyte Fraction (IRF) are indices for the RNA content of reticulocytes. The quantification is based on the determination of the RNA content in the reticulocyte. Immature reticulocytes have a higher RNA content than mature forms. A rise in the number of reticulocytes in the blood leads to an increase in RMI and IRF.

15.6.3.1 Indication

Assessment of erythropoietic activity:

  • In severe anemia (e.g., hemolytic anemia)
  • Following bone marrow transplantation and after chemotherapy.

15.6.3.2 Method of determination

Reticulocyte maturity index (RMI)

Hematology analyzers for reticulocyte counting utilize reagents that bind specifically and rapidly to nucleic acids, such as ethidium bromide, auramine O (Sysmex), CD4K530 (Abbott), oxazine 750 (Siemens), or new methylene blue (Beckman Coulter). The light energy of a laser is absorbed or scattered. Quantitative flow cytometric fluorescence measurements with the reagents like auramine O or ethidium bromide are also used. The reticulocyte population is generally classified, according to the corresponding threshold value that has been set, into 3 maturation stages: low fluorescence reticulocytes (LFR), medium fluorescence reticulocytes (MFR), and high fluorescence reticulocytes (HFR) /28/ (Fig. 15.6-4 – Differentiation of reticulocytes in maturation stages).

Healthy individuals normally have reticulocytes of the LFR fraction.

Immature Reticulocyte Fraction (IRF)

The IRF is calculated as follows:

IRF (%) = HFR (%) + MFR (%)

15.6.3.3 Specimen

EDTA blood: 1 mL

15.6.3.4 Reference interval

Refer to Tab. 15.6-5 – RMI and IRF reference intervals.

15.6.3.5 Clinical significance

The maturation stage of reticulocytes in peripheral blood is mainly dependent upon:

  • The severity of anemia
  • The iron, vitamin B12 and folic acid status
  • Stimulation with erythropoietin
  • The presence of systemic inflammation.

The RMI or IRF do not manifest a clear relationship to production of erythrocytes or to a reduction of their life span. However, under non-steady state conditions, they provide an early indication of incipient regeneration or suppression of erythropoiesis or its responsiveness to ESA therapy /15/. The diagnostic value of the RMI in combination with the reticulocyte count for clarification of anemia is shown in Tab. 15.6-6 – Diagnostic significance of reticulocyte count and reticulocyte maturity index /32/.

15.6.3.5.1 Acute hemorrhage

In association with a decreased Hb, the reticulocyte maturation time in bone marrow is shortened and increasing numbers of immature reticulocytes pass into the blood. RMI and IRF increase due to elevations in MFR and HFR fractions /10/.

Both of these indices increase within 5–8 hours of severe acute loss of blood, while a rise in reticulocytes only becomes significant after 24–48 hours.

15.6.3.5.2 Renal anemia, pernicious anemia, myelodysplastic syndrome

Reticulocyte maturation is delayed and patients with these diseases have a reduced reticulocyte count in association with elevated RMI and IRF /10/.

15.6.3.5.3 Bone marrow transplantation

The RMI is the earliest clinical indicator of engraftment following bone marrow transplantation /30/. If, on the 21st post-transplant day, the reticulocyte count reaches a value of 15 × 109/L and HFR a value of 0.5 × 109/L, this reflects to a degree of 100% the functioning of the transplant /32/.

In contrast to an increase in polymorphonuclear neutrophils and the reticulocyte count, however, the RMI and IRF manifest no obvious advantages /31/.

15.6.3.6 Comments and problems

There are various definitions of immature reticulocytes. In some studies, only the HFR fraction is considered, while in others the sum of HFR and MFR. For determination of the IFR, the HFR and MFR fraction are taken into consideration /31/.

Method of determination

The methods used by different hematology analyzers are not comparable, since no standard reticulocyte preparations are available and the manufacturers each have their own different calibration methods. Since the HFR fraction normally is only a small proportion of the reticulocytes, approximately 500 reticulocytes are found when 50,000 red blood cells are counted and only a few of these fall within the HFR group. Considerable disturbances with false positive HFR results can occur due to large blood platelets, leukocytes, erythroblasts, erythrocytes with malaria parasites, or Howell-Jolly bodies. Typically chronic lymphocytic leukemia may give spuriously RMI values /31/.

15.6.4 Reticulocyte indices

Reticulocyte analysis has been extended from the simple counting of reticulocytes to precise measurements of cellular indices such as volume, hemoglobin concentration, and content /33/.

15.6.5 Mean cell volume of reticulocytes (MCVr)

During maturation of erythropoietic progenitor cells in the bone marrow, their volume decreases continuously. Marked decreases occur:

  • In bone marrow from stage 0 (orthochromatic erythroblast) to stage 1 (reticulocyte with reticulum appearing in the form of a dense clump), due to loss of the cell nucleus
  • In blood during transition from stage 4 (reticulocyte with reticulum appearing as a few scattered granules or fragments) to the mature erythrocyte. The circulating reticulocyte has a diameter of 8.5 μm some 1–1.5 μm larger than the erythrocyte. The reticulocyte volume is on average 20% larger than that of the mature erythrocyte. Based on median mature erythrocyte volume (MCV) of 88 fL, median reticulocyte volume (MCVr) is around 106 fL.

15.6.5.1 Indication

  • Suspected stress erythropoiesis following acute bleeding, deficient oxygen supply, over stimulation of bone marrow through ESA-therapy
  • Assessment of therapeutic responsiveness in deficiency anemia (iron, folic acid, vitamin B12).

15.6.5.2 Method of determination

Principle: laser-based technology utilized in the Advia 120 hematology analyzer allows simultaneous measurement of volume and Hb concentration of both erythrocytes and reticulocytes. From the measurements of cell volume and Hb concentration, the Hb content of every cell is determined. EDTA blood is diluted with reticulocyte reagent. The reagent contains sodium dodecyl sulfate, which forces red blood cells into a spherical shape. The oxacine staining method used for reticulocyte analysis measures staining intensity and the indices such as MCVr, and Hb concentration (CHCMr). The mean Hb content of reticulocytes (CHr) is calculated from the product of the volume times Hb concentration of each reticulocyte /31/.

With Sysmex hematology analyzers, reticulocyte MCV is derived from forward scattering of fluorescence-labeled reticulocytes.

15.6.5.3 Specimen

EDTA blood: 1 mL

15.6.5.4 Reference interval

Adults /34/: 92–120 fL

15.6.5.5 Clinical significance

A decrease in MCVr is typical in iron deficiency, while a rise may indicate folic acid and vitamin B12 deficiency anemia. Macro reticulocytes are formed in erythropoiesis associated with stress situations (e.g., in acute hypoxia).

15.6.5.5.1 Stress erythropoiesis

The MCV of macro reticulocytes is ≥ 27% larger than that of erythrocytes /4/. In comparison with erythrocytes, MCV can be increased by a factor of 3. Macro reticulocytes are found under stress situations and are also designated as stress reticulocytes. In the blood smear they can be detectable as polychromatophilic erythrocytes. Stress reticulocytes are found:

  • 5–8 h after severe acute bleeding
  • During a stay at high altitudes
  • As a response to successful treatment of iron deficiency anemia in the early phase (first 2–3 days)
  • As a response to ESA therapy (ESA, erythropoiesis-stimulating agents). In individuals with normal iron status and without acute phase response, administration of ESA leads to a rise in the reticulocyte count and MCVr, however the reticulocyte Hb concentration (CHCMr) decreases. If functional iron deficiency develops, no increase in MCVr occurs and small reticulocytes are released in the blood.
15.6.5.5.2 Hemolytic anemia

In acute and massive hemolytic anemia (e.g., autoimmune hemolysis) increased production of erythropoietin stimulates erythropoiesis and development of iron-restrictive erythropoiesis. The result is an inappropriately low reticulocyte count. The MCVr is reduced and an inversion of the MCVr/MCV ratio, which is normally greater than 1.0, occurs /35/. It has been shown in phlebotomy investigations that this situation occurs some 2 days following a reduction in transferrin saturation. The same results are observed in over stimulation with ESA.

15.6.5.5.3 Folic acid and vitamin B12 deficiency anemia

Macrocytosis of erythrocytes and reticulocytes is present in both of these forms of anemia, and the MCVr/MCV ratio is greater than 1.0. With treatment of anemia, an inversion of the ratio with reticulocytes being smaller than erythrocytes is seen as a response to vitamin B12 therapy for megaloblastic anemia. The cause is a decrease in MCV which occurs sooner in the reticulocyte than in the erythrocyte. In a study /35/ the produced reticulocytes after 17 days treatment had an MCVr of 108.8 fL, while the MCV of erythrocytes was still 109.8 fL, because most of the circulating erythrocytes had been formed prior to the administration of vitamin B12.

15.6.6 Reticulocyte hemoglobin content (CHr or RetHe)

Reticulocytes have a higher fluid content, a 1–3 pg higher Hb content, and up to some 20% greater volume than erythrocytes. The results is that reticulocytes are more hypochromic than erythrocytes.

15.6.6.1 Indication

  • Evaluation the iron demand of erythropoiesis.
  • Assessment the therapeutic response of iron deficiency anemia within several days after the start of treatment
  • Monitoring of iron supply for erythropoiesis under ESA therapy.

15.6.6.2 Method of determination

On Advia 120, the Hb concentration of individual reticulocytes (CHCMr) is measured and the Hb content is calculated according to the equation CHr = MCVr × CHCMr /31/. In addition, the Hb content of the reticulocyte fraction (RFHb) can be determined according to the equation RFHb = Reticulocyte number × CHr.

On Sysmex NE-2100, the Ret-He is determined. The light intensity of the forward scattered fluorescence-labeled reticulocyte correlates with its Hb content. Results are expressed in pg /36/.

15.6.6.3 Specimen

EDTA blood: 1 mL

15.6.6.4 Reference interval

Tab. 15.6-7 – Reference interval for reticulocyte hemoglobin.

15.6.6.5 Clinical significance

CHr and Ret-He are markers for determining the actual iron demand of erythropoiesis. A value below 28 pg indicates iron-restricted erythropoiesis. When iron demand starts, CHr and Ret-He decrease within 48–72 h. Other markers such as %HYPO or biochemical markers of iron metabolism indicate changes following 10–20 days at the earliest, while erythrocyte MCV and MCH do so only after 2 to 3 months.

15.6.6.5.1 Iron deficiency

The detection of iron deficiency before microcytic hypochromic anemia develops is important to avoid systemic complications of the iron deficiency. Classical biochemical parameters for early diagnosis of iron deficiency are serum ferritin, the transferrin saturation and the soluble transferrin receptor. In children, in youth during the growth spurt, endurance athletes, women of menstrual age, and repeat blood donors, the ferritin value can be critical as an indicator of iron deficiency, since these individuals already have low values in spite of a still adequate supply of iron /41/.

In healthy individuals treated with ESA, it could be shown that decreases in CHr and Ret-He are early indicators of iron deficiency /42/. As early as 3–5 days following the start of ESA therapy, a significant decline in CHr or Ret-He was measured.

In the diagnostic investigation of iron deficiency in children, CHr was of higher predictive value than biochemical markers of iron deficiency /43/.

The fraction of hypochromic reticulocytes (CHCMr below 270 g/L), which is normally less than 25%, is also an early indicator of iron-restricted erythropoiesis. As iron deficiency progresses, CHr and Ret-He decrease and the hypochromic reticulocyte fraction increases.

15.6.6.5.2 Functional iron deficiency

Functional iron deficiency (ID) is a state of iron-poor erythropoiesis in which there is insufficient mobilization of iron from the stores in the presence of increased demands. Anemia with functional ID develops during increased erythropoiesis mediated either by endogenous erythropoietin responses to anemia, or by therapy with erythropoiesis stimulating agents (ESAs). In functional ID imbalance between the surging iron requirements of the stimulated erythroid marrow and iron availability is the pathophysiology. Iron is sequestered in macrophages of the reticulo-endothelial system. The iron stores are normal or increased (ferritin above 100–299 μg/L) in combination with transferrin saturation less than 20%.

Thus, in functional iron deficiency there is a situation in which the iron demand of erythropoiesis, due to overstimulated erythropoiesis, exceeds the rate of iron release from the stores and the transferrin iron transporting capacity. In consequence, hypochromic reticulocytes and erythrocytes are released from the bone marrow. The reticulocyte Hb content (CHr, RetHe) is decreased /44/.

In hemodialysis patients who are on ESA therapy, monitoring of CHr or RetHe is a better indicator of iron demand than biochemical markers of iron metabolism (ferritin, transferrin saturation).

CHr and RetHe below 28 pg are indicative of functional ID /41/:

  • In tumor patients, due to bleeding, hemolysis and cytostatic therapy
  • In anemia of chronic disease (ACD). IL-6 triggered by inflammation induces hepatocyte synthesis of hepcidin, which leads to augmented iron retention in the macrophages and reduced intestinal iron absorption. In this way, iron turnover is reduced, resulting in functional ID, which is developed in 5–10% of ACD patients. Reticulocytes and erythrocytes have a reduced Hb content, but their MCV value is usually sub-normal.
  • In dialysis patients, the threshold value of functional ID is ≤ 29 pg /45/.

Functional ID is also present if CHr or RetHe are smaller than the erythrocyte MCH value (CH inversion) /37/.

15.6.6.5.3 Diagnostic diagram

Development of iron deficiency and functional ID as a function of the iron supply can be identified and monitored with the aid of the diagnostic diagram shown in Fig. 15.6-5 – Diagnostic diagram for the assessment, monitoring and therapy of the iron status. In the diagram the ferritin index (sTfR/log10 ferritin) serves as indicator of the iron supply for erythropoiesis, since it correlates well with stainable iron of the bone marrow. The CHr or RetHe are indicators of the iron demand of erythropoiesis /41/. In a study /46/ the differentiation of anemia was not possible in 32% of the cases with classical serial determination of hematological and iron metabolism markers, but with the application of the diagram this percentage decreased to only 14%. In a treatment study of tumor patients, the concept of the diagram and the therapeutic recommendations for the different forms of anemia proved to be of value /47/.

15.6.6.5.4 Sickle cell anemia

In sickle cell anemia, reticulocyte Hb concentration (CHCMr) is elevated and, as a rule, greater than 380 g/L. CHCMr decreases within the first two weeks of treatment with hydroxyurea, due to improved hydration of the sickled cells; in consequence, the tendency of the cells to undergo sickling is attenuated /48/.

Compared with healthy individuals, patients with sickle cell anemia have a 2–3-fold higher Hb contained in the reticulocyte compartment (retHb) as normal controls. The Hb in the red cell compartment (rbcHb) is decreased. In a study /49/ the retHb values in controls were 1.76 ± 0.69 g/L and in SS4α 6.5 ± 4.2 g/L. The rbcHb/retHb ratio is an important indicator of sickle cell disease. In healthy individuals, as well as in iron deficiency anemia, the ratio is ≥ 50, in sickle cell anemia it is ≤ 50. RbcHb is the remainder obtained by the subtraction of retHb from total Hb.

15.6.6.6 Comments and problems

Method of determination

CHr and Ret-He are to be determined with the hematology analyzers Siemens Advia and Sysmex analyzers, but CHCMr only with Advia. All data, therefore, relate to the use of these analyzers.

Stability

Upon storage of the blood samples for 60 minutes, MCVr increases and CHCMr decreases, but CHr and Ret-He are stable for at least 24 hours /31/.

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23. Jelkmann W, Lundby C. Blood doping and its detection. Blood 2011; 118: 2395–404.

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26. Davis BH, Bigelow NC, Koepke JA, Borowitz MJ, Houwen B, et al. Flow cytometric reticulocyte analysis. Multiinstitutional interlaboratory correlation study. Am J Clin Pathol 1994; 102: 468–72.

27. Arbeitsgruppe Präanalytik der DGKC. Stellungnahme zu den Empfehlungen Stabilität der Messgröße in der Probenmatrix. DG Klinische Chemie Mitteilungen 1996; 27: 27–9.

28. Davis BH, DiCorato M, Bigelow NC, et al. Proposal for standardization of flow cytometric reticulocyte maturity index (RMI) measurements. Cytometry 1993; 14: 318–26.

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30. Kanold J, Bezou M, Coulet M, et al. Evaluation of erythropoietic/hematopoietic reconstitution after BMT by highly fluorescent reticulocyte counts compares favorably with traditional peripheral blood cell counting. Bone Marrow Transplant 1993; 11: 313–8.

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15.7 Hemoglobinopathies

Elisabeth Kohne

Human hemoglobin consists of the main component HbA and several minor components of which only HbA2 is genetically determined, whereas the so-called modified hemoglobins HbA1a, HbA1b and HbA1c are only formed secondarily within the erythrocyte under the influence of exogenous factors.

During the fetal period the embryonic hemoglobins Hb Gower 1, Hb Gower 2 and Hb Portland are synthesized first being replaced by HbF from the 5–7th gestational week. At term newborns have 80% HbF and 20% HbA. During the first 6 months of life a gradual shift in hemoglobin takes place until the adult pattern is reached after the 12th month.

The common structural principle of all hemoglobins is their composition of four heme molecules and the protein portions (globin) each consisting of two pairs of identical polypeptide chains (Tab. 15.7-1 – Normal human hemoglobins) /123/.

The umbrella term “hemoglobinopathy” includes all genetic hemoglobin disorders. These are divided into two main groups as follows:

  • Thalassemia syndromes
  • Structural hemoglobin variants (abnormal hemoglobins).

Both are caused by mutations and/or deletions in the α- or β-genes. When gene defects cause hemoglobin synthesis disorders, this gives rise to thalassemia. Hemoglobin structure in these cases is normal. When they change in the hemoglobin structure, this gives rise to abnormal hemoglobin. There are also mixed forms that combine features of both groups (e.g., β0+-thalassemias, HbSC disease and HbE α-thalassemias). The common features of the pathophysiology and various disease patterns are limited, and as a result so are the possibilities for summarizing them /24/.

Epidemiology of hemoglobinopathies

With about 7% of the worldwide population being carriers, hemoglobinopathies are the most common monogenic diseases and one of the world’s major health problems. They were originally found mainly in the Mediterranean area and large parts of Asia and Africa. Global migration has spread them from those areas all over the world. In many parts of Europe today, hemoglobin defects are classified as endemic diseases (Tab. 15.7-2 – Prevalence of hemoglobinopathy gene carriers in the world’s population) /45/. Germany is one of the countries in which hemoglobinopathies have increased in recent years and represent a relevant healthcare problem in German medicine. Most frequently occurring are thalassemias and sickle cell disease /456/. No epidemiological prevalence studies exist, but based upon calculations using currently available figures, it is assumed that genetic carriers of hemoglobinopathy make up 1% of the present German population /4/. An overview /5/of the most important hemoglobin diseases occurring in this country, listed according to prevalence, is found in Tab. 15.7-3 – The most important hemoglobinopathies in Germany.

Clinical laboratory investigations

The type and extent of laboratory investigations depend on the questions to be answered in each case based on the clinical and hematologic data and the history (ethnic background). The presence of a hemoglobinopathy is ruled out if all hematologic parameters are normal (exception: asymptomatic anomalies). If on the other hand hemoglobin analysis reveals normal values in the setting of very specific hematologic findings further laboratory investigations may have to be performed.

Peculiarities

A large variety of defects due to the varied ethnic composition of the population is typical for countries such as the USA or Germany, which have a significant proportion of immigrants. The clinical presentations are very different, varying from mild hypochromia and microcytosis with or without anemia to complex clinical presentations.

These peculiarities represent a challenge for the clinical laboratory in regard both to the methods of analysis and the knowledge required for the interpretation of the findings. In doubtful cases consultation with a specialized laboratory may be necessary.

15.7.1 Indication

  • Microcytic hypochromic anemia after iron deficiency has been ruled out
  • Chronic hemolytic anemia
  • Vascular obliteration crises of unclear etiology in patients from areas in which HbS and/or HbC is widespread
  • Drug-induced anemia
  • Erythrocytosis and/or cyanosis caused by hematological factors
  • Hydrops fetalis of unclear etiology
  • Prevention (testing of family members, diagnosis of partners for genetic counseling)
  • Prenatal diagnosis.

Generalized hemoglobin electrophoresis for all cases of anemia cannot be justified economically, particularly in those with no background of migration.

Indication for DNA analysis

Within the framework of thalassemia diagnostic investigation:

  • Genetic testing of β-thalassemia major
  • Molecular diagnosis of β-thalassemia inter media
  • Combination of different forms of thalassemia or of thalassemias with structural Hb anomalies
  • Suspected silent beta thalassemia trait carriers
  • Diagnostic investigation of α-thalassemia
  • Issues related to genetics and preventive medicine.

Diagnostic investigation of Hb structure anomalies

  • Identification of rare anomalies
  • For clarification in cases of pre-existing suspicion, but lacking electrophoretic or chromatographic separation
  • In the presence of issues related to genetics and preventive medicine
  • Forms of different hemoglobinopathies combined with one another or with thalassemia.

DNA analyses should and may be implemented only in cases where the issues cannot be resolved with the use of conventional hemoglobin analyses.

15.7.2 Method of determination

Laboratory examinations consist of the routine hematologic evaluation, cytologic tests, and hemolysate analyses including hemoglobin electrophoresis using different buffer and pH systems and chromatographic procedures. The stepwise approach shown in Tab. 15.7-4 – Program for the laboratory diagnostic investigation of hemoglobinopathies has proven itself for practical use.

The help of a specialized laboratory may be necessary since, particularly for economic reasons, even large laboratories have to contain the expenditures related to personnel and equipment in this special, originally purely hematological, field.

The plan that is depicted in Fig. 15.7-1 – Clinical laboratory diagnosis of thalassemia syndromes and hemoglobinopathies has proven itself as a practical procedure for hemoglobinopathy analysis.

15.7.2.1 Basic hematological diagnosis

In suspected hemoglobinopathy a complete red blood cell count, including reticulocyte count, is a requirement. The RDW (red cell distribution width) value also provides important evidence which is usually elevated as a measure of anisocytosis in iron deficiency, while in thalassemia minor normal RDW values are usually observed (Fig. 15.7-2 – Use of RDW values in diagnostic investigation of thalassemia minor).

The assessment of the blood smear elicits diagnostically valuable information because the thalassemias, as well as most structural hemoglobin anomalies, express characteristic changes in erythrocyte morphology /2/.

15.7.2.2 Clinical chemistry tests

These include biomarkers of iron status (ferritin, transferrin saturation) and the hemolysis parameters haptoglobin, LD, bilirubin, and the Coombs test.

15.7.2.3 Hemoglobin analyses

Hb electrophoresis

Routine electrophoresis is performed on cellulose acetate membranes with an alkaline Tris-EDTA borate buffer (pH 8.5) (micro zone electrophoresis). Fig. 15.7-3 – Separation schema for normal and abnormal hemoglobin on micro zone electrophoresis). Under these circumstances abnormal hemoglobins are only separated from normal ones if they have a difference in electrical charge. The relative concentrations of the separated bands can be determined by densitometry, or the fractions eluted and measured. For HbA2 both procedures have inherent errors. Using acid agarose gel electrophoresis with a maleic acid buffer at pH 6.1, as an additional technique, hemoglobins may be separated which migrate together on alkaline electrophoresis /27, 8, 9, 10, 1112/.

A well established procedure for qualitative and quantitative evaluations (HbA2) is starch block electrophoresis. However, it is worthwhile only for laboratories with a high demand on account of its complexity.

Depending on the experience in the laboratory isoelectric focusing may also be used.

HPLC

HPLC (cation-anion exchange systems) is the optimal method for separating normal and abnormal hemoglobins. HPLC is also the method of choice for quantitative analysis of all separable hemoglobin fractions /27, 8, 1011/.

15.7.2.4 HbF determination and HbF cell detection

Alkali denaturation is the classical method for quantification of HbF. A cyan hemoglobin solution is produced using a hemolysate followed by the addition of NaOH. During this process HbA2 is denatured. Subsequent precipitation using ammonium sulfate leaves HbF remaining in solution where it may be measured photometrically /2/.

The acid elution method serves to detect HbF cells in peripheral blood smear preparations. HbA is eluted from erythrocytes by a citric acid-phosphate buffer at pH 3.2 while HbF remains in the cells and is stained. In the case of a negative HbF cell result the process of alkali denaturation is superfluous.

15.7.2.5 HbS solubility test

The solubility test is used to differentiate between HbS and anomalous hemoglobins with identical migration patterns during electrophoresis, such as HbD and HbG. In a hemolysate to which dithionite has been added for removal of oxygen, HbS is the only hemoglobin to precipitate causing marked turbidity of the reaction mixture /2/.

15.7.2.6 Identification of abnormal hemoglobins

The common abnormal hemoglobins HbS, HbC, HbE and HbD represent more than 90% of all structural hemoglobin anomalies seen in daily laboratory practice /27, 8, 1011/. As a rule, these hemoglobins can be directly diagnosed electrophoretically or according to their chromatographic properties with, as necessary, chemical test procedures such as the solubility test. DNA analyses are employed for the identification of rare pathological hemoglobin variants. Molecular biologic testing should be restricted to those hemoglobin anomalies that cannot be clarified with conventional methods.

15.7.2.7 DNA analyses

The molecular biologic procedures are listed in Tab. 15.7-5 – Basic information on molecular biologic methodology /13/.

15.7.3 Specimen

Erythrocytes from blood with anticoagulants are required. EDTA blood (in coated sample containers) is best and is suitable for simultaneous determination of the blood cell count, RBC morphology and erythrocyte enzymes. EDTA blood may be stored for a few days without significant deterioration, the investigation of α-thalassemia being an exception. For routine examinations 5 mL of blood are usually adequate, larger volumes are needed in the case of severe anemia.

15.7.4 Reference interval

Refer to Tab. 15.7-6 – Reference intervals for hemoglobins.

15.7.5 Clinical significance

Basic forms of hemoglobinopathies are shown in Tab. 15.7-7 – Basic forms of hemoglobinopathies. Generally significant criteria are the age of the patient (e.g. for assessing an HbF value) the ethnic background, the family history, and the clinical hematologic findings. Hemoglobin analysis includes:

  • Assessment of quantitative changes of normal hemoglobins such as the increase in HbA2 and/or HbF typical of thalassemias
  • Exclusion or confirmation of an abnormal variant and its identification and quantification
  • In each case the question has to be addressed of whether the abnormal hemoglobin is responsible for the clinical symptoms or is simply a random finding without pathologic significance.

15.7.5.1 Thalassemia syndromes

This term includes all thalassemic hemoglobin synthesis disorders. Thalassemias are one of the most common single-gene disorders in the world. The presence of mutations in the globin genes cause the reduction or absence of globin chains and results in thalassemia. The mutations occur in the alpha, beta, gamma, and delta genes, and as a result, the synthesis of hemoglobin is disturbed. Different genotypes in thalassemia cause various phenotypes. The phenotypes vary from a thalassemia minor to thalassemia major with mild or severe anemia, respectively. The couples who are the carriers of thalassemia play an important role in the birth of infants with thalassemia major so that the chance of the birth of α-thalassemia major infant in a thalassemia couple is 25%. In autosomal recessive conditions alpha- and beta-thalassemias have the greatest significance. Heterozygous thalassemia carriers are not completely healthy: they always have symptoms that require differentiation with mild, iron-refractory, microcytic hypochromic anemia /1011, 14, 1516/.

15.7.5.1.1 α-thalassemias

α-thalassemias are a common monogenic gene disorder caused by a defect in α-globin genes located on the short arms of chromosome 16. α-thalassemias are caused by an α-globin chain synthesis defect. At the molecular level, they result from partial (α+) or total (α0) deletions, or more rarely mutations, of one or more of the α-globin genes (αα/αα). They occur mainly in Africa, Arab nations, and more frequently, South-East Asia. All become manifest perinatally. There are four clinical pictures of α-thalassemia, according to the number of genes affected by loss of function (Tab. 15.7-8 – Compilation of the most important criteria of α-thalassemias):

  • Clinically inapparent α-thalassemia minima (heterozygous α+-thalassemia, –α/αα). This can be identified on the basis of mild hypochromia with a barely reduction in hemoglobin concentration.
  • α-thalassemia minor (heterozygous α0-thalassemia, ––/αα, or homozygous α+-thalassemia, –α/–α) with mild anemia, hypochromia and microcytosis
  • HbH disease (compound heterozygous α+0-thalassemia with three inactive α-genes, ––/–α) moderate hypochromic hemolytic anemia with splenomegaly. Anemic crisis are caused by viral infections and oxidants (drugs). Complications include cardiac problems, gall stones, lower leg ulcers, and folic acid deficiency.
  • Hemoglobin (Hb)-Bart’s hydrops fetalis (homozygous α0-thalassemia) is the most severe form of α-thalassemias with very serious hemolytic anemia already present in utero and marked by a lack of any α-globin chain synthesis (––/––), with hydrops and ascites. Hb Bart’s hydrops fetalis is the most severe form of α-thalassemias and is incompatible with life. If both husband and wife are carriers of both α-globin gene deletions, they have a 25% chance of having a child with Hb Bart’s hyrops fetalis in each pregnancy /18/.
15.7.5.1.2 β-thalassemias

β-thalassemias are the result of insufficient (β+) or absent (β0) production of β-globin chains (Tab. 15.7-9 – Compilation of the most important criteria of β-thalassemias). Their molecular causes are β-globin gene mutations. Most patients come from Mediterranean countries, South-East Europe, Arab Nations, and Asia. Hematological changes become manifest from between the ages of 3–6 months onwards.

15.7.5.1.3 β-thalassemia minor

The starting point for the diagnosis of thalassemia minor (heterozygous β-thalassemia) is the complete blood count. The significant laboratory markers of β-thalassemia are increased HbA2 and/or increased HbF values. If the MCH is below 27 pg and the HbA2 above 3.5%, diagnosis of heterozygous β-thalassemia is made. The majority of β-thalassemia trait carriers have MCH reductions to 23–19 pg and HbA2 values of 4.0–6.0%; HbA2 increases to 6.5–8.0% can occur. In some 30% of the cases, HbF increase to 1–3%, occasionally to 3.0–15%, occurs simultaneously. To be noted are age-dependent higher HbF values in young children with β-thalassemia minor. The iron status (ferritin, transferrin saturation) is, as a rule, normal. Exceptions (i.e., iron deficiency in β-thalassemia minor) can occur in children and during pregnancy. Simultaneous iron deficiency can lead to the temporary underestimation of HbA2. If there is uncertainty, a reevaluation following correction of the iron deficiency is necessary.

15.7.5.1.4 β-thalassemia major

The homozygous or mixed heterozygous β-thalassemia presents no earlier than the age of some three to five months. At the time of diagnosis the anemia is variable; hemoglobin values are usually below 80 g/L. Anemia is always hypochromic, with an MCH ≤ 22 pg and an MCV between 50 and 60 fL. Thalassemia-like erythrocyte morphology with significant poikilocytosis can be seen on the blood smear. The hemoglobin analysis shows variable fractions of HbA, HbF and HbA2. In general it can be assumed that with an HbF increase between 20 and 98%, along with a typical hematological values, thalassemia major or thalassemia inter media is present.

The patient’s transfusion status must be considered during assessment. There is an increasingly frequent diagnostic problem that occurs in patients with treated thalassemia major, such as those receiving continuous transfusion therapy. As a result of treatment, the full clinical picture is no longer seen. The diagnosis can only be confirmed with DNA analysis. The issue concerns mainly young patients or young adults who come to Germany within the framework of a family reunion.

15.7.5.1.5 β-thalassemia inter media

The mild homozygous or mixed heterozygous β-thalassemia refers, primarily, to a clinical diagnosis in patients with a hemoglobin pattern resembling that of thalassemia major, who stand out due to a minimal or no transfusion requirement. Diagnostic differentiation with regard to thalassemia major is made over a period of time by regular clinical hematological monitoring. If necessary, DNA analysis is performed. Thereby, either a high level of residual β-globin gene activity is demonstrated, or classical thalassemia major is found, albeit with additional influencing factors, particularly hereditary HbF persistence or α-thalassemia.

15.7.5.1.5.1 Diagnostic significance of HbF persistence

Hereditary HbF persistence is a clinically harmless, frequently congenital proliferation of HbF. The specific diagnostic significance of elevated HbF values in β-thalassemia was mentioned. δβ-thalassemia is also characterized by high HbF values. In sickle cell disease, a high HbF value has a positive prognostic significance. Apart from hemoglobinopathies, HbF proliferation can occur as secondary phenomenon in many hematological diseases /2/.

15.7.5.2 Abnormal hemoglobins

This group of autosomal dominant inherited hemoglobin disorders are caused by structural defects resulting from an altered amino acid sequence in the α- or β chains. Clinicians must distinguish between clinical harmless hemoglobin anomalies and those that cause illness. These latter are divided into the following four groups /25, 10, 1117/:

  • HbS; variants with a tendency to aggregate and with sickle cell formation (e.g., sickle syndromes)
  • HbE; variants with abnormal hemoglobin synthesis (e.g., HbE)
  • Unstable hemoglobins, e.g. Hb Köln; variants with a tendency to precipitate and with hemolysis (unstable hemoglobins e.g., Hb Köln)
  • Variants with abnormal oxygen transportation and congenital polycythemia (e.g. Hb Johnstown), or with congenital cyanosis such as abnormal methemoglobins and HbM abnomalities (e.g. HbM Iwate).

The forms in both of the two last groups cause serious illness when heterozygous. When homozygous, they are fatal. The main hemoglobin abnormalities are HbS, HbE and HbC. The large groups of rare hemoglobin abnormalities that occur in isolated cases all over the world should also be monitored. These are often accompanied by hemolysis, polycythemia, and/or cyanosis. Identifying these is an important part of differential diagnosis of hematological diseases where other efforts towards diagnosis have proved inconclusive. They come first in the diagnostic investigation of hemoglobin defects and comprise more than 90% of the anomalies. Less common in routine laboratory practice are HbD and HbG variants, HbO-arab, HbG and HbJ. All other hemoglobin defects (e.g., the unstable hemoglobins Hb Köln, Hb Zürich) are extremely rare /5/. They occur with the same frequency in Germans and in foreigners (i.e., only in individual persons or families) and are not included in standard hemoglobin diagnostic investigation.

The clinical classification of pathological hemoglobin variants are shown in Tab. 15.7-10 – Clinical classification of the most important pathological hemoglobin variants.

15.7.5.2.1 HbS and sickle-cell disease

The term sickle cell disease includes all manifestations of abnormal HbS levels (proportion of HbS > 50%). These include homozygous sickle-cell disease (HbSS) and a range of mixed heterozygous hemoglobinopathies (HbS/β-thalassemia, HbSC disease, and other combinations). According to the International Nomenclature the previously commonly-used term sickle-cell anemia should not be used, as the dominant aspects of the disease are vascular obliterations and the organ damage they cause, not anemia /1217/.

Cardinal symptoms and diagnostic criteria

Symptoms begin before the age of 1 year, with chronic hemolytic anemia and developmental disorders. The main problems are pain crisis (sickle cell crisis) that can affect the back, extremities, thorax, abdomen, and the CNS in particular. Patients are also susceptible to infection. HbS is the most dangerous of all hemoglobinopathies. The sickle cells caused by a lack of oxygen lead to vascular obliterations, so infarctions with tissue death can occur in almost all organs (skin, liver, spleen, bone, kidney, retina, CNS). Chronic hemolytic anemia can usually be well tolerated. Aplastic crisis are seen with severe anemia following viral infections.

Molecular genetic diagnostic investigation in HbS and sickle cell disease: HbS is mainly diagnosed with conventional Hb analysis. Special indications for DNA testing are combined forms of HbS with other anomalies, with β- or α-thalassemia, and issues related to prenatal diagnosis.

HbS heterozygosity

Heterozygous HbS gene carriers are not affected clinically or hematologically and have a normal blood count. The diagnosis is based on the detection of HbS in typical position on electrophoresis, the proportion of which, as determined by HPLC, is quantitatively lower than that of HbA and accounts for 35–40% of total hemoglobin. HbS values of below 30% are suspicious with regard to the presence of iron deficiency or an coexisting α-thalassemia. The MCH is decreased in both cases.

HbS homozygosity

The hemoglobin level is usually 60–90 g/L. Sickle cells and target cells are found in the blood smear. In HbSS (homozygous form) no normal HbA is found on hemoglobin electrophoresis. The HbF fraction is variable, usually in the range of 5 to 15%. Higher HbF fraction often occur.

HbS-β-thalassemia

The hematological presentation resembles that of sickle cell disease. Differentiating characteristics in relation to HbS homozygosity are found in microcytosis and hypochromia. Differentiation of the HbS-βo and HbS-β+ forms is accomplished, in the simplest case, by demonstrating an HbA fraction in the HbS-β+ combination, while in the HbS-β0 form no HbA is present. The diagnosis can be confirmed by means of an Hb analysis, or molecular genetically (Tab. 15.7-11 – Diagnostic characteristics of sickle cell β-thalassemia).

Notes on HbS-β-thalassemia:

  • An elevated HbA2 value in HbS heterozygosity is not an attribute indicative of HbS-β-thalassemia
  • HbS-β-thalassemias are sickle cell diseases with variable clinical symptoms; they should not be interpreted as a type of thalassemia.
15.7.5.2.2 HbE and HbE disease

HbE is a common Hb variant native to South-East Asia. Its disease pattern is similar to that of β-thalassemias. HbE is also unstable, which means that hemolysis can be caused by viral infections and medications. HbE is often combined with thalassemias, which may result in serious major-form hemoglobinopathies /12, 1011/.

HbE heterozygosity

Mild, variable hypochromia (MCH 25 pg) and microcytosis are present. The HbE fraction is, as a rule, around 30–45%; the remainder is HbA. HbF is not elevated. At lower HbE concentration, the concomitant presence of iron deficiency or α-thalassemia must be considered.

HbE homozygosity (HbE disease)

Characteristic is hypochromia with microcytosis (MCH 20 pg, MCV 65 fL) in the presence of pronounced erythrocytosis. In addition, the presence of abundant target cells. The HbE fraction is approximately 95%; the remainder is HbF and HbA2. On electrophoresis, HbE migrates identical to HbO, HbC and HbA2. Molecular biologic or special electrophoretic, immunologic and chromatographic methods, particularly HPLC, permit the differentiation of these anomalies.

HbE in combination with other anomalies

The combination with β-thalassemia (HbE-β-Thal), leads to moderate to severe hypochromic and dyserythropoietic anemia, corresponding to thalassemia inter media or thalassemia major. In combination with α-thalassemia the HbE fraction, depending on the number of inactive α-globin genes, is significantly reduced, while hypochromia is more pronounced. Refer to:

15.7.5.2.3 HbC anomaly and HbC disease

HbC homozygosity, or HbC disease, progress in a similar way to sickle-cell disease, but is less serious. Variable hemolytic anemia is the most dominant form. Heterozygous HbC gene carriers enjoy complete clinical health /12, 1011/.

HbC heterozygosity

HbC trait carriers do not manifest anemia. Target cells are detectable in the blood smear. MCHC is elevated. On alkaline Hb electrophoresis, the HbC fraction migrates in a typical position, identical to HbA2, and in trait carriers the quantitative HbC fraction accounts for 30–40% of hemoglobin. Differentiation from hemoglobins with identical migration properties (i.e., HbO and HbE) is accomplished with acidic electrophoresis. HPLC is employed for quantitative analyses.

HbC homozygosity (HbC disease)

The blood count shows predominantly target cells (Tab. 15.7-12 – Characteristic features of the most important hemoglobin variants). The Hb level is 100–120 g/L. The MCHC value is above 350 g/L. Almost 100% HbC is seen on hemoglobin electrophoresis. HbF may be slightly increased.

15.7.6 Comments and problems

Method of determination

The initial emphasis during investigation of hemoglobin abnormalities is on excluding or confirming the presence of thalassemia or an abnormal hemoglobin. Usually electrophoresis, cytological and biochemical tests alone are sufficient for diagnosis of thalassemia. In certain cases structural studies may become necessary and this is usually undertaken in specifically designated laboratories. The same often holds true for other specialized procedures (e.g., methemoglobin) and spectral analyses, oxygen affinity and 2,3-DPG determinations.

Pre analytical factors

The laboratory should be informed about a preceding blood transfusion since interpretation of test results under these circumstances requires special experience.

In doubtful cases investigations must be performed after degradation of the foreign erythrocytes.

Analytical factors

Lack of carefulness during analytical procedures leads to error. For instance, inadequate removal of serum proteins results in spurious bands on electrophoresis, most commonly seen anodal to HbA, but occasionally on the cathodal side. Similar problems arise from denatured products from old, contaminated, or hemolyzed blood samples. It is advisable to conduct parallel examinations of normal control samples and if possible of reference samples.

The well known problem of identical speed of migration of the various hemoglobins must be addressed by using a combination of different methods.

Stability

Cooling of the specimen is not required unless extremely high temperatures are present; for some tests it may even be deleterious. It is recommended that the shortest possible transport time for the samples and optimal timing of the transport are arranged (e.g., on weekends when the laboratory may offer to process the incoming specimens). Samples are stable for 1 week at 8–12 °C for hemoglobin analysis but only for 24 h for the blood cell count and the peripheral blood smear.

15.7.7 Pathophysiology

Common causes of hemoglobinopathies are mutations and/or deletions in the α or β-globin genes. If the genetic defects lead to disorders of Hb synthesis, thalassemia develops. In such cases the hemoglobin structure is normal. If genetic defects provoke changes in the Hb structure, abnormal hemoglobins are formed. Among the individual groups, there are many combination and interactive forms (Tab. 15.7-7 – Basic forms of hemoglobinopathies).

Thalassemia syndromes /21415/

Common criteria: the inheritance is autosomal recessive. Nomenclature and classification of thalassemias are based on the globin chain that is affected in each case by the synthesis disorder. A common characteristic is reduced synthesis of the affected chain type, with a loss of synthesis balance. The pathological mechanisms that result are responsible for the disease symptomatology:

  • A decrease in hemoglobin synthesis causes anemia.
  • Severe forms of anemia are provoked mainly by ineffective erythropoiesis and hemolysis
  • A shortage of substrate leads to a reduced hemoglobinization of erythrocytes (i.e., to hypochromia and microcytosis).

Heterozygous thalassemia carriers are not completely healthy; rather, they have symptomatology, with mild, iron refractory hypochromic microcytic anemia, that at any rate necessitates clarification.

Homozygous or mixed (compound) heterozygous major forms are associated with severe hypochromic hemolytic anemia and complex diseases.

α-thalassemia /1415/

α-thalassemia is caused by a synthesis disorder at the level of the α-globin chains. The molecular basis is partial (α+) or complete (α0) deletion, less frequently mutations of one or more of the four α-globin genes (αα/αα). As a function of the number of genes that are affected by the loss of activity, there are 4 α-thalassemia phenotypes that are already fully expressed perinatally (Tab. 15.7-8 – Compilation of the most important criteria of α-thalassemias):

  • Clinically silent α-thalassemia minima (heterozygous α+ thalassemia; −α/αα), recognizable by mild hypochromia with barely measurable reduction in hemoglobin value
  • α-thalassemia minor (heterozygous α0-thalassemia; −−/αα) or homozygous α+-thalassemia; (−α/−α) with mild anemia, hypochromia and microcytosis.

In HbH disease (compound heterozygote α+0-thalassemia) with three inactive α-genes (−α/−−), the pathophysiology is provoked by deficient Hb synthesis and unstable excess of HbH, which is formed from β-chain tetrameres (β4). The result is an intermediate incidence of disease with hypochromic hemolytic anemia and splenomegaly. Anemia crises occur during viral infection and due to noxious oxidative agents (medication). Complications are cardiac problems, biliary calculi, lower leg ulcers and folic acid deficiency.

The Hb Bart’s hydrops fetalis syndrome (homozygous α0-thalassemia; −−/−−) is an hemoglobin synthesis disorder that occurs as early as the intrauterine period. Here hemoglobin is made up mainly of the non-functioning γ-chain tetramer Hb Bart’s (γ4). This syndrome is, consequently, not compatible with life. The pale and generally edematous children fall ill already at the intrauterine stage, during the last third of the fetal life, of severely hemolytic anemia with hydrops and ascites; without therapy they usually die before or shortly after birth (Tab. 15.7-8 – Compilation of the most important criteria of α-thalassemias).

β-thalassemia /214/

The β-thalassemia syndromes are the result of β-globin chain synthesis disorders. Molecular causes are β-globin gene mutations. Hematological changes are not manifested before the 3rd–6th months of life. The considerable degree of phenotypic heterogeneity is the result of the large number of different mutations that can affect any stage of gene expression with different pathophysiological effects. Depending upon whether β-chain deficiency is partial or complete, β+- or β0-thalassemias are distinguished (Tab. 15.7-9 – Compilation of the most important criteria of β-thalassemias).

Thalassemia minor: the deficient Hb synthesis causes the typical mildly-pronounced hypochromic microcytic anemia (i.e., Hb values are slightly decreased or lie within the lower reference interval).

Thalassemia major: homozygous β-thalassemia is a serious disease (thalassemia major). Here, not enough β-chains are synthesized while the unaffected α-chains are produced in excess. The α-chain excess acts on erythroid precursors to cause their precocious demise, thereby resulting in highly ineffective erythropoiesis. The correlate of this pathological mechanism is incipient severe anemia, which begins in parallel to the hemoglobin switch and, if left untreated, leads to death within a few years. An iron utilization disorder and increased iron resorption are threatening complications thereof. In consequence, and as a result of the obligatory continuous transfusion therapy, hemosiderosis with multiple organ defects develops during the course of the disease. For secondary hemochromatosis, refer to Tab. 7.1-9 – Iron overload not due to disorders of the hepcidin-ferroportin axis.

Thalassemia inter media: this thalassemia of moderate severity is a mild homozygous or mixed heterozygous thalassemia with varying need for transfusions.

δβ-thalassemia: due to a gene deletion, the δ- and β-chains are decreased or not synthesized at all. Heterozygous forms have a typical thalassemia minor constellation, with a 5–10% increase in HbF, but rather decreased HbA2. Homozygous δβ-thalassemia causes an intermediate syndrome, because mean Hb values are maintained via the reactivation of γ-chain synthesis.

Hb Lepore anomalies: Hb Lepore is an abnormal form of hemoglobin, the non-α-chain represents a fusion product of δ- and β-chain. Overall hemoglobin synthesis is significantly reduced. The clinical and hematological picture is similar to that seen in β-thalassemia. Hb Lepore homozygosity, or combined Hb Lepore/β-thalassemia, is similar to thalassemia major, while the heterozygous Hb Lepore constellation corresponds to thalassemia minor.

Abnormal hemoglobins (structural hemoglobin variants) /124/

Common criteria: these types of autosomal dominant inherited hemoglobin disorders arise on the basis of flawed genetic codes as products of modified amino acid sequences or deletions in hemoglobin α- and β-chains. Hemoglobin anomalies that are clinically harmless or that cause clinical disease must be distinguished. The latter are classified, based on their pathophysiology, into five well-defined groups (Tab. 15.7-10 – Clinical classification of the most important pathological hemoglobin variants).

Molecular biological principles: more than 90% of the over 1100 hemoglobin variants that have been discovered as yet are caused by point mutations or missense mutation in one of the globin genes. Furthermore, there are few variants with elongated or shortened globin chain due to mutations of the normal terminator codon or nonsense and frame shift mutations in the third exon of the β-globin gene. Fusion genes between adjacent genes result in fusion proteins (e.g., Hb Lepore) which is also related to β-thalassemia.

HbS and sickle cell disease: the pathophysiology is caused by the sickle cell hemoglobin HbS, the structural defect of which provokes a tendency for aggregation of Hb molecules, especially under conditions of oxygen deprivation. In consequence, erythrocytes take on a crescent shape, lose their deformability and alter their rheological properties. The clinical picture is characterized by a variety of phenotypic manifestations, of which homozygous sickle cell disease and sickle cell β-thalassemia are the most severe. The clinical symptomatology that develops following the first half year of life is comprised of two marked complexes of symptoms: vascular obstruction with multiple tissue and organ damage and chronic hemolytic anemia.

HbE anomaly and HbE disease: a characteristic feature is reduced hemoglobin production (i.e., HbE manifestation is similar to that occurring in β-thalassemias). Furthermore, HbE is slightly unstable so that hemolysis is triggered under the influence of oxidative substances.

HbC anomaly and HbC disease: the pathophysiology of HbC is, like that of HbS, based on disturbed solubility with formation of intracellular crystals in erythrocytes and increased tendency for cell aggregation.

Combinations of abnormal hemoglobins and thalassemias: these combinations form a special disease group. Combinations of β-thalassemia and abnormal β-hemoglobins are of the greatest practical relevance. In these cases, due to the interaction of different pathophysiological effects, characteristic biochemical constellations or clinical and hematological syndromes occur. Demonstration of a genetic predisposition to thalassemia must no lead to the interpretation of the clinical picture as a type of thalassemia, because clinically the thalassemia component plays no role.

Hemoglobinopathies with disturbed oxygen transport function: within this group, three disorders are distinguished:

a) Anomalies in permanent methemoglobin status (HbM anomalies)

b) Anomalies with elevated oxygen affinity

c) Anomalies with reduced oxygen affinity.

Characteristics of the hemoglobinopathies mentioned under (b) are polyglobulia and/or cyanosis. The ( c ) group is associated with anemia and cyanosis. Important features of abnormal hemoglobins are shown in Tab. 15.7-12 – Characteristic features of the most important hemoglobin abnormalities.

Unstable hemoglobins: certain amino acid substitutions provoke an instability of the hemoglobin structure, where the abnormal hemoglobin is denatured within the erythrocyte spontaneously or usually under the influence of oxidizing substances (medications) or also in viral infections. Different molecular structures of the over 150 various unstable hemoglobins lead to more or less different pathological mechanisms and diseases. The best known and most common anomaly is Hb Köln; other examples in the German population are Hb Tübingen, Hb Presbyterian, Hb Freiburg and Hb Zürich. The laboratory findings are chronic hemolytic anemia, erythrocytes containing Heinz bodies, and excretion of a brown pigment in the urine (mesobilifuscinuria).

References

1. Steinberg MH, Forget BG, Higgs DR, Nagel RL. Disorders of Hemoglobin: Genetics, pathophysiology and clinical management. Cambridge; University Press 2001.

2. Kleihauer E (ed), unter Mitarbeit von Kohne E, Kulozik AE. Anomale Hämoglobine und Thalassämiesyndrome: Grundlagen und Klinik. Landsberg; ecomed Verlagsgesellschaft: 1996.

3. Kulozik AE. Hämoglobinopathien. In: Ganten D, Ruckpaul K, eds. Monogen bedingte Erbkrankheiten. Berlin: Springer-Verlag 2000; 370–92.

4. Kohne E. Hämoglobinopathien: Klinische Erscheinungsbilder, diagnostische und therapeutische Hinweise. Dtsch Ärztebl 2011; 108: 532–40.

5. Kohne E, Kleihauer E. Hämoglobinopathien – eine Langzeitstudie über vier Jahrzehnte. Dtsch Ärztebl 2010; 107: 65–71.

6. Kulozik AE. Hämoglobinopathien nehmen zu: Dtsch Ärztebl Int 2010; 107: 63–4.

7. British Committeee for Standards in Haematology. Guideline. The laboratory diagnosis of haemoglobinopathies: Brit J Haematol 1998; 101: 783–92.

8. Wajcman H, Préhu C, Bardakdjian-Michau J, Promé D, Riou J, Godart C, Mathis M, Hurtrel D, Galactéros F. Abnormal hemoglobins: laboratory methods. Hemoglobin 2001; 25: 169–81.

9. Wild BJ, Bain BJ. Detection and quantitation of normal and variant haemoglobins: an analytical review. Ann Clin Biochem 2004; 41: 355–69.

10. Kohne E. Diagnostik von Hämoglobinopathien: J Lab Med 2004; 28: 400–9.

11. Taher AT, Musallam KM, Cappellini MD. β-thalassemias. N Engl J Med 2021; 384 (8): 727–43.

12. Wild B, Bain BJ. Investigation of abnormal haemoglobins and thalassaemia. In: Dacie and Lewis. Practical Haematology. 10th Edition. Elsevier Churchill Livingstone 2006; 271–311

13. Grody W, Nakamura R, Kiechle F, Strom C. Molecular Diagnostics. Techniques and Applications for the Clinical Laobratory. Elsevier Company 2009.

14. Weatherall DJ, Clegg JB. The Thalassaemia Syndromes: 4th Edition. Blackwell Science Ltd, Oxford 2001.

15. Higgs DR, Weatherall DJ. The Alpha Thalassaemias: Cell Mol Life Sci 2009; 66: 1154–62.

16. Kiani AA, Mohamadinejad M, Shokrgozar N, Abbasian S. Mutations in thalassemia carrier couples: The importance of prenatal diagnostic tests. Clin Lab 2022; 68: 1021–9.

17. Dickerhoff R. Sichelzellkrankheit. In: Leitlinien Kinderheilkunde und Jugendmedizin. I 1 2006; 1–7. Überarbeitet 03/2010.

18. Gan G, Li Y, Bai J, Jiang M, Zheng L, Li Y. Misdiagnosis of Hb Bart’s disease: prenatal screening and diagnosis of thalassemia in special population. Clin Chem Lab Med 2023; 61 (10): e210–e213.

19. Ramponi G. The diagnostic accuracy of carbon monoxide pulse oximetry in adults with suspected acute carbon monoxide poisoning: a systematic review. Front Med 2023. doi: 10.3389/fmed.2023.1250845.

15.8 Erythrocyte enzymes

Elisabeth Kohne

Erythrocyte enzymes play a key role in the regulation of the intracellular metabolism of erythrocytes. Enzyme deficiencies lead to impaired energy supply and may cause hemolytic anemias by decreasing the erythrocyte life span. These conditions were referred to in the past as congenital non-spherocytic hemolytic anemias /123/.

Most of the enzymatic deficiencies are not due to quantitative deficiencies of the enzyme protein but, in a similar way to the hemoglobinopathies, to defective enzyme proteins (enzymopathies). As a result of alterations in functional characteristics more rapid enzyme inactivation ensues equivalent to a reduction or lack in enzyme activity. Genetic causes of enzymopathies are mutations in the region of the coding genes. The inheritance of most enzyme defects is autosomal recessive (X linked). Heterozygous trait carriers manifest reduced enzyme activity but, as a rule, they are not sick.

Enzymopathies caused by homozygous (or compound heterozygous) inheritance can present with diverse clinical symptomatology, but hemolytic anemia comprises the largest group. Other important manifestations are polycythemia (erythrocytosis) and, additionally, syndromes of developmental disorders and severe neurological deficits /3/. The majority of enzymopathies can be detected in neonates by means of laboratory diagnostic investigation.

The most important erythrocyte enzyme defects are:

  • Pyruvate kinase (PK) deficiency, the most common enzyme defect of glycolysis /4/
  • Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzyme defect of the pentose phosphate cycle /2/.

Further rare but clinically relevant enzymopathies are:

  • Triose phosphate isomerase deficiency, which leads to neuromuscular symptomatology
  • Hexokinase deficiency, which is associated with chronic hemolytic anemia.
  • Glucose-6-phosphate isomerase deficiency, which causes severe hemolytic anemia
  • 2,3-diphosphoglycerate mutase deficiency, associated with polyglobulia.

Of the variety of erythrocyte enzyme defects, PK deficiency and G6PD deficiency are described in greater detail in the following. Regarding other or more rare enzymopathies, the reader is referred to the specialized literature /3/.

Prevalence and distribution

With more than 400 million genetic carriers, erythrocyte enzyme defects are among the most prevalent hereditary metabolic disorders. G6PD deficiency, the geographic distribution of which mostly coincides with malaria regions, ranks at the top of the list, because there is a selection advantage for carriers of the G6PD deficiency genetic endowment /2/. In Mediterranean countries, Southeast Asia and Africa, and in the Afro-American population, the frequency of the genetic G6PD deficiency is between 10–20%. In Central and Northern Europe this enzyme deficiency was originally rare, but the number of affected individuals has greatly increased due to immigration of a large number of persons from endemic regions /123/.

The second most prevalent enzyme defect is PK deficiency, the origin of which is mainly Central and Northern Europe as well as North America. In the current German population, it is to be expected that at most 10% of all patients with congenital hemolytic anemia have an enzymopathy /1/.

15.8.1 Indication

Chronic hemolytic anemia, the etiology of which remains uncertain following hematological pre-diagnostic investigation.

15.8.2 Method of determination

Determination of enzyme activity

For routine purposes commercial automated enzyme analysis systems are normally used /123/. The enzyme activities are measured spectrophotometrically in the hemolysate. The latter is incubated at 37 °C with a mixture of substrate and reagents designed to make the enzyme activity to be determined rate-limiting. The parameter to be determined spectrophotometrically is the change in concentration of the pyridine nucleotides NADH or NADPH. The enzyme activities are calculated by using standardized formulae.

In some of the enzymopathies the functional deficit is associated with characteristic alterations in a wide array of physicochemically properties of the enzyme. In these settings the sole determination of enzyme activities no longer suffices for establishing the diagnosis; instead, additional parameters (e.g. enzyme kinetics, enzyme electrophoresis, pH optimum, and thermostability) have to be considered.

DNA analyses

G6PD: if the G6PD gene mutation in the patient under evaluation is known, a PCR-based DNA analysis can be performed. In the meantime, individual laboratories have begun to offer complete sequencing of the G6PD gene, with which the known G6PD mutations can be ruled out or confirmed /12/. The indication is limited to the diagnostic confirmation of heterozygous enzyme deficiency carriers (e.g., the question of the carrier status or, in extreme cases, of prenatal diagnosis).

PK: in patients of Central and Northern European origin, targeted PCR-restriction enzyme analysis can be performed due to the high prevalence of type 1529 G to A mutations. This method can also be utilized for prenatal diagnosis in afflicted families if a 1529 G to A PK defect is present in the parents. For molecular diagnosis of PK, only a few special laboratories that can confirm or rule out currently known PK variants exist in Europe /45/. This also applies to other erythrocytic enzymopathies.

15.8.3 Specimen

EDTA blood: 5 mL

15.8.4 Reference interval

Refer to Tab. 15.8-1 – Reference intervals for erythrocyte enzymes.

15.8.5 Clinical significance

The investigation of enzyme deficiency states is usually indicated by decreased or minimal residual enzyme activities. Making a defined diagnosis from the laboratory findings may pose difficulties on account of the large variety of erythrocyte enzymatic defects which may occur. It must be noted that:

  • Reticulocytes and young erythrocytes have a higher level of enzyme activity than senescent cells. In consequence, during hemolytic crises of any etiology, a false normal finding may be obtained in a markedly red cell population. Particular attention must therefore be paid to relative activities of various enzymes and their cell distribution.
  • Falsely normal enzyme activities may be measured after recent blood transfusion. Patients with hypochromic anemia have seemingly elevated enzyme activity if the hemoglobin level is taken as reference for enzyme activity.

15.8.5.1 Glucose-6-phosphate dehydrogenase deficiency

A classification of abnormal enzyme findings according to the following categories is helpful /2/:

1. Slightly enzyme deficiency

2. Moderate enzyme deficiency

3. Severe enzyme deficiency

4. Most severe enzyme deficiency.

Group 1 is insignificant from a clinical point of view. In group 2, hemolysis is triggered only by oxidative stress. In group 3, extremely severe hemolytic crises occur under oxidative stress. Group 4 is characterized by permanent hemolysis which in addition can worsen critically.

Peculiarities resulting from the X-linked inheritance of G6PD deficiency must be taken into account.

Depending on the genotypes involved which differ in males and females the following manifestations occur:

  • Men can be hemizygously normal or hemizygously deficient
  • Women may be homozygously normal, homozygously deficient or heterozygous. Heterozygous women have normal, intermediate, or very low G6PD activities according to the timing of X-chromosome inactivation (Lyon hypothesis).

At the time of acute hemolysis the diagnosis of G6PD deficiency may be very difficult since only those cells with relatively high enzyme activity remain following the destruction of cells deficient in enzyme activity. A follow-up examination may be warranted.

In a few cases of G6PD deficiency the life span of the erythrocytes is markedly shortened, even without exposure. The resulting chronic non-spherocytic hemolytic anemia is distinguished from the forms known to be associated with defects of glycolysis (e.g., PK deficiency) only insofar as, additionally, hemolytic crises can be triggered by noxious oxidative agents. In all cases that have been examined closely, enzyme variants with particularly unfavorable kinetic properties, poor stability and low activity were found. With regard to further details, see Tab. 15.8-2 – Findings and enzymatic characteristics associated with the most important erythrocyte enzymopathies.

15.8.5.2 Pyruvate kinase deficiency

The level of enzyme activity by itself is an unreliable diagnostic parameter since there is no exact correlation between the enzyme activity and severity of the hemolytic anemia /478/. The following observations, based on experience, provide useful indications. The majority of PK mutants associated with severe hemolytic anemia have enzyme activities of less than 30% of normal. In mild clinical course the enzyme activity may be significantly higher or, more rarely lower than they. Reticulocytes must always be taken into consideration. In every case monitoring of enzyme activity and reticulocyte counts, are advisable. The types of clinical-hematological presentations and the enzyme patterns found in cases of G6PD and pyruvate kinase deficiency are summarized in Tab. 15.8-2– Findings and enzymatic characteristics associated with the most important erythrocyte enzymopathies.

15.8.6 Comments and problems

Stability

Storage of samples for 3–4 days at 4–6 °C possible.

15.8.7 Pathophysiology

Glucose-6-phosphate dehydrogenase deficiency /126/

The coding sequence of the polypeptide chain comprising 515 amino acids of the G6PD wild-type GdB+ is localized in the nucleotides of exons 2–13 of the G6PD gene. The GdA variant of black Africans is based on mutation 376 A G (structure; 126 Asn Asp); the Mediterranean type GdB is caused by mutation 563 C T (Structure; 188 Ser Phe). Many other variants with different phenotypic expression have been found and it is considered to be a proven fact that the population of Southeast Asia is also affected to a great extent.

The clinically most important enzyme defects are the Mediterranean type (Gd Mediterranean, almost inactive) or the abnormal variants of the black population type A+ (GdA+, moderate reduction in activity) or type A (GdA, residual activity 5–15%).

In the absence of oxidative damage, the vast majority of the G6PD deficiency carriers have a normal Hb concentration and reticulocyte number. Hematological crises are triggered by a range of oxidative injuries, in particular infections, medications (Tab. 15.8-3 – Medication and chemicals that cause hemolysis in G6PD deficiency), fava beans, acidosis and other factors.

Hydrogen peroxide or other free radicals oxidize reduced glutathione, which due to the enzyme deficiency cannot be reduced again. The resulting precipitation of hemoglobin in the form of Heinz bodies causes massive intravascular hemolysis. This can, following destruction of the aged cells with the minimal enzyme activity, be self-limiting. An exception is the Mediterranean type deficiency (e.g.; favism).

Pyruvate kinase (PK) deficiency /4578/

PK deficiency is based on a variety of heterogeneous mutations that, in turn, generate very different functional enzyme properties. The different molecular PK forms are coded by two genes:

  • The gene M or muscle type gene, detectable in leukocytes and in many tissues
  • The gene L or liver type gene, which also controls erythrocyte PK and is termed R type.

Only variants of the PK-L gene cause hemolytic anemia. It is assumed that as a result of reduced ATP synthesis the cell membrane is damaged. The problem especially concerns reticulocytes and young erythrocytes. Due to the loss of energy sources, the cell membrane integrity is disturbed, resulting in increased rigidity and shrinking of the erythrocyte. In some cases, irregularly shaped cells are observed. They are sequestered by the spleen and phagocytosed by the reticuloendothelial system. The spleen eliminates mainly young cells, because following splenectomy the reticulocyte number increases considerably.

The anemia can be so mild that diagnosis is not made before adulthood. In severe forms, already a newborn can become severely ill, manifesting the whole clinical picture of neonatal hemolytic disease. Splenomegaly usually does not develop before the age of 4 to 6 months. Pallor and anemia are the rule in the first weeks of life. Later hemolytic crises, particularly when infection is present, alternate with periods in which anemia is only moderately severe.

Other hereditary red cell enzymopathies are only of secondary significance in the pathogenesis of enzymopenic hemolytic anemia, and are only detected in individual cases (overview Ref. /12/).

References

1. Jacobasch G. Hereditäre Membrandefekte und Enzymopathien roter Blutzellen. In: Ganten D, Ruckpaul K (eds). Handbuch der Molekularen Medizin. Berlin: Springer- Verlag, 2000; 6: 392–441.

2. Beutler E. Glucose-6-phosphate dehydrogenase deficiency and other red cell enzyme abnormalities. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ, Seligsohn U (eds). Williams Hematology 6th ed. New York: McGraw-Hill, 2001: 527–45.

3. Prchal JT, Gregg XT. Red Cell Enzymes. Hematology Am Soc Hematol Educ Program 2005; 19–23.

4. Zanella A, Bianchi P. Red cell pyruvate kinase deficiency: from genetics to clinical manifestations. Baillieres Best Pract Res Clin Haematol 2000; 13: 57–81.

5. Pissard S, Max-Audit I, Skopinski L, Vasson A, Vivien Pascal, Bimet C, Goossens M, Galacteros F, Wajcman H. Pyruvate kinase deficiency in France: a 3-year study reveals 27 new mutations. J Haematology 2006; 133: 683–9.

6. Mehta A, Mason PJ, Vulliamy TJ. Glucose-6-Phosphatdehydrogenase deficiency. Baillières Best Pract Res Clin Haematol 2000; 13: 21–38.

7. Beutler E, Blume KG, Kaplan JC, Löhr GW, Ramot B, Valentine WN. International Commitee for Standardization in Haematology: Recommended methods for red-cell enzyme assays. Brit J Haemat 1977; 35: 331–40.

8. Pekrun A, Schröter W. Erythrozytenenzymdefekte als Ursache angeborener hämolytischer Anämien. In: Huber H, Löffler H, Faber V, eds. Methoden der diagnostischen Hämatologie. Berlin: Springer, 1994.

15.9 Enzymopenic methemoglobinemia

Elisabeth Kohne

Enzymopenic methemoglobinemia is a group of rare diseases with autosomal recessive inheritance that are provoked causally by NADH-cytochrome B5 reductase deficiency /12/. The general clinical term for all forms is congenital recessive methemoglobinemia.

Cytochrome B5 reductase exists in two variants, a soluble erythrocyte form that is involved in metHb reduction in erythrocytes and a membrane-bound form in different somatic cell systems that is integrated into numerous metabolic processes. Accordingly, enzyme deficiencies can exert different effects and can be associated with two different diseases:

  • Hereditary enzymopenic methemoglobinemia type I is limited to erythrocytes. Homozygous or mixed heterozygous patients become ill with methemoglobinemia, while heteroyzgotes remain normal.
  • Hereditary enzymopenic methemoglobinemia type II is the generalized form. Apart from congenital methemoglobinemia, there are progressive neurological symptoms with very severe psychomotor disorders and death in early childhood.

15.9.1 Indication

  • Differential diagnosis of methemoglobinemia
  • Uncertain situation with regard to cyanosis
  • Cyanosis does not improve under oxygenation
  • In conditions of cyanosis with normal or slightly reduced (about 85%) arterial blood oxygen saturation.

15.9.2 Method of determination

Firstly, the blood metHb concentration is measured (see Section 15.5 – Dyshemoglobins). Determination of metHb reductase (cytochrome B5 reductase) activity in erythrocyte hemolysate is performed spectrophotometrically /3/. To confirm the diagnosis, determine the hereditary status and classify the type of hereditary methemoglobinemia that has been demonstrated, an DNA analysis is required.

15.9.3 Reference interval

Cytochrome B5 reductase activity in erythrocytes /3/

  • Neonates

9.61 ± 1,91 IU/g Hb

  • Adults

19.2 ± 3.9 IU/g Hb

15.9.4 Clinical significance

In patients with congenital persistent methemoglobinemia and enzymatically determined cytochrome B5 reductase deficiency, a diagnosis of enzymopenic methemoglobinemia is made. In heterozygosity, clinical symptoms are absent. With the demonstration of the underlying basic molecular defect, the disease type can be classified. The investigation, which includes parental genetic counseling, is a component of the diagnostic program. If necessary, prenatal diagnosis can be performed.

15.9.4.1 General symptomatology

The patients attract attention due to cyanosis that begins at birth. Methemoglobin values in newborns may exceed 40%. In older children and adults, the values are usually around 10–25%, but values as high as 40% may occur. The intake of food with different vitamin C content is the explanation for seasonal fluctuations. Some patients develop moderate compensatory polyglobulia.

15.9.4.2 Enzymopenic methemoglobinemia type I

Type I cytochrome B5 reductase deficiency is characterized by the fact that it is limited to erythrocytes. Homozygous or mixed heterozygous patients become ill with uncomplicated methemoglobinemia; heteroyzgous trait carriers are normal but they are, nonetheless, sensitive to oxidizing substances.

15.9.4.3 Enzymopenic methemoglobinemia type II

Type II is the generalized and lethal form of cytochrome B5 reductase deficiency, which, apart from the methemoglobinemia, is associated with progressive neurological symptoms. Neurological changes include severe disorders of mental development, microcephaly, dwarfism, cataract formation, as well as seizures, opisthotonus and generalized hypertension. The defect affects not only erythrocytes but also microsomal cytochrome B5 reduction in the liver, brain, muscles, leukocytes, thrombocytes and fibroblasts. Severe disorders of lipid metabolism with a reduction in brain cerebrosides, elevation of palmitic acid and decrease in linoleic acid in fatty tissues and characteristic changes in phospholipids, phospho glycerides and cholesterol in the liver, kidneys, spleen, muscle and adrenal glands also occur. The disease usually causes death in early childhood.

References

1. Kleihauer E, Kohne E, Kulozik AE. Anomale Hämoglobine und Thalassämie-Syndrome: Grundlagen und Klinik. Landsberg; ecomed Verlagsgesellschaft, 1996.

2. Percy MJ, Lappin TR. Recessive congenital methaemoglobinaemia: cytochrome b5 reductase deficiency. Brit J Haemat 2008; 141: 298–308.

3. Beutler E. Red cell metabolism. A manual of biochemical methods. 3th ed. Grune and Stratton, Inc 1984.

15.10 Erythropoietin (EPO)

Lothar Thomas

The organism’s red blood cell mass is kept constant under physiological conditions to assure optimal oxygen supply to the tissues. Every day some 2 × 1011 erythrocytes are lost due to blood cell turnover and without replacement hemoglobin (Hb) values would drop by 1 g/L in 24 hours. Since both the Hb content and the life span of the erythrocyte are genetically determined by its volume, red blood cell mass can only be preserved by dynamic adjustment of erythropoiesis.

This occurs through a sensitive homeostatic mechanism that links the production of red blood cells to the oxygen requirements of the tissues. This process is mediated by EPO, an interleukin that is synthesized in the kidneys. EPO maintains the red blood cell mass by promoting the survival, proliferation, and differentiation of erythrocytic progenitors. If oxygen supply is reduced, increased quantities of EPO are produced, resulting in hyper proliferation of erythropoiesis /1/. Inhibition of EPO synthesis leads to the development of hypo proliferative, normocytic, normochromic anemia.

In patients with anemia increase in erythropoietic activity stimulated by endogenous or exogenous EPO facilitates compensatory iron acquisition during recovery from hemorrhage-induced anemia. The erythroid regulator erythroferrone (ERFE) is produced by erythroid precursors in the marrow and the spleen and acts directly on the liver to decrease hepcidin production, and thereby increase iron availability for new red blood cell synthesis /2/.

Refer to:

15.10.1 Indication

  • Normocytic anemia of uncertain etiology
  • In hypo regenerative erythropoiesis for the differentiation of inadequate EPO synthesis from intrinsic hypo proliferation of bone marrow (see also Section 7.4 – Soluble transferrin receptor (sTfR))
  • Differentiation of erythrocytosis (polycythemia), see also Section 15.4 – Hematocrit
  • Suspicion and progression monitoring of para neoplastic EPO formation
  • Prior to treatment of non-renal anemia with erythropoiesis-stimulating agents (ESAs)
  • Recognition of a fetal emergency.

15.10.2 Method of determination

Radioimmunoassay

As tracer 125J-labeled human recombinant EPO is used; the primary antibody is generated in rabbits, the secondary antibody for the precipitation of the immune complex is a goat antibody.

Immunometric assay

EPO is detected with the use of two monoclonal antibodies that are directed against recombinant EPO. The secondary antibody is enzyme labeled or with a chemoluminescent label /3/. Reference for calibration is the WHO 2nd IRP 67/343.

15.10.3 Specimen

Serum, plasma (heparin): 1 mL

Additionally, EDTA blood should be collected so that a reference of the EPO level to the hematocrit or the Hb value can be made.

15.10.4 Reference interval

Refer to References /14/ and Tab. 15.10-1 – Reference intervals for erythropoietin.

15.10.5 Clinical significance

Tissue hypoxia stimulates the synthesis of EPO, while normal tissue oxygen supply suppresses its formation.

15.10.5.1 Assessment of erythropoietic activity

The EPO concentration must be assessed relative to the red blood cell mass, the indirect measure of which is the hematocrit (HCT) or the Hb concentration. In this way it can be established, in chronic anemia, whether a stimulation of erythropoiesis is adequate for a decrease in HCT or Hb. There is an inverse relationship between the Hb level or the HCT and the decadic logarithm of serum EPO concentration. This is the case only in anemia, but not within the reference interval of the Hb or HCT /7/. With a slight decline in the HCT to 0.38–0.35 or in Hb to 125–115 g/L, EPO begins to increase slightly in the reference interval. However, a marked rise in EPO occurs only as of a HCT≤ 0.30 or an Hb ≤ 100 g/L (Fig. 15.10-2 – Expected range of EPO concentration as a function of the hematocrit /5/.

For the same degree of anemia, however, an adequate increase in EPO does not always occur; this depends, rather, on the cause of the anemia /6/. Thus, the most marked increases in EPO are seen in aplastic anemia, and the minimal rises are observed in anemia of chronic disease and in the final stages of chronic renal insufficiency; iron deficiency anemia lies in between (Fig. 15.10-3 – Relationship between HCT and EPO concentration).

EPO levels are lower in fetuses than in adults, in spite of the very regenerative nature of erythropoiesis. The mean fetal EPO concentration is ≤ 5 U/L until the 37th week of gestation. Due to fetal stress during birth, the values increase 10-fold on the average and then decrease during the first week of life. At the age of 7–12 weeks, infants manifest physiological anemia with an Hb value of 90–110 g/L; EPO values are also low, with the exception of a slight increase in the Hb nadir /8/.

The assessment of deficient EPO formation is based on the EPO level in comparison with reference patients (iron deficiency anemia, hemolytic anemia, thalassemia inter media) /7/ with comparable Hb values or HCT (Fig. 15.10–3 – Relationship between HCT and EPO concentration).

With values in the range of diminished EPO formation, the following causes are to be considered:

  • Neonatal anemia
  • Anemia of inflammation (rheumatoid arthritis, chronic infection, AIDS, inflammatory intestinal disease, autoimmune disease, critically ill patients)
  • Cancer-related anemia with or without chemotherapy (solid tumors, multiple myeloma, malignant lymphoma) /8/
  • Erythrocytosis (polycythemia), see Section 15.4 – Hematocrit.

The behavior of serum EPO in different forms of anemia is shown in:

15.10.5.2 Chronic kidney disease

According to the National Kidney Foundation in the USA, the term chronic kidney disease includes /10/, patients with chronically diminished kidney function, dialysis-dependent patients and those with a malfunctioning kidney transplant. In untreated cases, renal anemia leads to the following disorders: reduced oxygen availability to the tissues, increased cardiac output, enlargement of the heart, ventricular hypertrophy, angina pectoris, congestive heart failure, decreased mental alertness, reduced immune response, and menstrual disorders. In children, delayed growth and diminished intellectual capacity occur.

Renal anemia is based on inadequate EPO formation due to reduction of functional kidney tissue. Additional factors are iron deficiency, loss of blood, acute and chronic inflammation, aluminum toxicity, and a reduction in erythrocyte life span.

A clarification of chronic kidney disease-associated anemia should be performed if /10/:

  • In premenopausal women and prepubertal female individuals the Hb value is < 110 g/L (HCT < 0.33)
  • In men and in postmenopausal women, the Hb value is < 120 g/L (HCT < 0.36).

Increasing the Hb value to 130–150 g/L by ESA therapy does not reduce the risk of cardiovascular events within 3 years /11/. Iron metabolism and ESA in end-stage renal disease, see Tab. 7.3-4 – Diseases and conditions associated with elevated serum ferritin concentrations.

15.10.5.3 Chronic inflammation

In anemic patients with chronic inflammatory disease, the inflammatory decrease of EPO formation is the key cause of normocytic, normochromic anemia /12/.

Rheumatoid arthritis

These patients have, at times, Hb values < 80–90 g/L, but hardly need banked blood. In severely anemic patients with rheumatoid disease, anemia is linked to iron deficiency. ESA therapy is not recommended in general, but can be important in individual cases.

AIDS

Approximately two thirds of these patients have normocytic, normochromic anemia, which is aggravated under treatment with AZT. ESA therapy may be justified in symptomatic patients.

Tumor-associated anemia /13/

Anemia is common in tumor patients (see also Tab. 15.3-11 – Classification and differentiation of normocytic anemia). The inflammatory inhibition of EPO formation is of particular relevance and depends on the release of inflammatory cytokines and hepcidin. Chemotherapy and radiotherapy amplify the anemia due to the inhibitory effect on the bone marrow and the reduction in EPO formation (Fig. 15.10-4 – Activation of the immune system in acute phase response).

15.10.5.4 Critically ill patients

In the critically ill, anemia is multifactorial and corresponds to the anemia of chronic disease. It is contingent upon inadequate low EPO formation, hypo proliferation of erythropoiesis (hepcidin increase and functional iron deficiency), due to intrinsic factors, and a reduced erythrocyte life span /14/.

15.10.5.5 Therapy with erythropoiesis-stimulating agents (ESA)

Erythropoiesis and ESA

Approximately 90% of hemodialysis patients receive ESA therapy (7000–8000 units rHuEPO per week), and 70% receive iron substitution (300 mg per month) in addition. Thus the erythron is extended and fatty marrow is replaced through hematopoiesis. The stimulation leads mainly to proliferation of early erythroid cells of the colony forming unit erythroid (CFU-E) (Fig. 15.1-2 – Erythropoiesis is separated into a proliferation pool and a maturation pool). In contrast, the pool of late erythroid progenitor cells is increased in cases of chronic red blood cell requirement (e.g., in chronic hemolytic anemia). If the marrow is already hyperproliferative due to stimulation by endogenous EPO, ESA does not lead to a meaningful further increase in regenerativity. Thus, erythropoiesis enhanced 2.9-fold can only be increased to 3.6-fold with high doses of ESA /15/.

The stimulatory effect of ESA is dependent upon:

  • The extent of regenerative bone marrow (no intrinsic hypo proliferation)
  • The iron supply of erythropoiesis
  • The presence of an acute phase reaction (CRP level).

Iron availability

Due to enhanced proliferation of erythropoiesis, ESA stimulation leads to an increased requirement for iron and to the distribution of iron from the stores into the bone marrow. With normal iron reserves in healthy individuals, intrinsic EPO stimulation can only increase erythropoiesis by 3-fold above the normal rate, without the generation of hypochromic red blood cells. If erythropoiesis is overstimulated by the administration of ESA, iron requirement exceeds the supply; this condition is termed functional iron deficiency. In consequence, hypochromic red blood cells are formed. This situation occurs particularly in the early regeneration phase of erythropoiesis following administration of ESA. If the iron stores are repleted and if erythropoiesis is adequately stimulated with ESA, functional iron deficiency only occurs if the ferritin value is below 100 μg/L. Under ESA therapy, the ESA dose and iron supply must be coordinated with one another.

Acute phase reaction (APR)

Systemic inflammatory changes in the organism are termed APR. Besides functional iron deficiency, APR is one of the most important reasons for the different responsiveness of patients to ESA and for a elevated ESA requirement /15/.

Patients with inflammation have elevated serum concentrations of IL-6, CRP, fibrinogen and ferritin. Transferrin and transferrin saturation are reduced. The cause of reduced bone marrow responsiveness in inflammation is an enhanced expression of IFN-γ, IL-6 and hepcidin /16/. In bone marrow, these mediators antagonize the anti-apoptotic effect of EPO on CFU-E and lead to ineffective erythropoiesis (Fig. 15.10-4 – Activation of the immune system in acute phase response).

In addition, an increase in hepcidin leads to a disturbance in iron distribution due to inhibited iron release from macrophages and enterocytes via the ferroportin receptor (see also Section 7.6 – Hepcidin). The resulting functional iron deficiency intensifies the ESA resistance, leading to anemia.

Patients with chronic kidney disease, with malignancy, with chronic inflammatory disease, as well as critically ill patients, often suffer from anemia which affects, to a significant degree, morbidity, mortality and the quality of life. The majority of these patients have normocytic, normochromic erythrocytes. ESA treatment is advantageous for these patients, particularly the use of banked blood is reduced. Additional parenteral administration of iron enhances the erythropoietic response to ESA.

ESA treatment is expensive. It is therefore important to select patients who are likely to respond to ESA and monitor the erythropoietic response. Tests before starting ESA therapy and for therapeutic monitoring are listed in Tab. 15.10-5 – Tests for the assessment of erythropoiesis under ESA therapy.

Treatment with ESA is not free of risks. Thus, investigations show that ESA therapy may be associated with /9/:

  • An elevated risk of venous thrombosis
  • Enhanced tumor progression and, possibly, a shortening of survival.

ESA therapy in chronic renal insufficiency

Anemia is a strong predictor of cardiovascular disease and mortality in patients with chronic kidney disease /10/. The correction of anemia with values of < 110 g/L with ESA therapy, to target values of 110–120 g/L, improves the condition of the patients. Optimal iron reserves are present with ferritin values of 200–500 μg/L and transferrin saturation (TSAT) of > 20% (Tab. 15.10-5 – Tests for the assessment of erythropoiesis under ESA therapy). According to the National Kidney Foundation of the USA, intravenous iron therapy should be implemented in patients with functional iron deficiency with a TSAT < 20% if the ferritin value is ≤ 800 μg/L.

Refer also to Tab. 15.10-6 – Erythropoiesis stimulating agents.

ESA therapy in tumor-associated anemia

ESA therapy should only be initiated in patients with symptomatic anemia with an Hb level below 100 g/L and in patients with asymptomatic anemia with an Hb level below 80 g/L. The Hb target is a stable level of about 120 g/L /17/.

Effectivity of ESA therapy

The effectivity of ESA therapy depends on the cause of the anemia and the presence of inflammation. Patients with end stage renal disease have the best response rate, which is some 70%. Tumor patients have a response rate of 30–70%, but in those with myelodysplastic syndrome it is only 20%. A rise in the Hb value of 10 g/L within 4 weeks after initiation of therapy is considered to be a positive response.

15.10.6 Comments and problems

Sampling

Should be performed during the morning due to daily fluctuations of EPO concentrations. Maximal values are seen at midnight, the nadir occurs during the morning hours.

Method of determination

Values obtained with commercials assays are comparable /1/. The measurement of synthetic EPO preparations elicits very different results due to variability in structure and immunogenetics.

Precision: depending on the test CV% is 7–24 in the range of 5–10 U/L. Only at 50 U/L is CV% below 10.

Accuracy: when tested with standard 87/684, the accuracy of 6 assays was ± 25% and with one assay it was + 200% /18/.

Reference interval

Since the reference interval is very broad, and a significant increase beyond the upper value of the reference interval can only be detected with a HCT of less than 0.30 or a Hb of below 100 g/L, reference must be made to one of these two parameters. The reference curve relating the HCT or Hb to the EPO concentration can be generated based upon an investigation of cohorts that include patients with iron deficiency anemia, hemolytic anemia or thalassemia inter media /7/. EPO concentrations are not age dependent /19/.

Half-life

With normal Hb values, the half-life of endogenous EPO is 5.2 hours; in patients with anemia it is 1.5–2.9 hours.

Stability

In serum at least 2 weeks at room temperature /20/.

15.10.7 Pathophysiology

In combination with other hematopoietic growth factors, EPO is a physiological regulator of erythropoiesis (Fig. 15.10-5 – Site of action of erythropoietin and other hematopoietic growth factors in erythropoiesis/20/. Depending on the developmental stage of erythroid progenitor cells, EPO can be a mitogen, an apoptosis inhibitor, or a differentiating factor. Thus the CFU-E cells require contact with EPO molecules in order not to become apoptotic /21/.

In healthy individuals, circulating EPO originates from neuronal fibroblasts near the proximal tubular cells. The synthesis of EPO is controlled at a transcriptional level due to dynamic changes in the oxygen tension. The production increases under hypoxic conditions (Fig. 15.10-6 – Erythropoietin feedback control loop/1/. In minor amounts EPO is produced in the liver and brain.

EPO exerts its influence by binding to a specific receptor, the EPO receptor (EPOR) on the cell surface of erythroid progenitor cells, resulting in erythroid proliferation, differentiation and inhibition of apoptosis (Fig. 15.10-7 – Homodimer erythropoietin receptor following showing phosphorylated tyrosines). The EPOR homodimerizes in the presence of EPO and auto phosphorylation of the Janus tyrosin kinase 2 (Jak2) occurs. Once JAK2 is activated, specific EPOR tyrosines are phosphorylated and form docking sites for adapter molecules such as Grb 2, the signal transducer and activator of transcription 5 (STAT5) and phosphatidylinositol-3-kinase (PI3-K). Activated STAT5 forms dimers and trans locates into the nucleus where it induces transcription of genes involved in proliferation and cell survival. PI3-K, via activated Akt, also induces the expression of several anti-apoptotic proteins, such as Bcl-2 and Bclx, to prolong cell survival by Janus kinase 2 (JAK 2) and the subsequent transfer by STAT 5 to the cell nucleus.

Two groups of EPO receptor mutations are clinically relevant (see also Section 15.4 – Hematocrit (HCT)/22/:

  • Replacement of arginine with cysteine at position 129 of the extracellular portion of the EPOR with the consequence of spontaneous homodimerization of the EPOR in the absence of EPO. The continuous stimulation of the progenitor cells, with development of erythrocytosis, is the result.
  • The modification of the intracellular C-terminal portion of the EPOR, with deficiency of the binding site for phosphatase SHP-1. Physiologically, this enzyme removes phosphate groups from the tyrosine residues of the C-terminus of the EPOR thereby inactivating signal transduction. The removal of the SHP-1 binding site increases the sensitivity of the cell for EPO, leading to erythrocytosis.

EPO is a glycoprotein of MW 30–34 kDa. Its biological activity is dependent upon its tertiary structure, which is comprised of 4 α-helices with 2 long loops and 1 short loop. Endogenous and recombinant EPO have the same sequence of 165 amino acids, but they differ from one another with regard to their glycosylation. Endogenous EPO is more acidic than rHuEPO and they can be distinguished from one another through isoelectric focusing of a urine sample. Four oligosaccharide chains make up 35–40% of the molecular mass.

Recombinant HuEPO that is formed in mammalian cell cultures (e.g. of the Chinese hamster) is fully glycosylated, but it is not glycosylated if synthesized in E. coli cultures /23/.

The fetal liver is the main site of EPO production from the 20th week of gestation. Following birth, the renal interstitial cells increasingly take over the role of EPO formation, and in adults 85% of the daily EPO synthesis takes place in the kidneys. Under normal conditions, EPO synthesis is activated as a response to a reduction in the red blood cell mass (anemia) or to reduced O2 saturation of erythrocyte hemoglobin (hypoxemia) /24/. The result of hypoxic stimulation is increased formation of hypoxia-inducible factor-1 (HIF-1) in the kindey. HIF-1, as the most important EPO gene regulating factor, stimulates EPO synthesis. HIF-1 is a global regulator of cellular and systemic O2 homeostasis. HIF-1 also regulates vessel formation, promotes the survival of ischemic cells and plays a role in carcinogenesis.

Hypoxia-driven EPO gene expression in peritubular fibroblasts results from an array consisting of the kidney inducible element(KIE) and a negative regulatory element (NRE) /23/.

15.10.7.1 Roxadustat

Roxadustat an oral hypoxia-inducible factor prolylhydrolase inhibitor increases endogenous EPO concentration through the stabilization of hypoxia inducible factor (HIF; refer to section 15.4), which is achieved by inhibiting the activity of its pan-prolylhyroxylase domain. A study /54/ showed that roxadustat increased the hemoglobin concentration and reduced ferritin and transferrin saturation after patients undergoing peritoneal dialysis were switched from an Erythropoietin stimulating Agent (ESA).

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43. Beguin Y, Loo M, R’Zik S, Lejeune F, Sautois B, Rovrie G, Fillet G. Quantitative assessment of erythropoiesis in hemodialysis patients demonstrates gradual expansion of erythroblasts during constant treatment with recombinant human erythropoietin. Br J Haematol 1995; 89: 17–23.

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15.11 Thrombocyte count and thrombocyte indices

Lothar Thomas

Thrombocytes, also called platelets, are anucleic, granula rich cells, the functions are:

  • Maintenance of hemostasis
  • Initiation of tissue repair following vascular injury and in inflammation.

In healthy individuals, the bone marrow releases 1× 1011 thrombocytes daily, approximately one half of the daily erythrocyte production. Megakaryopoiesis and thrombocyte formation are regulated by thrombopoietin. Thrombocytes arise from the fragmentation of megakaryocytes, a process in which the formation includes elongated strands of cytoplasm, also called prothrombocytes. Refer also Section 15.1 – Hematopoiesis)

Thrombocytes in the blood are normally in the resting stage. Following physiological stimulation, they undergo a change in shape, with subsequent adhesion to a surface, followed by aggregation. The changes in shape that thereby occur can be observed as surface changes in vivo using physical methods, and with immunological methods by measuring the expression of receptors. In platelet aggregation or following marked stimulation, the platelets are degranulated and the granula membrane is transferred to the thrombocyte surface. In this way, neoantigens are expressed on the activated thrombocyte in the form of glycoproteins. These processes can be identified diagnostically in the laboratory with the use of immunofluorescence and flow cytometry. But flow cytometry alone is also capable of recognizing changes in platelet shape. Thus, changes in forward and side scattering of light reveal, respectively, changes in platelet volume and in granularity.

In addition to this chapter refer also to

15.11.1 Indication

  • Bleeding of uncertain etiology
  • Exclusion of a bleeding tendency
  • Monitoring during radiotherapy and under treatment with cytostatics and heparin
  • In the presence of erythrocytosis
  • In splenomegaly. Suspicion of bone marrow disease (myelophthisis, myeloproliferation).
  • Suspicion of destruction, consumption (e.g., sepsis) or reactive proliferation of thrombocytes.

15.11.2 Thrombocyte count

15.11.2.1 Microscopic counting

Principle: venous or capillary blood is diluted with a hypotonic solution containing a platelet aggregation inhibitor (1% ammonium oxalate solution). This is performed using a pipette into which blood is aspirated up to the 1 mark, and the diluting solution (1% ammonium oxalate solution) up to the 101 mark. For the purpose of hemolysis, the sealed pipette is rotated for 15 minutes. Counting is performed in the Neubauer chamber or the Thoma chamber. Before starting counting, the thrombocytes are to sediment in a chamber for 10 minutes /1/. The reference method is a hemocytometer count with the use of a phase contrast microscope /2/.

15.11.2.2 Hematology analyzer

The determination can be performed using the impedance method, the optical method, or with immunoplatelet counting. One-dimensional and two-dimensional optical methods are distinguished. Some hematology analyzers measure according to the impedance and the optical method.

Impedance method

The platelets are counted using the electric resistance measurement (impedance) method. If a thrombocyte that migrates through a capillary interrupts the applied electrical circuit, an impulse is triggered. Thrombocytes and erythrocytes from the same cell suspension are determined. For thrombocytes the pulses are counted with a window of 2–20 fL and for red blood cells in one > 36 fL. The average size of all pulses in the thrombocyte histogram is expressed as the mean platelet volume (MPV) /3/.

One-dimensional optical method

A laser diode is used for generation of monochromatic light. The light is directed to the flow cell at an angle of 2–3°. According to the Mie scattering theory, the intensity of the monochromatic light that is scattered from a homogeneous particle depends on its volume and the difference in the refraction index between the particle and its surrounding medium. The thrombocyte count is determined by scattered light impulses that are generated, while the MPV is determined by the scattered light intensity /3/.

Two-dimensional optical method

The principle corresponds to that of the one-dimensional method. With the use of a detergent, however, a spherical structure is imposed on the platelets, and the scattered light is measured at two angles, a low angle of 2–3° and a high angle of 5–15°.

With the two-dimensional light scattering technology, normal-sized platelets, large platelets (20–30 fL), red blood cell (RBC) fragments, erythrocyte ghosts, microcytes, and cellular debris can be differentiated /4/.

Combination of impedance and optical methods

Some hematology analyzers use both counting methods to distinguish between platelets and other particles, particularly with low platelet counts. With low thrombocyte count and in the presence of other particles, the impedance measurement manifests a bias toward higher values than optical measurement because other particles are also recognized /5/.

Immunoplatelet counting

Antibodies against CD41 (GPIIb), CD42 (GPIb) and CD 61 (GPIIIa) are used to identify platelets which are measured by flow cytometry. The thrombocyte count is calculated from the relationship between the detected fluorescence and the number of red blood cells (RBC ratio) that are identified with a blood cell analyzer by means of impedance counting. The platelet count is calculated by multiplying the RBC ratio with the number of red blood cells. The advantage of the RBC ratio is that the thrombocyte count is independent of dilution and pipetting errors /6/. An alternative method involves the addition of a fixed quantity of latex particles to the sample. The number of latex particles is determined by impedance counting and the platelet-dependent fluorescence is derived from that number /7/.

15.11.3 Thrombocyte indices

Apart from the thrombocyte count, hematology analyzers determine or establish the mean platelet volume (MPV), the platelet crit (PCT), and the platelet distribution width (PDW). The Advia 120 determines, in addition, the mean platelet component concentration (MPC), the platelet component distribution width (PCDW), the mean platelet mass (MPM), and the platelet mass distribution width (PMDW). Changes in the MPC are a gauge of platelet activation. The broadening of the PDW indicates hyper regenerative thrombopoiesis.

15.11.3.1 Immature Platelet Fraction (IPF)

Determination of the IPF (Immature Platelet Fraction) is useful for the clarification of thrombocytopenia.

Procedures: the flow cytometric determination is performed (e.g., with the Sysmex XE-2100) using the fluorescent dyes polymethine and oxazine. Both dyes penetrate the thrombocyte cell membrane and label thrombocyte and erythrocyte RNA. The labeled cells are directed by a semiconductor diode laser beam and the resulting forward scattering (platelet volume) and fluorescence intensity (platelet RNA content) are recorded. The IPF and mature thrombocyte fraction are distinguished using a computerized algorithm. The IPF is expressed as a relative fraction of the total thrombocyte fraction.

15.11.4 Specimen

EDTA blood: 1 mL

Capillary blood (EDTA-coated capillaries): 0.02 mL

15.11.5 Reference interval

In thrombocyte count sex and genetic background are not important for reference intervals. The effect of age is higher as of sex and genetic background. Reference intervals (2.5 to 97.5 percentiles) in population setting are as follows (x 109/L): Below 15 years 176–452; females 15–64 years 165–405; males 15–64 years 141–364; females > 64 years 140–379, males > 64 years 122–350 /22/. Reference intervals for platelet indices are published in Ref. /60/. The coefficient of variation (CV) from day to day is 6.7%, and from month to month 10.6%, respectively /21/.

15.11.6 Clinical significance

The hemostatic system consists of coagulation factors, the vascular wall and thrombocytes. The circulating lifespan of thrombocytes that are released from the bone marrow is 7–10 days, until they are removed by macrophages of the reticuloendothelial system. In healthy individuals approximately one third of all thrombocytes are contained in the spleen, while the remainder is in the circulation. The thrombocyte pool in the spleen is readily available and there exists a free exchange of thrombocytes between the spleen and the circulation.

To fulfill their hemostatic function thrombocytes must not only be fully functional, in addition their number must lie within certain limits. Clinicians consider a thrombocyte count of (100–400) × 109/L to be normal, and only occasionally do clinically normal individuals manifest values that are outside this range. With thrombocytopenia ≤ 10 × 109/L there is a danger of bleeding, and in thrombocytosis ≥ 450 × 109/L the risk of thrombotic events is increased.

Since, in many diseases, determination of the complete blood count is performed with the use of hematology analyzers, identification of thrombocytopenia and thrombocytosis is far more common than the clinical cases with clinical hemostaseological symptomatology.

15.11.6.1 Thrombocytosis

The terms thrombocythemia and thrombocytosis are used synonymously and are not defined unambiguously. Commonly, they are taken to mean a rise in the thrombocyte count to above 450 × 109/L. Individuals with values of (350–450) × 109/L should be monitored. The extent of thrombocytosis is classified arbitrarily according to the thrombocyte count as /12/:

  • Mild with (450–700) × 109/L.
  • Moderate with (700–900) × 109/L.
  • Severe with more than 900 × 109/L.

Based on their etiology, thrombocytosis is classified as:

  • Hereditary or familial
  • Clonal forms, which are associated with myeloproliferative or myelodysplastic disease
  • Secondary forms: these are reactive forms of thrombocytosis.

Clonal forms of thrombocytosis are termed primary thrombocytoses. Since thromboembolic events occur more frequently in primary thrombocytoses, the differentiation of primary and secondary forms is important.

15.11.6.1.1 Primary thrombocytosis

Primary thrombocytoses are the consequence of myeloproliferative and myelodysplastic disorders or are genetically or are familial. Hereditary causes are activating mutations in the MPL gene, which codes for the megakaryocyte and thrombocyte receptors of the same name, or the TPHO gene, which codes for thrombopoietin.

15.11.6.1.2 Secondary thrombocytosis

Either there is increased formation of thrombocytes due to a stimulus or thrombocytes are released into peripheral circulation from the splenic pool. Increased release from the splenic pool occurs (e.g., in physical effort, stress, or administration of catecholamines).

Bone marrow thrombocytopoiesis is stimulated by the peripheral loss of thrombocytes (e.g., due to immunological factors, sepsis, blood loss, or oncogenes). Secondary thrombocytosis hardly ever triggers thrombosis. Following recovery, the thrombocyte count decreases once again. In the bone marrow, the megakaryocytes are increased and are seldom dysmorphic. The large thrombocytes that appear in peripheral circulation are round, their functionality is normal, and the platelets do not tend to aggregate spontaneously, as do the primary thrombocytes /12/. Approximately 88% of thrombocytoses over 500 × 109/L are secondary in nature and are usually caused by an inflammatory event /13/.

Differentiation

The differences between primary and secondary thrombocytosis in laboratory diagnostic investigation are listed in Tab. 15.11-2 – Differences between primary and secondary thrombocytosis.

15.11.6.2 Thrombocytopenia

Thrombocytopenia refers to a decrease in the count to < 100 × 109/L. Threatening complications of bleeding do not occur, however /14/:

  • In ambulatory patients with aplastic anemia and counts of ≥ 5 × 109/L
  • Following minor surgical procedures in patients with fever > 38 °C or following thrombocyte transfusion due to recent grade 3 bleeding according to WHO, if the count was ≥ 10 × 109/L.

An important point for such a decision is the accuracy of the thrombocyte count with values of this order of magnitude /5/. The benefits of the administration of banked thrombocytes in patients with a thrombocyte count ≥ 5 × 109/L without bleeding is not documented.

Frequent clinical findings in thrombocytopenia are petechiae, purpura, mild to moderate mucosal bleeding, bilateral epistaxis, and gastrointestinal, pulmonary and urogenital bleeding. Symmetrical petechiae and purpura, which affect both the trunk and the extremities are characteristic.

In routine clinical practice, thrombocytopenia is frequently associated etiologically with the ingestion of medication, chemotherapy, sepsis, disseminated intravascular coagulation, or massive blood transfusions. All cases of thrombocytopenia require laboratory confirmation by means of an additional method of counting, and the investigation of a blood smear.

Etiologies of thrombocytopenia are:

  • Reduced thrombocyte formation.
  • Elevated thrombocyte turnover.
  • Disturbed thrombocyte distribution or a dilution-associated increase of plasma volume.
  • Pseudo thrombocytopenia.

Findings for differentiation of etiologies are shown in Tab. 15.11-3 – Differentiation of the mechanism of thrombocytopenia.

15.11.6.2.1 Reduced thrombocyte formation

Reduced formation is relatively rare (children < 5%; adults < 10%) the cause of thrombocytopenia. A distinction is made between /15/:

  • Hereditary forms (e.g., Wiskott-Aldrich syndrome, Chediak-Higashi syndrome, thrombocytopenia-absent radius syndrome, Alport syndrome, Fechtner syndrome, Trousseau Syndrome, May-Hegglin anomaly, type IIB von Willebrand disease, Bernard-Soulier syndrome, gray platelet syndrome, platelet type von Willebrand syndrome, Mediterranean macro thrombocytopenia), and congenital thrombocytopenia (macro thrombocytes).
  • Disease and therapy associated transient thrombocytopenia following chemotherapy for malignant hematological neoplasia or following bone marrow infiltration of solid tumors, following stem cell injury due to radiation or medication. The thrombocyte count should be ≥ 10 × 109/L; in necrotizing tumors ≥ 50 × 109/L.
15.11.6.2.2 Increased thrombocyte turnover

Destruction of the circulating thrombocytes is the most frequent cause of thrombocytopenia. Two essential forms are distinguished:

  • Immune thrombocytopenia (ITP). This refers to enhanced clearance of thrombocytes from circulation, due to thrombocyte-associated IgG and complement activation. ITPs are complications of HIV and hepatitis C viral infections and following the treatment of patients with Helicobacter pylori infection. Medications also cause ITP /16/. A selection is listed in Tab. 15.11-4 – Medication-associated autoimmune thrombocytopenia.
  • Non-immune associated: disseminated intravascular coagulation (DIC), sepsis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, intraoperative, following multiple blood transfusions.

Acquired thrombocytopenia due to destruction or peripheral consumption are, in most cases, severe forms of the disease. The thrombocyte count is markedly reduced, the MPV and thrombocyte function are normal, but the lifespan of the thrombocytes is reduced. The bone marrow manifests hyper regenerative megakaryopoiesis.

15.11.6.2.3 Distribution and dilution associated thrombocytopenia

Patients with splenomegaly sequester thrombocytes. Hypersplenism usually leads to mild thrombocytopenia of (50–100) × 109/L. The cause is the storage of up to 90% of the thrombocytes in the enlarged spleen. The lifespan of the thrombocytes is only slightly shortened, which shows that they are only sequestered and not destroyed. In patients with splenomegaly and liver cirrhosis, thrombocytopenia is due to the sequestration of the platelets in the spleen to a lesser extent than it is to decreased hepatic TPO formation /17/.

Dilutional thrombocytopenia is caused by transfusion therapy in massive blood loss.

15.11.6.2.4 Thrombocytopenia and thrombocyte transfusion

The following banked thrombocyte concentrates (TC) are available as thrombocyte substitutes /17/:

  • Pooled units from 4–6 donors with (240–360) × 109 platelets in 200–350 mL of plasma or plasma replacement solution
  • Apheresis TC from a single donor with (200–400) × 109 thrombocytes in 200–300 mL of plasma.

A TC contains < 3 × 109 erythrocytes and < 1 × 106 leukocytes. The thrombocyte recovery rate in peripheral blood is only 60–70%, since the remainder is kept in the spleen. In sepsis, in disseminated intravascular coagulation, or in the presence of thrombocyte antibodies, the recovery rate is even lower. Fresh donor thrombocytes are detectable for 7–10 days in peripheral blood. The thrombocyte count should be determined prior to transfusion, as well as 1 and 20 hours post-transfusion. If a refractory condition is present, the increase following 1 hour is < 7,5 × 109/L and following 20 hours < 4,5 × 109/L. Recommendations for transfusion are provided in Tab. 15.11-5 – Recommendation for thrombocyte transfusion.

Critically ill patients often have disorders of hemostasis. Thrombocytopenia < 50 × 109/L was observed in a multicenter study /18/ in 13.7% of the cases and 35.4% of the patients with severe thrombocytopenia died in intensive care. The administration of thrombocyte concentrates was very inconsistent. Approximately 40% of the transfusions were administered with a thrombocyte count > 50 × 109/L and 34% in spite of the fact that with this thrombocyte count on the day of the transfusion there was no significant bleeding. With a transfusion of, on the average, 1.7 units, the mean thrombocyte increase was:

18.5 × 109/L [interquartile range (2.0–35.5) × 109/L].

In patients with severe thrombocytopenia (platelet count 10–50) × 109/L witholding prophylactic platelet transfusion before central venous catheter (CVC) placement did not meet the predefined margin for noninferiority and resulted in more CVC-platelet bleeding events than prophylactic platelet transfusion /63/.

15.11.6.3 Diseases and conditions with thrombocytosis or thrombocytopenia

15.11.6.4 Mean platelet volume (MPV)

The MPV can be used, in combination with the thrombocyte distribution width, to distinguish conditions of reduced formation from those with increased thrombocyte destruction.

15.11.6.4.1 Acute bleeding

In the presence of a significant decrease in the thrombocyte count, the MPV is increased and the platelet distribution width (PDW) is broadened.

15.11.6.4.2 Disturbances of thrombocyte formation

In aplastic anemia, megaloblastic anemia, chemotherapy of malignant tumors, acute leukemia, and systemic lupus erythematosus, the thrombocyte count and the MPV are decreased, and the PDW is broadened. With improvement of the clinical symptomatology, or following chemotherapy, a rise in the MPV occurs before the thrombocyte count.

15.11.6.4.3 Immune thrombocytopenia

The MPV and the PDW are normal.

15.11.6.4.4 Hereditary thrombocytopenia

All forms of thrombocytopenia with x-linked recessive inheritance have a low MPV with a left shift of the log-normal volume distribution (e.g., Wiskott-Aldrich syndrome).

Hereditary thrombocytopenia with macro thrombocytes and elevated PDW are: Alport syndrome, May-Hegglin anomaly, Sebastian anomaly, type IIB von Willebrand disease, Bernard-Soulier syndrome, Mediterranean macro thrombocytopenia, and autosomal dominant thrombocytopenia /15/.

15.11.6.4.5 Reactive thrombocytosis

Under reactive conditions with an elevated thrombocyte count due to release from the splenic pool, which is the case with infection, tumor, rheumatoid arthritis, pancreatitis, or following a surgical procedure, MPV and PDW are normal. In myeloproliferative syndrome, the MPV may be elevated and the PDW may be broadened.

15.11.6.4.6 Splenectomy

The thrombocyte count and the MPV may be elevated, and the PDW may be broadened.

15.11.6.4.7 MPV, metabolism and ischemic heart disease

In comparison with normal thrombocytes, those with an increased MPV have increased metabolic activity and an elevated potential for thrombosis. Increased MPV is associated with obesity, diabetes mellitus, and ischemic cardiovascular events. In one study /20/, patients with acute coronary syndrome had a higher MPV value and a lower thrombocyte count than patients with stable angina pectoris and healthy controls.

Thus, the mean values in 60 individuals in each group were:

  • Healthy controls: 257 × 109/L, MPV 9.1 fL
  • Stable angina pectoris: 267 × 109/L, MPV 10.0 fL
  • Acute coronary syndrome: 201 × 109/L, MPV 11.0 fL.

15.11.6.5 Immature Platelet Fraction (IPF)

In thrombocytopenia it is important to differentiate whether the cause is failure or suppression of the bone marrow, increased peripheral platelet consumption, or peripheral platelet destruction. In the two latter cases, the bone marrow releases immature thrombocytes with high RNA content. These thrombocytes are analogous to the reticulocytes in erythropoiesis, and are also termed reticulated platelets. Reticulated platelets are identical to the IPF.

The fraction of reticulated platelets, or the IPF, reflects the rate of thrombopoiesis. The IPF reference interval is 1.1–6.1%. A particularly high IPF is demonstrated in autoimmune thrombocytopenia (9.2–33.1%) and acute thrombocytopenic purpura (11.2–30.9%). With adequate therapy the IPF falls, and the thrombocyte count increases /19/.

15.11.7 Comments and problems

Method of determination

In thrombocytopenic patients, variations are seen between thrombocyte determinations performed on hematology analyzers with different counting methods. Causes are:

  • The inability to distinguish between thrombocytes, fragmented erythrocytes and microcytes. In chronic lymphocytic leukemia, nuclear and cytoplasmic lymphocyte fragments may also be counted as thrombocytes
  • Macro thrombocytes are excluded from the platelet count with impedance counting and one-dimensional optical methods.
  • Pseudo thrombozytopenia, caused by platelet clumping, is often found in clinical routines. However pseudo thrombocytosis resulting from fragmentation of red blood cells is a very rare phenomenon.

Lipemia

Platelet count can be measured by fluorescence method, optical method, and impedance method. The fluorescence method can more accurately reflect the true platelet count of lipemia specimens compared with impedance method and optical method /61/.

Immunoplatelet counting

Likewise, immunoplatelet counting is not free of disadvantages. Thus /6/:

  • Platelet aggregates, platelet complexes with leukocytes, and macro thrombocytes can lead to gating problems with flow cytometry
  • Due to antibodies (CD 41, CD 42b, CD 61), thrombocytes are not counted in Bernard-Soulier syndrome and in Glanzmann’s thrombasthenia
  • Thrombocyte autoantibodies in patients receiving therapy against glycoproteins (e.g., anti-GP IIb/IIIb; Reopro), can interfere with the determination.

Repetition of thrombocyte count

In thrombocytopenia of clinically uncertain etiology, the laboratory must repeat the determination using a different procedure.

Reference interval

Age, sex and genetic background modulate platelet count in healthy people. The effect of aging is much bigger than those of sex and ethnicity. Reference intervals (2.5th and 97.5th percentiles) estimated on the global population were as follows (× 109/L): all < 15 years 176–452; women 15–64 years 156–405; men 15–64 years 141–364; women > 64 years 140–379; men > 64 years 122–350 /22/.

Pseudothrombocytosis

Abnormally shaped erythrocytes in the form of spherocytes, microspherocytes, and cells with budding projections (asterisks) can be the reason of pseudothrombocytosis in patients with severe burn injuries. Heat-induced disruption of red cell membranes can generate smaller cells that may be incorrectly counted as platelets by automated blood counters /62/.

Intraindividual variation

CV over the course of the day: 6.7%; from day to day: 11.5%; from month to month: 10.6% /21/.

Stability

The thrombocyte count is stabile at room temperature for at least 24 hours; and with certain hematology analyzers, for up to 168 hours.

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15.12 Leukocyte count

Lothar Thomas

Leukocytes are produced in the bone marrow and the lymphoid organs, they use the blood stream as their route of transport and exert their effects in the tissues.

A distinction is made between the following forms:

  • Neutrophils: the cells are divided into two pools of approximately the same size in the vascular system. Only the cells belonging to the circulating pool are captured with blood sampling, not those of the marginated pool, in which the cells adhere loosely to the vascular intima. There is a continuous exchange of cells between the circulating and the marginated pools. The lifespan of the neutrophils in the blood is 21 hours and in the tissues it is 4–5 days. Neutrophils are phagocytic cells, and they are in the front lines of the defense against infectious pathogens. See Section 19.7 – Polymorphonuclear neutrophil function).
  • Eosinophils: the synthesis of their granular proteins begins with their transformation from eosinophilic myeloblasts to promyelocytes. Interleukin-5 is an important survival factor in the maturation of eosinophils and prevents apoptosis. Eosinophils react to immunological stimuli and are important effector cells in allergic and parasitic inflammatory events. They circulate in the blood for only a few hours before passing into the tissues.
  • Basophils: like eosinophils, basophils differentiate from agranular progenitor cells, and leave the bone marrow as mature cells. In the tissues, they reside in the small blood vessels and the post-capillary venules, and following a stimulus they migrate into the interstitium. They play an important role in the early phase of an IgG-mediated allergic reaction. In contact allergies, basophils located in the skin release the content of their granules over a period of days.
  • Monocytes: this cell population circulates in the blood with a transit time of 14 hours and, like the neutrophils, are distributed into two pools. Following their passage into the tissues, the cells can be activated and are transformed into metabolically active macrophages. The macrophage-supported immune defense ensues through the phagocytosis of inflammatory pathogens as well as the formation of pro-inflammatory cytokines and activation of the immune system.
  • Lymphocytes: these mononuclear cells include B cells, T cells and natural killer (NK) cells. Morphologically, these cells are similar. Refer to Section 21 – Immune system.

15.12.1 Indication

Determination of the leukocyte count, including the sub populations, is indicated in:

  • Initial investigations within the framework of the automated blood count
  • Suspicion of hematologic disease e.g., abnormal blood count, hemolysis, thrombosis, lymph node swelling, splenomegaly
  • Inflammation, infection, tissue necrosis, toxic disorders of the hematopoietic system (medication, radiation)
  • Fever, shock, breathing difficulties, abdominal pain, urogenital symptoms, headache, disturbance of consciousness
  • Frequent bacterial infections
  • Allergic disease and helminthiasis
  • Assessment of the progression and treatment of the symptoms mentioned.

15.12.2 Method of determination

Automated hematology analyzer with a broad number of parameters and a high throughput are used in clinical routines.

15.12.2.1 Manual counting method

In a micro collection device 1 part blood and 10 parts dilution solution (acetic acid 1–3% or Türk’s solution) are mixed. Thereby, the erythrocytes are hemolyzed and the leukocyte nuclei become more refractive. The counting chamber (e.g., the Neubauer chamber) is filled with diluted sample, and 4 corner squares (4 mm2) are counted. Leukocyte count (109/L) = sum of all counted cells × 25 × 106. The coefficient of variation of the hemocytometer count is around 15% with low leukocyte counts, and improves to 6.5% with normal or increased numbers. The counting chamber method continues to be indispensable for verifying the accuracy of determinations with hematology analyzers.

15.12.2.2 Hematology analyzer

The counting and differentiation of the leukocytes is performed optically, with a combination of impedance and optical methods, or of optical methods with a cytochemical reaction. Beforehand, however, the erythrocytes are destroyed with the use of a detergent /12/.

Volume conductivity light scattering technology

The cell counting and three part differential is based on the principle of cell counting and sizing by detection and measurement of changes in electrical resistance as cells pass through an aperture between two electrodes suspended in a conductive diluent. If a leukocyte passes through the aperture, a momentary rise in impedance, in the form of an electric pulse, is recorded. The pulse amplitude is proportional to the cell volume. Pulses from cells with a volume of greater than 35 fL are recorded.

The different leukocytes are distinguished electronically by the pulses they generate. Using a special reagent that results in different cell shrinkage of leukocytes, these cells are differentiated and expressed on a histogram (Fig. 15.12-1 – Hematogram of leukocyte differentiation using volume, conductivity and scatter technology).

Flow cytometry cytochemical technology

These systems perform the differentiation of leukocytes in two separate channels, which are termed the peroxidase and basophil/lobularity channels. The differentiation of the leukocytes, based upon their size and myeloperoxidase (MPO) content, is accomplished in the peroxidase channel. In the leukogram, the leukocytes are represented in a system of coordinates as clouds, whereas the MPO and the volume are plotted on the x and y axes, respectively Large leukocytes with high MPO content, such as neutrophils, are shown in the upper right part of the leukogram, while small MPO-free cells such as lymphocytes are found in the lower left section. The differentiation of the basophils is accomplished following the removal of the cytoplasm with acidic buffer, and the counting of the nuclei (nucleogram) in the basophil/lobularity channel. Refer to Fig. 15.12-2 – Hematogram of leukocyte differentiation with combined flow cytometry and cytochemistry methodology.

15.12.2.3 Blood smear

See Section 15.13 for cell differentiation.

15.12.3 Specimen

EDTA blood: 1 mL

Hemocytometer count method, capillary blood: 0.1 mL

15.12.4 Reference interval

Refer to references /345678/ and Tab. 15.12-1 – Leukocyte reference intervals. Reference intervals of lymphocyte subpopulations refer to Ref. /73/.

15.12.5 Clinical significance

Stepwise diagnostics in hematology is important in patients with an abnormal white blood cell (WBC) count.

15.12.5.1 Classifying white blood cell disorders

In patients with abnormal WBC count, review the CBC with differential and examine the peripheral blood smear e.g., atypical cells, neoplastic cells, parasites. Characterize the WBC count as normal, high or low according to the leukocyte differentials expressed in absolute numbers.

White blood cell count

Leukocyte counts of (4–10) × 109/L within the framework of a screening evaluation, are considered to be definitely normal; values of (2.5–4) × 109/L are borderline, and those below 2.5 × 109/L are classified as definitely pathological. Smokers may have values as high as 12 × 109/L, and in heavy smokers these may reach 15 × 109/L. In a study, non-smokers had mean values of 6.1 × 109/L, while those of smokers were 10% higher /7/.

Changes in the WBC count are primarily due to a change in the number of polymorphonuclear neutrophils (PMN) or of lymphocytes. The relative PMN fraction in healthy individuals is 40–75% of the leukocyte count. Infections are the primary cause of leukocytosis. Thus, a typical acute infection is characterized by the following processes: neutrophilic combat phase, monocytic recovery phase, and eosinophilic healing phase. In chronic infection each of the 3 phases can be present, depending upon whether the acute (neutrophilia), subacute or remittent (monocytosis) or chronic (lymphocytosis) phase persists. Viral infections and certain bacterial infections, such as enteric fever, generally do not follow the depicted course of events.

A low WBC count is often drug-induced, caused by bone marrow disease (e.g., neoplasia and leukemia), a hereditary disorder of development, immunosupressive diseases or sepsis.

Peripheral blood smear

When the WBC count is high the microscopic differential may evaluate:

  • Granulocytosis, lymphocytosis, monocytosis?
  • Left shift and toxic granulation?
  • Eosinophilia, basophilia?
  • Atypic cells, progenitors, blasts?
  • Hairy cells?
  • Red cell morphology?
  • Nucleated red blood cells?

Useful further information

Initial questions to be answered are:

  • Any clinical information?
  • Infection (antibodies to Epstein-Barr virus)?
  • Inflammation (C-reactive protein)?
  • Thrombocytes?
  • Hemoglobin level?
  • LD, urea, uric acid (increased cell turnover)
  • Haptoglobin (hemolysis)?

15.12.5.2 Neutrophile granulocytosis

Polymorphic nuclear cells (PMN)

The PMN progenitor cells in the bone marrow are differentiated into those that are capable or incapable of cell division. Cells that are capable of cell division are found in the mitotic pool, while those that are no longer capable of cell division undergo their post mitotic maturation in the storage pool. Myeloblasts, promyelocytes and myelocytes belong to the mitotic pool, while metamyelocytes, bands and mature granulocytes are part of the post mitotic pool. The newly formed and mature cells dwell in the pools for some 10 days, before they are released into the blood stream. The storage pool contains 15–20 times as many granulocytes as the peripheral blood /10/. The kinetics of the PMN is shown in Tab. 15.12-2 – Kinetics of the neutrophil granulocytes.

During the first hours of life, the newborn has marked neutrophilia with peak values after 12 hours of over 10 × 109/L; as of the third day there is a continuous fall, with stable granulocyte values in the range of (2.0–7.0) × 109/L. There is a distinct left shift in comparison with children of 2 years of age. Healthy children with very low birth weight and without perinatal or neonatal complications manifest considerable variation in their leukocyte counts, and some 95% are rated as neutropenic according to the Manroe criteria (Fig. 15.12–3 – Neutrophil count in the newborn/4/.

Normally, only PMN are released into the blood. On the other hand, the bone marrow contains more banded than PMN. If the requirement for PMN cannot be met by the bone marrow, more banded neutrophils and metamyelocytes will be continuously released. Thus, if the fraction of banded neutrophils and, perhaps, that of the metamyelocytes as well, increases in the blood, that is a sign of augmented release of granulocytes from the marrow and a reduction of the storage pool. This course of events is also termed a left shift.

The PMN determination in peripheral blood provides only limited information on the neutrophil mass, because the circulation contains only 5–10% of the body’s neutrophil pool, and only 2% of the PMN neutrophil life cycle is captured.

Three processes can cause neutrophile granulocytosis /11/:

  • A shift in the PMN from the vascular marginal pool into the circulating pool. This is the case with hard labor, psychological stress, catecholamine release and the administration of noradrenaline. A rise of maximally 2-fold above the initial value occurs. This course of events is also termed pseudo neutrophilia, since a true increase in the blood neutrophil count does not occur.
  • The enhanced release of PMN from the storage pool. This form of leukocytosis (e.g., in response to endotoxin) lasts for only some minutes to a few hours.
  • Increased granulopoiesis. This takes place in the maturation pool and, following stimulation of the bone marrow, at least 2–3 days are required before proliferation of the cells in the storage pool occurs. With proliferative stress (e.g., infection) granulopoiesis can be increased by a factor of 20. In this case, the PMN maturation time is shortened from 10 to 2 days. In conditions of high peripheral requirements and an empty storage pool, cells of the mitotic pool such as myelocytes and promyelocytes are also released directly into the peripheral blood (leukemoid reaction). If granulopoiesis does not respond sufficiently to the requirements of the tissues (e.g., with systemic infection) neutropenia may result; this occurs in sepsis, in some 20% of the cases.

Fc receptor antigen CD64

Neutrophils express the Fc receptor antigen CD64. The antigen is expressed by myeloid stem cells, and is maintained up to the metamyelocyte stage. PMN and banded neutrophils have only approximately 1000 of these antigens on their cell membrane. Newborn and premature infants express a higher proportion of the antigen. Upon activation, PMN increase the number of antigens by 5–10-fold, and the measurement of neutrophil CD64 thereby permits a distinction between healthy and inflammation /12/.

In systemic inflammation, as in cases of infection and sepsis, pro-inflammatory cytokines like IFN-γ , IL-1 and IL-6, as well as GCSF, activate the up-regulation of CD64 on the granulocytes. In the diagnostic investigation of a systemic infection, CD64 is believed to have a diagnostic sensitivity of 90% with a specificity of 90–100%; this in comparison with the leukocyte count which has a sensitivity of 60% with a specificity of 51% / /13/.

Neutrophilia

In school children and adults, neutrophilia is present if the number of PMN and their precursors is greater than 7.5 × 109/L. Elevated granulocytes are an indication of:

  • Inflammation due to acute inflammation like infection and acute tissue necrosis. In chronic infection neutrophilia may be present due to enhanced granulopoiesis. The peripheral requirements for granulocytes is increased, but there is overcompensation of granulopoiesis with the development of an enlarged storage pool.
  • Myeloid leukemia
  • The ingestion of glucocorticoids. These lead to neutrophilia due to an increase in granulopoiesis, augmented migration from the marginal to the circulating vascular pool, and reduced migration of the granulocytes from the blood stream.

The Monroe and the Rodwell criteria are utilized for the diagnostic investigation of neonatal sepsis (Tab. 15.12-3 – Criteria of neonatal sepsis). Diseases and conditions with neutrophilic granulocytosis are listed in Tab. 15.12-4 – Diseases and conditions with neutrophilia.

15.12.5.3 Neutropenia

Neutropenia is defined as a decrease of the PMN including the band forms. The number is determined with a hematology analyzer, and if a decrease of below 0.5 × 109/L is indicated, a blood smear is counted as a control and the percentage of PMN and band forms is multiplied with the leukocyte number. Normally, in healthy adults and children over 5 years of age, the number of PMN is greater than 1.5 × 109/L. In persons of African origin and in other ethnic groups (e. g,. in Israel and Jordan) values as low as 1.0 × 109/L are also normal. These individuals also have a low leukocyte count. Thus, only 25.2% of male Caucasians aged 3–74 years have a leukocyte count of below 5.0 × 109/L, while for blacks the corresponding figure is 48.1%. In females, the corresponding figures are 27.1% and 42.6% /15/.

Neutropenia is classified as /16/:

  • Mild; the neutrophil count is 1.0–1.5 × 109/L
  • Moderate; the neutrophil count is 0.5–1.0 × 109/L
  • Severe; the neutrophil count is below 0.5 × 109/L.

This classification predicts the risk of a pyogenic infection, if the neutropenia has been present for more than 2–3 months. Only patients with severe neutropenia are at risk for bacterial infections /10/. The infection is usually due to the patient’s own flora. The most common symptoms are gingivitis, ulcerations and oral thrush.

Febrile neutropenia is present if /17/:

  • The oral temperature, with a single measurement, is ≥ 38.3 °C, or ≥ 38 °C for at least 1 hour and
  • The neutrophil count is < 0.5 × 109/L or < 1.0 × 109/L with a tendency to decline to ≤ 0.5 × 109/L.

In severe neutropenia following chemotherapy for solid tumors, treatment with hematopoietic growth factors (G-CSF, GM-CSF) is indicated if the neutropenia has been present for longer than 10 days or if there is fever of > 38.1 °C.

Causes of neutropenia

15.12.5.4 Lymphocytosis

Lymphocytes

Lymphocytes are formed in the bone marrow and in the secondary lymph organs such as the spleen and the lymph nodes. The lymphocytes of the blood represent only some 2% of the lymphocytes of the organism. These are found in the spleen, the lymph nodes and the organ-associated lymphatic system. In contrast to the granulocytes, the lymphocytes enter continuously the circulation and migrate in the fluid compartments before they return to the lymph nodes; this process is known as homing /18/. The lymphocyte count in the blood is constant only 2% of the lymphocytes circulate in the blood, and in any case for less than 1 hour /10/.

Most of the lymphocytes in the blood are small, intermitotic cells in the resting stage. A small proportion of the cells is medium-sized, apparently originating from the small lymphocytes, and in an activated state. In addition, large lymphocytes with coarse eosinophilic granulation (large granular lymphocytes, LGL) occur during infection.

Lymphocytes are immune cells which express, due to immunophenotyping, specific identifiable features on their surface.

Based upon their functional activity in immune defense, and their surface features (CD classification), three classes of lymphocytes are distinguished:

  • T cells that are released from the thymus gland, and are primarily responsible for cell-mediated immunity
  • B cells, the origin of which is the bone marrow and the secondary lymph organs. They are the precursors of the immunoglobulin-forming plasma cells, and are responsible for the humoral immune response.
  • Natural killer (NK) cells. They express no surface lymphocyte features, and are active within the framework of the non-specific immune defenses. The NK cells are also distinguishable morphologically since, in the blood smear, some of them are visible LGL’s.

In the peripheral blood, 65–80% of the lymphocytes are T cells, 8–15% are B cells and 10% are NK cells. NK cells do not recirculate, like the other lymphocytes, from the blood to the lymph nodes and back.

The lymphocyte count is subject to influencing factors to a considerable degree; it is higher in the late afternoon and in the evening than during the morning. Lymphocytosis caused by increased lymph flow occurs after brief physical exertion. Following longer and more marked exertion, and in cases of systemic infection such as sepsis, lymphopenia and eosinopenia develop along with a rise in PMN.

Large granular lymphocytes (LGL)

In response to pathogens that attack the organism, two different cell types react: cytotoxic T lymphocyte and natural killer (NK) cells /19/. These are morphologically identical, and they appear microscopically as large lymphocytes with azurophilic cytoplasmic granules. They are, however, functionally different, and they recognize antigens via T cell receptors and NK cell receptors.

In healthy individuals, only a small fraction of the circulating lymphocytes are LGL’s. The majority of the LGL’s are cytotoxic T cells of phenotype CD3+ CD8+ CD4, while the few NK cells are of phenotype CD3CD16+.

The polyclonal proliferation of LGL’s occurs in a reactive and transient manner within the framework of viral infection (EBV, Cytomegalo virus), autoimmune disease, malignancies and also, in some cases, splenectomy.

The clonal proliferation of LGL’s is maintained over the long term, independently of whether or not the patient is symptomatic. The proliferation can affect T cells and NK cells, in spite of the fact that the WHO has designated NK proliferation as a separate disease, defined as chronic lymphoproliferative disease of the NK cells. NK cell lymphoproliferative disease is poorly understood.

T cell LGL leukemia is a clonal proliferation of end-differentiated cytotoxic T cells, with a functional α/β+T cell receptor and a pattern CD8+ CD4 or, occasionally CD4+CD8–/+dim.

Lymphocytosis

In adults, lymphocytosis is present with a cell count of > 4.0 × 109/L. In children, the reference interval is age-dependent (Fig. 15.12-4 – Age-dependency of the lymphocyte count in children). Thus, the lower reference interval value at the age of 8 months is 4.5 × 109/L, and at the age of 18 years it is 1.0 × 109/L /20/.

Disorders in association with lymphocytosis are:

  • Viral infections such as infectious mononucleosis. Many of the lymphocytes are transformed by the Epstein-Barr virus and manifest a colorful image in the blood smear. The virus infects only B cells, not T cells. The lymphocyte count is (6–15) × 109/L. A similar picture, with transformed lymphocytes, is also seen with Cytomegalo virus and viral hepatitis. In these infections the lymphocyte count is only slightly elevated, if at all.
  • Infections like toxoplasmosis, enteric fever, brucellosis or pertussis. In pertussis, the lymphocyte count increases over 20 × 109/L, while the lymphocytes are small cells of normal appearance /10/.
  • Neoplasias of the lymphocytic system, such as in acute and chronic lymphatic leukemia and, occasionally, in non-Hodgkin lymphoma.

Indicators of a lymphoproliferative disease are:

  • If lymphocyte count exceeds 5 × 109/L
  • If accompanied by > 5% smudge cells
  • Atypic cells e.g., pro lymphocytes, mantle cells, Sezary cells
  • Clinical signs of lymphoproliferation (lymphadenopathy, splenomegaly, B symptoms)

Further procedure: immunophentyping of peripheral blood, nodal histopathology.

15.12.5.5 Lymphopenia

Depending on the literature source, lymphopenia in adults is defined as a cell count of below 1.5 × 109/L, or below 1.0 × 109/L /20/.

In children, the lower threshold is age-dependent (Fig. 15.12–4 – Age-dependency of the lymphocyte count in children). Diseases and conditions with lymphopenia are shown in Tab. 15.12–9 – Diseases and conditions with lymphopenia.

Small children of less than 3 months of age with no previous underlying disease, who are admitted to hospital with acute symptoms on an emergency basis, often require life-saving measures if lymphopenia is present. Thus, out of 42 lymphopenic children, 26 had to be admitted to the pediatric intensive care unit; by way of comparison, this was only necessary for 1 out or 42 non-lymphopenic children /22/.

15.12.5.6 Monocytosis

Monocytes

Monocytes emerge from the bone marrow and are part of the inflammatory response and immune response to infection. Monocytes share a common stem cell with the granulocytes. During maturation they go their own separate ways, but in the bone marrow the morphology of the pro monocytes and the promyelocytes is almost identical, and their small population is integrated into that of the neutrophil progenitor cells during counting. They can only be differentiated with the use of esterase staining. The monocytes are released into the blood following two to three cell divisions. No storage pool analogous to that of the neutrophils exists. Following a transit time of 14 hours they leave the blood and migrate into the tissues. They mature in the tissues, and the nature of the maturation (i.e., their enzyme pattern) is a function of the tissue where the maturation process takes place. Thus, as alveolar macrophages of the lungs, they have a different protein make-up than the Kupffer stellate cell of the liver or the peritoneal macrophages. Fusion of macrophages can occur in the tissues, with the formation of large cells such as Langhans giant cells, which are found in granulomatous inflammation such as that occurring in tuberculosis. Like lymphocytes, macrophages can also divide in situations of increased need /10/. Important macrophage tasks are phagocytosis and the killing of microbes. The internalized pathogens are processed, and are presented to helper T cells in association with an HLA class II antigen (see Section 21.1.2 – Immune recognition).

In inflammation, extravasation of the PMN initially precedes that of the monocytes at the affected site. Thus, the PMN granules release proteins that stimulate the expression of vascular β-integrin and formyl peptide receptors, to which activated monocytes bind. At the site of the inflammation, the PMN that are subject to apoptosis release lysophosphatidylcholine, which binds to the monocyte G2A receptors and attracts them to the affected site /23/.

Monocytosis

In adults and school children, an increase in the monocyte count to over 0.9 × 109/L is termed monocytosis. Newborns and small children have a higher upper reference interval value. Monocytes are found in infection, autoimmune disease, systemic hematological disease, with solid tumors and for various other reasons. In infectious diseases monocyte distribution width is increased. Diseases and conditions with monocytosis are shown in Tab. 15.12-10 – Diseases and conditions with monocytosis.

Signs of neoplastic monocytosis:

  • Immature Monocytes (promonocytes) and/or blasts
  • Thrombocytopenia and/or anemia
  • Organ infiltration (skin, lung, spleen)
  • No conclusive medical explanation for monocytosis

Bone marrow diagnostics (cytology, histopathology, cytogenetics) are important investigations.

15.12.5.7 Eosinophilia

Eosinophils

The eosinophil granulocyte arises from a pluripotent stem cell as a cell of the myeloid series, which differentiates under the influence of hematopoietic growth factors. In individuals with healthy bone marrow, the eosinophil fraction of the white blood cells is some 3%, of which one third are promyelocytes and myelocytes, 26% are metamyelocytes, and one third are mature eosinophils that dwell in the storage pool. The polyclonal synthesis of eosinophils takes place following stimulation with GM-CSF, IL-3 and IL-5. The mature eosinophil enters the blood after approximately 4 days, where it circulates for 6–18 hours before migrating into the tissues.

The eosinophil stays for 2–5 days in the mucosal membranes of the respiratory and gastrointestinal tracts, as well as in the skin, before undergoing apoptosis: unless this is delayed by GM-CSF, IL-3 and IL-5. The daily turnover of the eosinophils is 2.2 × 108 cells/kg BW; the post mitotic storage pool contains (9–14) × 108 cells/kg BW /24/.

Eosinophilia

Eosinophilia in the blood entails a 100-fold more marked eosinophilia in the tissues. The tissue concentration can be high even if the eosinophil count in the blood is low.

The blood eosinophil count manifests diurnal fluctuations. Highest values are measured during the evening, and the lowest values are found in the morning.

Eosinophilia is present with a cell count of > 0,5 × 109/L.

Eosinophilia is classified as:

  • Mild, with up to 1.5 × 109/L
  • Severe, with > 1,5 × 109/L.

If eosinophilia is present, the following should be considered /25/:

  • A reactive event such as an allergic/atopic disease, urticaria, parasitic infection, malignant disease or vascular collagen disease
  • If the afore mentioned causes are ruled out and the hyper eosinophilia persists, the diagnosis of idiopathic hyper eosinophilic syndrome is likely, in particular if the hyper eosinophilia has been present for longer than 6 months and is above 1.5 × 109/L.

The accumulation of eosinophils in the blood and the peripheral tissues can be due to /26/:

  • A disorder of the myeloid cell lineage (primary eosinophilia). This can occur late in the eosinophil differentiation process, and can lead to the rare diagnosis of eosinophilic leukemia. If the disorder occurs early in the differentiation process, and if the eosinophils are a component of the malignant clone that also forms other myeloid cells, or even lymphoid cells, then the eosinophilia is an event within the framework of a myeloproliferative disease.
  • The increased formation of eosinophil-stimulating cytokines by non-myeloid cells (secondary eosinophilia). The result is the polyclonal formation of eosinophils. Stimulated by inflammatory mediators like IL-5, eotaxin, platelet-activating factor, C5a and C3a, eosinophils migrate to the site of inflammation in the tissue and release from their granule proteins that are destructive to tissue, such as eosinophil cationic protein (ECP) and reactive oxygen radicals /27/.

For the differential diagnosis of eosinophilia see Tab. 15.12-11 – Differential diagnosis of eosinophilia.

15.12.5.8 Basophilia

Basophils

Basophils make up some 0.3% of the circulating leukocytes, and are related functionally to the mast cells /2829/. Both cell types express the functionally active IgG receptor, and form the same effector molecules, such as histamine, lipid mediators, (leukotrienes, prostaglandins), serine proteases and interleukines (IL-4, IL-13, IL-6).

Basophils leave the bone marrow as mature cells, and are characterized by the expression of FcεRI, CD49b, and the high affinity IL-3 receptor CD123. Their concentration in the bone marrow, the liver, the spleen, and the peripheral blood is low under basal conditions. Within the framework of the specific immune response, and in types of inflammation, they pass into the peripheral tissues and the lymph nodes.

Mast cells accomplish their maturation in the peripheral tissues such as the skin, the small intestine and the peritoneal cavity. They are characterized by the expression of FcεRI and c-Kit. Under receptor-mediated stimulation (FcεRi), basophils and mast cells release effector molecules.

Basophils accelerate the TH2 immune response, since they release IL-4 within minutes of activation. The activation occurs through the cross-linking of two surface-bound IgE molecules, or by means of a large number of substances in an antigen-independent manner.

Basophilia

Elevated basophils are commonly associated with immediate type hypersensitivity reactions. Total IgE is often also increased. There is, however, no correlation between the rise in IgE and the basophil count. Little increase (> 2–3%) may be indicative for a myoproliferative neoplasia.

Basophilia can occur in:

  • Allergic inflammation such as hypersensitivity to medication or food, erythroderma, urticaria, and rheumatoid arthritis
  • Parasitic infection
  • Stem cell disease like myeloid leukemia, myeloproliferative syndrome, M. Waldenstroem
  • Diabetes mellitus, myxedema, estrogen-containing medication
  • Infectious disease such as tuberculosis, chicken pox, influenza
  • Post-splenectomy syndrome.

15.12.6 Comments and problems

Method of determination

The lysis of the erythrocytes is time-critical. If the lysis time is too short, or if relatively lysis-resistant red blood cells are present (e.g., reticulocytes or erythrocytes of newborns) then the leukocyte count will be overestimated. If the lysis time is too long, or if the leukocytes have previously been damaged (e.g., in chemotherapy or sepsis) because their volume is smaller than normal and they will not be counted. Giant thrombocytes may be counted along with the leukocytes. For the determination of the leukocyte count in the blood smear, see Section 15.13.

Blood sampling

Reliable values are obtained with EDTA blood; capillary blood sampling should only be performed as an exception. With capillary blood sampling, the first two drops of blood following puncture are to be discarded.

In newborns and small children, the leukocyte count depends upon the blood sampling. Compared with capillary blood sampling from the heel, venous and arterial sampling yield only 82 ± 3,5% and 77 ± 5,3% of the leukocyte count, respectively. Blood sampling following violent screaming leads to a rise in capillary-sampled leukocytes to 146 ± 6,1% of the resting value /30/.

IgM-mediated neutrophil agglutination

A case that caused pseudo neutropenia has been described. The neutrophils were loaded with IgM antibodies /31/.

Cryoglobulins

At room temperature and with counting by means of hematology analyzers, cryoglobulins lead to pseudo leukocytosis. Within this temperature range protein crystals, the size of leukocytes, are formed, and these are captured with automated counted as leukocytes. The protein crystals disappear if the sample is warmed to 37 °C.

Intraindividual variation of leukocyte counts

The CV within 1 day is 19.9%, the day-to-day CV is 16.3%, and the month-to-month CV is 17.3% /32/.

Diurnal variation of leukocyte counts

The diurnal variation shows lower values during the morning and higher values in the late afternoon and at night; at 12 o’clock at night 9.5 × 109/L and 7 × 109/L between 8.00 and 11.00 o'clock /72/.

Pseudoleukocytosis

Pseudoleukocytosis may occur under several conditions: presence of nucleated red blood cells (NRC), cryoglobulins, cryofibrinogen, platelet clumps, microorganisms, lysis-resistant red blood cells, lipids. NRC are especially common in hemolytic anemia, massive blood loss, severe oxygen deficiency, acute and chronic leukemias, malignant tumor, osteomyelofibrosis.

Stability

In EDTA blood at room temperature and refrigerated within 72 hours, dependent upon the hematology analyzer /33/:

  • Total leukocytes: decrease of 0.6–5.1%
  • Neutrophils: increase of 1.3–10.3%, with one analyzer, a decrease of 1.2% is reported
  • Monocytes: with one analyzer, an increase of up to 31%, with another, a decrease of 28–78%
  • Lymphocytes: on one analyzer at room temperature an increase of 5%, on another, a decrease of 3–14%.

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22. Adamski JK, Arkwright PD, Will AM, Patel L. Transient lymphopenia in acutely unwell young infants. Arch Dis Child 2002; 86: 200–1.

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24. Giembycz MA, Lindsay MA. Pharmacology of the eosinophil. Pharmacological Reviews 1999; 51: 213–339.

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34. Weimann A, Lun A, Lun S, Zimmermann M, Borges AC, Ziebig R, et al. Leukocyte, neutrophil, immature granulocyte counts and interleukin-6 are superior to procalcitonin, C-reactive protein and delta He for detection of mild inflammation: data from marathon runners producing mild systemic inflammation visible immediately after the run. J Lab Med 2010; 34: 53–9.

35. Rochlitz CF, Herrmann R. Rauchen und Leukozytose. Dtsch Med Wschr 1990; 115: 756.

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40. Dale DC, Link DC. The many causes of severe congenital neutropenia. N Engl J Med 2009; 360: 3–5.

41. Boztug K, Appaswamy G, Ashikov A, Schäffer AA, Salzer U, Distelhorst J, et al. A syndrome with congenital neutropenia and mutations in G6PC3. N Engl J Med 2009; 360: 32–43.

42. Dale DC, Bolyard AA, Aprikyan A. Cyclic neutropenia. Semin Hematol 2002; 39: 89–94.

43. Kannourakis G. Glycogen storage disease. Semin Hematol 2002; 39: 103–6.

44. Cham B, Bonilla MA, Winkelstein J. Neutropenia associated with primary immunodeficiency syndromes. Semin Hematol 2002; 39: 107–12.

45. Juul SE, Calhoun DA, Christensen RD. Idiopathic neutropenia in very low birthweight infants. Acta Paediatr 1998; 87: 963–8.

46. Boxer L, Dale DC. Neutropenia: Causes und consequences. Semin Hematol 2002; 39: 75–81.

47. Palmblad JEW, von dem Borne AEG. Idiopathic, immune, infectious, and idiosyncratic neutropenias. Semin Hematol 2002; 39: 113–20.

48. Bux J, Hofmann C, Kauth T, et al. Serological and clinical aspects of granulocyte antibodies leading to alloimune neonatal neutropenia. Transfus Med 1992; 2: 43–9.

49. Conway LT, Clay ME, Kline WE, et al. Natural history of primary autoimmune neutropenia in infancy. Pediatrics 1987; 79: 728–33.

50. Starkebaum G. Chronic neutropenia associated with autoimmune disease. Sem Hematol 2002; 39: 121–7.

51. Nossent JC, Swaak AJ. Prevalence and significance of hematological abnormalities in patients with systemic lupus erythematodes. QJMed 1991; 80: 605–12.

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15.13 Blood smear examination

Lothar Thomas

The blood smear examination is the cornerstone of the diagnosis of diseases of the hematopoietic system and of other organ or systemic disorders which compromise hematopoiesis.

Despite automated hematology analyzers, flow cytometry, cytogenetics and molecular diagnostics. The efficacy of automated analyzers in generating reliable results and flagging the ones that need to be verified by additional means including a blood smear examination has been widely documented. Hematology analyzers are highly efficient because of the improved precision and turnaround time, but they can not eliminate the need for the blood smear examination in selected cases. The blood smear provides the investigator with insight into the structure of the blood cells, something that is not possible with automated counting and differentiation /1234/.

Blood smear examinations serve two important objectives:

  • It often provides the most useful information
  • It serves as a quality control tool in verifying the results generated by automated hematology analyzers.

The International Consensus Group for Hematology Review has elaborated 41 criteria for when an automated blood count should be supplemented with a blood smear. In a modification of the criteria, the blood smear fraction is, in many laboratories, 10% in ambulatory patients and 20% in in-patients /5/.

15.13.1 Indication

Historically, leukocyte differentiation was perfomed microscopically. The most modern hematology laboratories use automated hematology analyzers.

15.13.1.1 Decision-oriented laboratory testing

Decision-oriented laboratory testing using microscopic differentiation is still performed in some situations following automated CBC and WBC /5/.

Red cell morphology

Because many morphologic red cell shape changes cannot be recognized by hematology analyzers it is necessary to examine a blood smear in patients presenting with anemia /1/.

Reasons for examining a blood smear even in the presence of a normal automated complete blood count include the following:

  • Clinical information that the patient had a prior red cell abnormality and its current status is questioned
  • A family history of a morphologic red cell abnormality
  • Investigation whether the patient has a hypo splenic or post splenectomy blood picture
  • Determination of the presence of parasites.

Leukocyte morphology

The evaluation of leukocytes consists of three parts /4/.

  • Verification of the white blood cell count
  • Differential leukocyte count
  • Detection of morphologic abnormalities within a population of cells

Thrombocyte morphology

Thrombocyte evaluation consists of two parts /4/:

  • Verification of the automated blood count
  • Evaluation of the platelet morphology (verification of the automated mean platelet volume, identification of (e.g., aggregates, giant platelets their differentiation from erythrocyte fragments).

None blood cells

Other important cells are /4/:

  • Endothelial cells, epithelial cells, tumor cells
  • Organism; intracellular (plasmodium, babesia), extracellular (trypanosomas, micro filariae).

15.13.1.2 Clinical indication /2/

  • Anemia, unexplained jaundice, or both
  • Sickle cell disease (dactylitis or sudden splenic enlargement and pallor in a young child or, in an older child or adult, limb, abdominal, or chest pain)
  • Thrombocytopenia (e.g., petechiae or abnormal bruising) or neutropenia (e.g., unexpected or severe infection)
  • Lymphoma or other lymphoproliferative disorder (lymphadenopathy, splenomegaly, enlargement of the thymus or other lymphoid organs, skin lesions suggestive of infiltration, bone pain, and systemic symptoms such as fever, sweating, itching, and weight loss)
  • Myeloproliferative disease (splenomegaly, plethora, itching, or weight loss)
  • Disseminated intravascular coagulation
  • Acute or recent-onset of renal failure or unexplained renal enlargement, particularly in a child
  • Retinal disorders, hemorrhages, exudates, signs of hyper viscosity, or optic atrophy
  • Bacterial or parasitic disease that can be diagnosed from a blood smear
  • Disseminated non hematopoietic cancer (weight loss, malaise, bone pain)
  • General ill health, often with malaise and fever, suggesting infectious mononucleosis or other viral infection or inflammatory or malignant disease.

15.13.2 Sample preparation

Anticoagulation

K2or K3-EDTA or sodium citrate, but not heparin, are acceptable for venous or capillary blood sampling. Heparin is obsolete because of /3/:

  • Developing thrombocyte clumps that interfere with the morphologic interpretation of the smear
  • Heparin causes the development of a purple-blue hue on stained films.

Sample processing

Films should be prepared within 2 hours after blood collection. If this is not possible, the sample should be stored for less than 8 hours at 4 °C but storage at room temperature (22 °C) is also possible. Long-term storage should be at 4 °C. After prolonged storage blood samples should always be mixed by a minimum of 10 complete (180 degree) inversions /3/.

Preparation of the film

Approximately 5 μL of blood should be used to allow for a wedge blood film of appropriate thickness and of 2.5–4 cm length /3/. The blood drop should be placed about 1 cm from the end of the slide (opposite labeling end). After the blood drop has been placed on the slide, the spreader slide is moved slowly backward at about a 30 to 45 degree angle toward the blood drop (Fig. 15.13-1 – Blood smear method). The faster the blood is spread on the slide the thicker the film. The smear is thicker at the front, becomes thinner and has a short feathered edge towards the back (Fig. 15.13-2 – Relevant areas for the evaluation of the blood smear). The thickness and the length of the smear are influenced by /3/:

  • The size of the blood drop and the HCT (the bigger the drop and the higher the HCT, the thicker the smear)
  • The angle of the spreader (the greater the angle, the thicker and shorter the film)
  • The speed of spreading (fast spreading produces thicker films).

Drying of the film

Air drying for 1 hour without forced air circulation is sufficient /3/. At higher relative humidity (≥ 70%), forced air drying is recommended. If slides cannot be stained immediately, fixation with methanol is necessary within 4 hours, but preferably less than one hour, after air-drying; otherwise the plasma causes gray-blue background effects.

15.13.2.1 Staining

Blood films are typically stained by Romanowsky dyes. A Romanowsky stain is any stain containing oxidized methylene blue and/or its products of oxidation (azure B), and a halogenated fluorescein dye, usually eosin B or Y. The most commonly used procedures which ensure that all cell types in a blood film can be identified reliably include Wright-Giemsa stain and the Pappenheim stain.

Wright-Giemsa stain

The stain is composed of basic dyes (methylene blue and its derivatives) and an acidic dye (eosin) in a 2 : 1 ratio /6/. Eosin is termed acidic because it binds to cellular components such as hemoglobin, eosinophilic granule contents, and basic granular proteins. Methylene blue a member of the blue staining thiazin dyes (azure A, azure B, methylene violet) are termed basic dyes. They bind to acidic components, such as DNA, RNA, neutrophil granules and acidic cellular proteins.

The use of basic and acidic mixtures of dyes leads to a panoptic (multicolored) staining of the cells. The best panoptic staining is obtained, according to Pappenheim’s specifications, with follow-up staining of the smear, initially with the May-Grünwald dye, and then with the Giemsa stain. Basic stains (blue) bind to acidic cell components such as DNA, RNA and acidic cytoplasmic and granular proteins. Eosin is an acidic dye (red) and it binds to basic cellular components like hemoglobin and basic proteins in the cytoplasm and in certain granules (eosinophil granules). For staining instructions according to May-Grünwald-Giemsa, Pappenheim, or Wright-Giemsa, see Ref. /367/.

Pappenheim stain

May-Grünwald’s eosin methylene blue and Giemsa’s azure methylene blue are used for staining. A lot of modifications are described /7/.

Requirements for acceptable blood film

Desirable qualities are:

  • Minimum 2.5 cm in length terminating at least 1 cm from the end of the slide
  • Gradual transition in thickness from the thick to thin area, ending in a squared or straight edge
  • No artifact introduced by the technique
  • Macroscopically, pale red and violet-blue in the thin and thicker sections of the smear, respectively. Color shifts in the staining process due to the predominance of alkaline (blue cast) or acidic (red cast) constituents are prevented with the use of buffered distilled water at pH 6.8–7.2 as the rinsing solution.
  • Microscopically, the red blood cells should be pale red, and the leukocyte nuclei should be more purple than blue. The blood cells should be free of vacuoles and other artifacts, and the glass slides should not be contaminated with precipitates from dye residues /3/.

15.13.2.2 Cytochemical staining

Cytochemical stains serve to demonstrate enzymes and other components of the blood cells. They are useful in the differentiation of immature hematopoietic cells and the differentiation of lymphocytes and are indicated:

  • To differentiate between myeloid and lymphocytic leukemias
  • To differentiate precursors of the granulocytic and the monocytic lineage
  • To identify ALL subtypes
  • To characterize cells in CLL and hairy cell leukemia
  • To differentiate between reactive leukocytosis, leukemoid reaction and neoplastic myeloproliferative disease
  • To detect enzymatic defects, especially in neutrophilic granulocytes (e.g., partial peroxidase defect in the case of the myelodysplastic syndrome)
  • To differentiate between a rise in reactive and a rise in neutrophil granulocytes by means of the neutrophil alkaline phosphatase.

NAP score

100 neutrophil leukocytes are scored from 0 to 4+ on the basis of the intensity of the precipitated dye in their cytoplasm. The sum of the cells in each category multiplied by its category factor yields the score (reference interval 13–130). Because of a time-dependent activity loss, the smears must be processed within 3 days. The determination of the NAP score is useful for differentiating CML (low score) from other myeloproliferative diseases and from leukemoid reactions. In polycythemia vera, the NAP score is elevated in approximately 70% of the cases and thus supports this diagnosis versus a reactive erythrocytosis.

15.13.2.3 Artifacts

Artifacts can arise at all steps of the blood smear procedure.

Anticoagulant

With the use of EDTA blood for the smear, the following artifacts may arise: agglutination of leukocytes, satellitism (e.g., platelet coating of granulocytes) as well as agglutination of platelets. IgG autoantibodies may induce agglutination of blood cells in EDTA-containing blood but not in citrated blood. IgM autoantibodies are often EDTA independent. Some but not all autoantibodies are of the cold-reactive type. EDTA-associated artifacts include not only agglutination of granulocytes, but also of lymphocytes and circulating lymphoma cells /7/.

Age of the sample

Best results are obtained when films are prepared within 2 hours of blood collection because of degenerative artifacts that may manifest (e.g., vacuolization of granulocyte and monocyte cytoplasm, or fragmentation and lobulation of granulocytes). The lymphocytes take on an apoptotic appearance, so that only with difficulty they can be attributed to a viral infection or to a lymphoproliferative disease /3/.

Preparation of the film

When the film is prepared, certain cell types may be damaged especially in conditions with large numbers of atypical lymphocytes. In chronic lymphocytic leukemia ruptured cells may be produced or are present primarily (smudge cells or Gumprecht Kernschatten) /3/. In order to minimize the cellular changes that occur during the preparation of the smear, the addition of 1 drop of albumin (22%) to 5 drops of blood, followed by usual spreading, is recommended.

Drying and fixation

Optimal results are obtained if the fixation and the staining are performed directly after the air drying of the film. If it is not possible to stain the smears immediately, they should be fixed in methanol within 1 hour of air drying. If this does not occur, the background of the smear takes on a grey-blue appearance, due to changes that have occurred in the blood plasma. Air drying that proceeds too slowly causes the contraction of the cells, a reduction in the fine structure of the nucleus, and the formation of cytoplasmic vacuoles. The staining of a smear that is still moist leads to the irregular spread of the blood film and to poorly differentiable cell morphology /3/.

If the water content is in excess of 3% in the methanol fixation solution, artifacts occur, such as decreasing crispness of cellular appearance and the development of artificial cytoplasmic vacuoles /3/.

15.13.3 Microscopy

The systematic approach to macroscopic examination of the blood smear is to select an area near the feathered edge and away from the thick portion and looking for a zone where no more than 50% of the erythrocytes are in contact with one another /9/. Thin areas of the smear, in which the erythrocytes form discrete strips (feathered edge), should not be analyzed. As a rule, the optimal analysis site is found towards the last third of the smear (Fig. 15.13-2 – Relevant areas for the evaluation of the blood smear). The evaluation of the cells includes:

  • The verification of the cell counts provided by the hematology analyzer
  • The differentiation of the leukocytes
  • The analysis of the morphology of the red blood cells and the thrombocytes
  • Consideration of cell aggregates and satellite phenomena
  • The search for toxic or degenerative cellular changes and infectious pathogens.

The smear is initially scanned with a low magnification (100 to 250-fold), for a reliable quantitative estimate of the cell counts. After finding the best suited area for evaluation individual cells should be investigated at higher magnification /10/.

15.13.3.1 Verification of the cell counts

Scanning the smear at low (100x) magnification a reliable quantitative estimate of the counts from the blood smear is attainable by the following calculations:

  • For normal erythrocyte count 600/field
  • For normal thrombocyte count 30–60/field; for a count of (400–1000) × 109/L, 75–125/field; for a count of (50–100) × 109/L, 25–30/field and with a count of (20–50) × 109/L, 10–15/field
  • For normal leukocyte count, 1–2/field; for a leukocyte count of (20–50) × 109/L, 5–6/field; for a leukocyte count of (50–100) × 109/L, 10–15/field; for a leukocyte count above 100 × 109/L, 20–30/field.

With the use of a 1000-fold magnification, only approximately one sixth of the cells is counted in each case. For the verification of the cell count, the mean value from 10 view fields should be used.

15.13.3.2 Leukocyte differentiation

The differentiation of the leukocytes is performed at a 400 to 1000-fold magnification. Destroyed or pyknotic cells are not included in the count. If, however, their fraction is large, this should be noted in the laboratory report. Smears from patients with chronic lymphocytic leukemia often contain a large number of smudged cells. In these cases a repeat smear on a blood sample to which albumin has been added should be made. In the laboratory results the fraction of individual leukocytes, in relation to 100 counted leukocytes, should be expressed in percent. If other nucleated cells (normoblasts, megaloblasts, megakaryocytes) or cell nuclei are present, these should be counted and their fraction should be expressed per 100 leukocytes /4/. Gumprecht Kernschatten are counted as lymphocytes. Morphological changes in the leukocytes must be characterized and interpreted. Refer to:

15.13.3.3 Platelet morphology

Thrombocyte evaluation is usually performed under 100x oil immersion objective lens (1,000 × magnification) /4/. A blood smear from a healthy individual usually shows 1 thrombocyte per 10 to 30 erythrocytes, or 7 to 21 thrombocytes per field at 1000 × magnification. Platelet satellitosis may cause falsely low automated thrombocyte counts and is detected by microscopy. The platelet morphology is of importance; giant platelets are seen in hereditary disorders and thrombocytopenia in cases with increased thrombocyte consumption.

Refer to:

15.13.3.4 Red blood cell morphology

Four features of red cells should be evaluated on a peripheral blood film: size, shape color and inclusions /1/.

Red cell size

In the thicker areas the cell size is underestimated and in the thin areas it is overestimated.

Red cell shape

Abnormalities in red cell shape are described by the term poikilocytosis. Red cell shape abnormalities are shown in:

15.13.3.5 Blood smear in infection

In addition to leukocytosis, a variety of morphologic changes in neutrophils may be seen in reactive states, which are collectively designated as toxic changes. The main changes are toxic granulation, Döhle bodies and vacuolization. Toxic granulation is present in two thirds of patients with sepsis, but lacks specificity for infectious states. In noninfectious reactive conditions toxic granulation may be also seen. Toxic vacuolization is a useful morphologic change during bacterial sepsis /11/.

The monocytes manifest nonspecific morphological changes.

The morphological changes of the lymphocytes in EBV infection and in cytomegaly are listed in Tab. 15.13-1 – Artifacts and morphological changes of leukocytes in the blood smear.

The blood smear is suitable only in rare cases, or not at all, as the primary diagnostic method for the identification of infections. A summary is shown in Tab. 15.13-4 – Morphological blood smear findings in infectious disease.

15.13.3.6 Blood smear in hypo- and hypersplenism

In hematology the terms hypersplenism and hyposplenism refer to changes in the blood cell count that are due to altered function of the spleen.

Normal splenic function consists of storing reticulocytes for maturation that are released into the circulation, the removal of cell particles and nuclear fragments, and the final configuration of erythrocytes. Immature cells that are released by the bone marrow are eliminated. Aged or damaged erythrocytes are taken up and degraded by the spleen. The enhanced degradation of erythrocytes in a hemolytic reaction leads to splenomegaly.

The spleen also maintains the thrombocyte count constant. In acute complete release of thrombocytes the increase in peripheral thrombocyte count is about 50 × 109/L.

Hyposplenism

This hematologic symptom may result from splenectomy, and from extensive immunological and lymphoproliferative diseases, such as hairy cell leukemia or amyloidosis of the spleen. Associated changes can be /12/:

  • Transient or long-time increased thrombocytosis
  • An abnormal blood smear: target cells, Howell-Jolly bodies, and red blood cells of irregular shape (spiculated cells). Additionally, nucleated red blood cells, immature granulocytes and megakaryocyte residues are seen in small numbers.

Hypersplenism

This term refers to a clinical symptom and not to a pathological or a histological entity. The spleen can be enlarged, or it may simply manifest hyper function. The latter results in cytopenia that can concern one or more hematopoietic cell lineages. The affected cell lineage manifests bone marrow proliferation, and can also express morphological changes or the interruption of maturation.

Changes that may be associated with hypersplenism are /12/:

  • Permanent thrombocytopenia
  • Spherocytic cells, teardrop cells, and schistocytes in the blood smear.

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24. De Braekeleer E, Douet-Guilbert N, Rowe D, Brown N, Morel F, Berthou C, et al. ABL1 fusion genes in hematological malignancies: a review. Eur J Haematol 2011; 86: 361–71.

25. Foa R, Chiaretti S. Philadelphia chromosome – positive acute lymphoblastic leukemia. N Engl J Med 2022; 386: 2399–2411.

15.14 Bone marrow examination

Torsten Haferlach

The laboratory diagnostic investigation of leukemia and lymphoma has evolved considerably. A large part of the progress that has been made is related, on the one hand, to the increasing understanding of the pathophysiological mechanisms of these diseases and, on the other hand, to the further development of laboratory techniques and instruments, including computer supported procedure for the analysis of the findings. Today, a prompt and comprehensive laboratory diagnostic investigation often leads to targeted therapy and includes best possible prognosis. At the same time the available methods should be utilized in the laboratory work flow in a targeted and cost effective way, in the sense of a step-wise diagnostic investigation. This presupposes strict algorithms with regard to sample collection and laboratory examinations.

With this swift further development of diagnostic investigation, it is difficult for the requesting physician to always ask his laboratory the appropriate question. Furthermore, it is a difficult challenge for the laboratories to keep abreast of the swift methodological developments regarding primary diagnosis and, similarly, the demonstration of minimal residual disease, which is increasingly required. For these reasons, an up-to-date orientation for the routine diagnostic investigation of leukemia and lymphoma is provided herein to the referring physician and the investigating laboratory.

15.14.1 Bone marrow specimens

Bone marrow histology and aspiration

A bone marrow evaluation is generally indicated on the basis of the clinical findings, and only upon detection of changes in the blood cell count or its composition. If there is evidence of disease of the hematological system, or of lymphoma, an investigation of the bone marrow aspirate, often supplemented with a biopsy/histology, is necessary /1234/.

Site of bone marrow aspiration

The posterior iliac crest (spina iliaca posterior superior) is the preferred site of specimen collection. The puncture of the iliac crest at this site is a technically more simple and less painful procedure than sternal puncture, which should be performed only in exceptional cases and which is only suitable for aspiration; in fact, this procedure should be considered obsolete. Prior pelvic radiation, punctio sicca, or a patient who is too obese constitute such exceptions. But in the latter case as well, or in mechanically ventilated patients in supine position, pelvic puncture can, in fact, be performed at the spina iliaca anterior superior or the anterior iliac crest. Here, the os ilium at the spina iliaca posterior superior is at its widest (approximately 3 cm) so that, injuries to vital organs or to large vessels are largely ruled out if the correct technique is applied (Fig. 15.14-1 – Transverse section at the level of the biopsy site).

Whenever possible, the procedure and its associated risks of complications are explained to the patient on the previous day. The puncture can also be performed on an out-patient basis on patients at risk of bleeding, but in such cases a sufficiently long follow-up period of observation must be assured. In special cases, the administration of banked thrombocytes must be considered prior to puncture, in order to achieve thrombocyte counts of above 20 × 109/L. Premedication with tranquilizers is only necessary for extremely sensitive patients, while in children the use of a short anesthesia may be recommended, following discussion with the anesthetist. Puncture is possible in the supine or the abdominal position; the author recommends the abdominal position since the patient is, thereby, stable and safe, even with the use of considerable exertion, and does not have to be stabilized further.

Bone marrow biopsy

In patients without leukemia, or with bone marrow aspirates containing very few cells, a biopsy for histology, in addition to the aspiration, is necessary. In myeloproliferative disease and chronic lymphocytic leukemia, as well as for the staging of lymphoma, bone marrow histology is always required.

In order to obtain artifact-free tissue, the histology should be performed prior to aspiration. In the abdominal position, a minimum of 10 mL of local anesthetic is administered following thorough disinfection and the sterile covering of the skin to the periosteum of the spina iliaca posterior superior. The time required to achieve satisfactory anesthesia is at least 5 minutes. The original sternal puncture needle (to be used at the pelvis without the spacer) and a histology needle (e.g., the so-called Jamshidi needle) are to be used for the puncture (Fig. 15.14-2 – Jamshidi needle in comparison with a usual puncture needle). Disposable instruments are highly suitable.

In children, a short anesthesia is, in some cases, to be recommended, following discussion with the anesthetist.

Bone marrow histology

An 8-gauge needle is preferred; in young patients with strong bones, the 11-gauge needle has proven successful. For particularly obese patients, a longer needle is available. The Jamshidi needle is placed on the middle of the posterior iliac crest with a mandrel and, following removal of the mandrel, is inserted through the bony cortex in the direction of the spina iliaca anterior, which is usually palpable. In this way, a biopsy core length of up to 4 cm is possible, allowing for a representative assessment of the bone marrow. Furthermore, a large biopsy permits easier rotation and better adhesion of the punched bone marrow cylinder in the canula. Once the cylinder has been obtained, imprint smears can be made if necessary and, depending upon the planned embedding method, the biopsy is fixed according to the instructions of each laboratory.

If, in cases of punctio sicca and non leukemic disease, material for the cytogenetic analysis cannot be aspired, a punch cylinder can also be placed directly following sampling in a physiological saline solution containing heparin. The tube is then dispatched, together with the punch biopsy, to the genetics laboratory. In these cases, the markers of immunophenotyping can be performed with the histology of a second punch biopsy following paraffin embedding.

Bone marrow aspiration

The bone marrow aspiration at the iliac crest follows the sampling of the biopsy material. Usually, a Klima and Rosegger puncture needle without arresting disc is used. Here as well, disposable needles are preferred, due to the fact that they are sharper as well as for hygienic reasons. The bone marrow puncture is made through already existing skin incision, approximately 1 cm from the histology biopsy site and at an angle to the direction of the biopsy. Prior to aspiration the patient is informed that short-lived pain which, in any case, cannot be prevented with the careful use of a local anesthetic will be felt. Aspiration is made quickly and strongly with a full stroke of a 10 mL syringe. Thereby, 2–3 mL of bone marrow suffice for the first EDTA sample for further cytomorphological analysis, in order to avoid dilution with peripheral blood. A second and a third syringe, with heparin as anticoagulant, can then be withdrawn, up to 5 or 10 mL in each case. If all the samples are taken from a single site, the last aspirates may manifest a different cell composition than the first one, due to increasing dilution with blood. If the aspiration is unsatisfactory, the position of the puncture needle must be altered by rotating under suction, or by means of a repeated puncture. In punctio sicca the rotating of the needle in the bone while continuing to aspirate often does help to achieve a successful sampling. If the puncture is still unsuccessful the other side as well, or the anterior iliac crest, can be punctured following appropriate anesthesia. A sternal puncture (ultima ratio) is also possible, but in such a case a depth stop must definitely be used. In infants, the tibial crest below the attachment of the M. quadriceps can also be considered for puncture in exceptional cases.

Following the application of an adhesive bandage, the puncture site is compressed by lying on a sand bag. The site is monitored at least 30 minutes later for bleeding.

Sample distribution

Aspirates for the following analyses are taken routinely, with the additives for the syringes in each case in brackets:

  • Aspiration cytology: 0.5 mL EDTA ad 2–3 mL bone marrow aspirate (maximum)
  • Immunophenotyping: 0.5 mL heparin or EDTA ad 5 mL bone marrow aspirate (maximum)
  • Cytogenetics: 0.5 mL heparin ad 5–10 mL bone marrow aspirate (maximum)
  • Molecular genetics: 0.5 mL heparin or EDTA ad 5 mL bone marrow aspirate (maximum).

The aspiration cytology with EDTA always comes first, since potential heparin contamination from the cone of the syringe can cause serious staining artifacts in the Pappenheim staining.

All syringes must be marked prior to puncture with the patient’s name, the material, and also with the type of anticoagulant, and then immediately sent to the corresponding laboratories. As a general rule, the smaller the sampling volume, the less the contamination with peripheral blood, and the more representative is the aspired bone marrow. If heparinized samples are used for immunocytolgy, EDTA smears that have not yet been stained are to be provided as well. Unlimited sample quality become increasingly important as the level of difficulty of the diagnostic issue increases (e.g., for the monitoring of minimal residual disease). This signifies that for cytomorphology, the smear should be made, if at all possible, within 3 hours of aspiration, and then sufficiently (for 30 minutes) air-dried, before it is packaged for dispatch. These also still allow for gut staining results over a number of days. The material for cytogenetics, immunophenotyping and molecular genetic analytic procedures should reach the investigating laboratory within 24 hours. In summary, the proposed algorithm for the sampling of bone marrow and its dispatch to the individual laboratories is presented in Tab. 15.14-1 – Methods for investigation of bone marrow in primary diagnosis or suspected diagnosis of leukemia and lymphoma.

The biopsy cylinder should be dispatched within one day, in the fixing solution recommended by the laboratory.

15.14.2 Processing and analysis of blood smear and bone marrow aspirate

Analysis of peripheral blood

If peripheral blood is examined, the blood count, including thrombocytes, MCV and MCH, reticulocytes, and the differential, are indispensable basic tests. In hematological diseases in particular, interfering factors can be present in EDTA blood, and these interfere with the widely used automated measurements of the blood count, leading to erroneous results. In consequence, a few examples:

  • Leukocytes of over 100 × 109/L manifest, in many instruments, falsely high hemoglobin levels (10–20 g/L too high)
  • Fragmentocytes e.g., in disseminated intravascular coagulation or the splitting of cytoplasma from blasts, e.g. in AML M4 or M5 can lead to falsely high thrombocyte counts (up to 30 × 109/L too high)
  • In all cases of reduced platelet counts as determined by automated counting, thrombocyte agglutinins or EDTA-induced pseudo thrombocytopenia should be excluded by the investigation of a blood smear
  • In almost all cases, the differential count is compiled primarily by automated hematological systems. Attention should be paid to the fact that the instruments are focused on the differentiation of normal blood from pathological complete blood counts. In the presence of a pathological cell population, the analyzer documents a signal, which of necessity leads to a blood smear and microscopic analysis. Since hematology analyzers produce more or less reliable results, the microscopic analysis of peripheral blood is an absolute requirement in every case of suspicion of hematological disease or for its monitoring /6/. For preparation and microscopy of peripheral blood refer to Section 15.13 – Blood smear examination.

Processing and assessment of the bone marrow aspirate

The examination of the bone marrow is part and parcel of the basic diagnostic investigation of hematological disease. There are different opinions as to whether or not aspiration cytology alone is sufficient in acute leukemia, or whether, rather, supplementary biopsy is indicated. For the assessment of leukemic cells in acute leukemia, good quality blood and bone marrow smears, at a minimum, are most suitable. These should be sufficiently dried (more than 30 minutes) before dispatch and staining. The additional assessment of a bone marrow biopsy is certainly indicated in cases of an unsuccessful aspiration (punctio sicca); it allows for immunohistological analysis as well. In myeloproliferative disease, the bone marrow histology plays an important role at the time of diagnosis because, among other things, of the necessary evaluation of the degree of fibrosis.

The diagnostic value is crucially dependent upon the subsequent processing of the collected material. The best smears for semi quantitative estimate of cell density are obtained when a cover slide is placed on the bone marrow fragments on the glass slide (following filtration or collected from a watch glass), and then the sample is smeared with the cover slide as a single stroking motion using moderate pressure to thin out clumps (Fig. 15.14-3 – Preparation of a smear of bone marrow fragments).

The bone marrow preparations must then be air-dried for 30–60 minutes. The subsequent Pappenheim or May-Grünwald-Giemsa staining procedures represent the standard methods for blood and bone marrow imaging. Bone marrow smears that are suitable for assessment contain bone marrow fragments at the center of the smear, and mixed bone marrow and blood at the periphery. Following careful observation of the entire preparation at low magnification, the cellularity and different distribution patterns, e.g., chronic lymphatic leukemia or lymphoma, even with nodular infiltrates, or the detection of tumor cells can be assessed. Thereon, a single analysis of at least 200–500 cells from 2 representative sections of the smear is performed. The distribution of bone marrow cells from healthy individuals is shown in Tab. 15.14-2 – Percentage of bone marrow cells in healthy individuals

The additional examination of at least two smears is performed according to the following criteria:

  • Assessment of the cell density: a decrease can be a function of the sampling and the smearing procedures. A true decrease in cellularity may only be assumed if marrow fragments with fat and stromal cells are detectable. The age of the patient is to be taken into consideration; cellularity decreases physiologically with age.
  • Assessment of the erythropoiesis to granulopoiesis ratio (EP:GP). The normal ratio is approximately 1 : 3 to 4. The cytological examination of the smear only allows for a relative quantitative assessment. Additionally, the distribution of the different stages of maturity, including in particular the percentage of blasts, as well as changes in the cytoplasm and cell nuclei and signs of dysplasia. The eosinophil, basophil and monocyte fractions are specified. The quantitative and qualitative assessment of the megakaryocyte number, and the distribution and fine structure of the lymphocytes and the plasma and reticular cells are necessary.
  • Assessment of absolute cell density and possible heterogeneous cell distribution in medullary cavities, histological slices must be evaluated, likewise in an age-dependent manner. Iron storage in the reticular cells as well as the quantification of sideroblasts and ringed sideroblasts (stained with Berlin blue) can be better performed cytomorphologically than histologically.

For the demonstration of blasts, or with regard to the question of myelodysplastic syndrome, the following cytochemical staining procedures are performed on blood and bone marrow smears for further morphological differentiation:

  • The myeloperoxidase staining is obligatory, and, possibly, for histology, chloracetate esterase or Sudan Black as a hint regarding affiliation with the granulocyte lineage, as well as non-specific esterases. The latter provides an indication of affiliation with the monocyte lineage (Tab. 15.14-1 – Methods for investigation of bone marrow in primary diagnosis or suspected diagnosis of leukemia and lymphoma).
  • The PAS staining as an indication of affiliation with the lymphatic and erythrocyte series in AML-M6, as well as acid phosphatase as a hint of the T cell nature of an acute lymphatic leukemia, are optional and, today, no longer necessary because of the use of immunophenotyping.

With the obligatory use of immunophenotyping, fluorescence in situ hybridization (FISH) or molecular genetics (PCR), the following tests are not applied due to deficient sensitivity and specificity:

  • Alkaline leukocyte-neutrophil phosphatase (ANP); previously in connection with the exclusion of CML (today BCR-ABL)
  • Tartrate resistant acid phosphatase for the diagnosis of hairy cell leukemia (today CD103 as APAAP or immunophenotyping/immunohistology)
  • Terminal deoxynucleotidyl transferase (TdT) on the smear in acute lymphocytic leukemia (today immunophenotyping).

15.14.3 Processing and assessment of bone marrow histology

Bone marrow biopsy allows not only for an assessment of the individual cellular components of the bone marrow but also, for a true quantitative assessment of the cell content, as well as for an analysis of the topographic distribution of the cells and their iron content, and statements concerning the bone marrow stroma and osseous changes. The significance of the bone marrow biopsy in the diagnostic investigation of hematological disease is closely related to the particular clinical issue. Thus, bone marrow histology indispensable to the diagnosis in, e.g., primary myelofibrosis and in aplastic anemia. In other diseases, such as, e.g., acute leukemia, it can provide valuable supplementary information on cytomorphology. Paraffin embedding employed in the diagnosis of suspected leukemia/lymphoma allows a precise diagnostic expert appraisal of the tissue /157/.

The following standard stains are used for all biopsies:

  • Giemsa for cellular details
  • PAS for cellular details and for the demonstration of memory cells
  • Gomori silver for the display of filaments
  • Berlin blue for evaluation of the iron content
  • Naphthol-AS-D chloroacetate esterase reaction for the display of granulopoiesis and of mast cells.

Immunohistochemistry is used, in addition, for the display and characterization of the cells. The following markers are routinely employed for the investigation of hematological disorders:

  • Myeloperoxidase and CD15 for granulopoiesis
  • CD61 for megakaryopoiesis
  • Glycophorin A for erythropoiesis
  • CD34 for progenitor cells.
  • CD68 for cells the monocyte-macrophage system.
  • CD117 for mast cells.

Furthermore, immunohistological investigations are employed, particularly in the characterization of lymphatic aggregates, in hairy cell leukemia, in the sub typing of infiltrates of malignant lymphoma, e.g., CD19, CD20, CD3, CD103, CD138, and in the identification of metastases of non-marrow tumor cells, e.g., cytokeratin and hormone receptors.

References

1. Mufti GJ, Flandrin G, Schäfer HE, Sandberg AA, Kanfer EJ: An Atlas of Malignant Haematology. London; Verlag Martin Dunitz, 1996.

2. Löffler H, Rastetter J, Haferlach T. Atlas der klinischen Hämatologie, 6. Aufl. Heidelberg; Springer, 2004.

3. Theml H, Diem H, Haferlach T. Taschenatlas der Hämatologie, 5. Aufl. Stuttgart; Thieme, 2002.

4. Haferlach T, Labordiagnostik bei Leukämien und Lymphomen, 2. Aufl.. Bremen; UNI-MED, 2007.

5. Bain JB, Clark DM, Lampert IA, Koch S: Knochenmarkpathologie. Atlas und Lehrbuch. Blackwell; Berlin, 2000.

6. Haferlach T, Haferlach C, Kern W, Labordiagnostik in der Hämatologie, Köln, Deutscher Ärzte-Verlag, 2011

7. Swerdlow SH, Campo E, Harris NL, Jaffe ES. WHO Classification of tumours of haematopoietic and lymphoid tissues. Lyon, International Agency for Research on Cancer (IARC), 2008.

8. Löffler H, Haferlach T. Hämatologische Erkrankungen, Heidelberg, Springer, 2010.

15.15 Acute leukemias

Torsten Haferlach

In acute leukemia, the diagnostic possibilities have been extended in a fundamental manner. On the one hand, numerous methods of diagnostic investigation have been established and, on the other hand, insights from correlations of biological parameters and clinical course have led in many cases to diagnostic findings becoming basic prerequisites for the initiation of appropriate therapy. Furthermore, important prognostic biomarkers are provided by these diagnostic investigations. These reasons suffice, therefore, in order that the diagnostic investigation be carried out in a comprehensive and, at the same time, goal-oriented manner. The insights have also led to new classifications /1/. Thus, classifications according to morphology (FAB classification), cytogenetics (WHO classification to a certain extent) and, in addition, results from immunophenotyping (similar to the EGIL classification), are interconnected and must be taken into consideration /23, 4, 56/. This leads to potential algorithms in the diagnostic investigation of leukemia, not only for the primary investigations but also for the follow-up examinations for the monitoring of treatment (minimal residual disease, MRD). At the same time, much new data, especially from molecular methods such as, gene expression analysis and next-generation sequencing (NGS), are to be expected in the short term. These will, precisely in the field of hematology, supplement current diagnostic procedures in the short term and, in some cases, perhaps even completely replace them.

Sample collection

The parallel investigation of blood and bone marrow should be striven for in all cases of suspected acute leukemia. For the creation of smears for cytomorphology and cytochemistry (at least 6–8 smears should be prepared). If alkaline phosphatase anti-alkaline phosphatase (APAAP) or fluorescence in situ hybridization (FISH) are to be determined, EDTA or citrate must be used as anticoagulant. The material that has been rendered incoagulable in this manner can also be utilized, as required, for immunophenotyping and for molecular analysis (e.g., with the polymerase chain reaction). The last two methods mentioned, as well as FISH, can also be implemented, as an alternative, with heparin blood or bone marrow. Heparin must be used as the anticoagulant for cytogenetics, since only the vital cells that go into metaphase are available for the chromosome analysis. Thus, a current diagnostic investigation of leukemia requires, optimally, EDTA and heparin blood and bone marrow.

In total, 5–20 mL of bone marrow should be collected. In addition, in acute leukemia, sampling for histology by means of an iliac crest puncture, must be taken into consideration as a function of the suspected diagnosis /7/. An orientation is provided in Tab. 15.15-1 – Methods for the diagnostic investigation of acute leukemia.

Classification

For the first diagnosis and classification of acute leukemia, the morphological criteria of the FAB classification, die EGIL classification and the WHO classification as well, are still employed in parallel today on account of their clinical relevance and feasibility /12, 3, 4, 56/. Above and beyond that, ALL is classified in an even more precise manner according to immunophenotyping and cytogenetics/molecular genetics. This renders the orientation complicated at the present time, but in the midterm it will contribute, through better understanding of the biology of individual leukemia subgroups, to a more specific diagnosis and, finally, to targeted treatment decisions as well. To make a diagnosis is, thus, not always to be considered the same as classification in the sense of the WHO document.

15.15.1 Diagnostic investigation of acute leukemia

15.15.1.1 Acute myeloid leukemia (AML)

Depending upon the leukemia subtype, the methods and classification models are of special importance in the primary diagnostic investigation and in follow-up investigations. According to the FAB and WHO classifications, the bone marrow smear should be analyzed. The counting of 200–500 bone marrow cells is recommended. According to the FAB classification, the blast fraction should be greater than 30% of the nucleated cells of the bone marrow; WHO has set the limiting value at ≥ 20%, and this is generally considered to be valid. Furthermore, according to WHO, AML with specific genetic aberrations [i.e., AML with t(15;17) or t(8;21)or inv(16) or 11q23] are also ruled out with this limiting value.

One also speaks of AML if the corresponding chromosomal changes are demonstrated, and the bone marrow blast fraction is under 20%. The cases are, however, very rare. The algorithm for the FAB classification, which is further helpful in daily clinical practice, and the FAB AML subgroups, are shown in:

For the diagnosis of ALL, it should initially be demonstrated purely cytomorphologically, as well as with cytochemistry (myeloperoxidase below 3%, nonspecific esterase negative) that AML L-M1–M6 is not present. Immunophenotyping is required for further goal-oriented classification; it separates, on the one hand, AML-M0 and AML-M7 according to FAB as well as bilineal and biphenotypic acute leukemia according to EGIL and WHO from the classical ALL of B and T lineage. These are then divided into additional subgroups based upon their marker profiles. See

First Assessment of the bone marrow in AML and ALL

The assessment includes:

  • Cell content on the smear, small fragments present
  • Description of the blast morphology according to size, nucleus-to-plasma ratio, cytoplasm color, inclusions, Auer rods, pseudo Chediak bodies
  • Fraction of promyelocytes, myelocytes and metamyelocytes, rods, segmented, eosinophils, abnormal eosinophils, basophils, monocytes
  • Fraction of nucleated cells of the erythrocyte series, by stage of maturation
  • Fraction of mature lymphocytes, plasma cells
  • If necessary, available tissue mast cells
  • Number and form of the megakaryocytes.

Extended definition of the blasts

It has often been attempted to subdivide the myeloblasts further according to shape and, in particular, to the number of cytoplasmic granules.

From the current standpoint it seems to make sense to proceed according to the following classification:

  • Type I blasts: myeloblasts with immature cytoplasm, agranular, the nucleus may contain a few nucleoli
  • Type II blasts: similar to type I, but with 20 (other classifications 15) azurophilic granules in the cytoplasm.

The subtype that was previously differentiated as a type III blast, containing more than 15 or 20 granules, should be omitted from routine use. It should be remembered that the reproducibility of this blast classification is extremely limited, and is lacking in clinical or classificatory relevance. It can, therefore, be ignored in most cases in the classification of AML and MDS. Furthermore, according to morphology and in particular in the absence of cytochemistry as well as (if necessary) with the assistance of APAAP or immunophenotyping it should be possible to subdivide the blasts as follows:

  • Atypical promyelocytes in AML-M3 and M3 variants with translocation (15;17) and the demonstration of PML-RARA
  • Monoblasts and promonoblasts, especially following nonspecific esterase reaction in AML-M4, AML-M5a und AML-M5b
  • Magakaryoblasts following measurement of CD41 or CD61 in AML-M7 with APAAP or immunophenotyping.

Proerythroblasts are not added to the true blasts, since it is difficult to ascribe them to the normal or the malignant populations.

Assessment of dysplasia

Apart from the blast fraction – especially in AML – which makes possible a demarcation relative to myelodysplasia, the detection of dysplastic changes in the three cell lineages has also become important in AML, according to WHO 2008 (WHO classification, Tab. 15.15-3 – WHO classification of AML). The Goasguen and Bennett criteria are to be used for the assessment of dysplasia in AML according to WHO /18/:

Dysgranulopoiesis

≥ 50% of the segmented cells (at least 10) are agranular or hypogranular, or

  • manifest pseudo-Pelger-Huet changes or
  • peroxidase defect
  • More than 100 cells should be assessed

Dyserythropoiesis

≥ 50% of at least 25 nucleated cells of the erythropoietic series manifest one of the following morphological abnormalities:

  • Karyorrhexis
  • Megaloblastoid changes
  • Multinuclearity
  • Nuclear fragmentation

Dysmegakaryopoiesis

≥ 50% of at least 6 megakaryocytes manifest one of the following abnormalities:

  • Micromegakaryocytes
  • Multiple individual nuclei
  • Large mononuclear core form

In the assessment of dysplasia, attention should be paid in particular to the fact that in AML, at least 50% of all cells of a given series have to manifest one or more of the above-mentioned changes. Only in such cases is the series considered to be dysplastic. This is in contrast to the assessment of the same dysplasia criteria in myelodysplastic syndromes, in which only 10% of the observed cells of each series has to fulfill these criteria, in order that the condition be considered to be dysplasia.

In AML, dysplasia according to WHO 2008 is required, because with the demonstration of 2 or 3 dysplastic lines a patient could be allocated to the subgroup “AML with myelodysplastic-like changes.”

Morphological classification of AML according to the FAB criteria

The FAB classification is based upon the stage of maturity of the blasts, the cell lineage affiliation and the number of blasts, as well as the assessment of the cytochemistry, particularly myeloperoxidase (MPO) and the nonspecific esterase (NSE) reaction in the bone marrow smear. Even if they, in fact, should no longer be used in the times of the WHO 2008 classification, they continue to be of practical relevance from the clinical point of view (one cannot wait 7–10 days for a complete diagnosis according to the WHO criteria).

The maturation of the blasts and their respective differentiation with regard to normal and to abnormal promyelocytes (AML-M3) can be assessed according to Tab. 15.15-4 – Differentiation of the blasts from abnormal promyelocytes and from normal promyelocytes. The further sub-classification of AML, based upon morphological properties according to FAB, is shown in Tab. 15.15-2 – Previous FAB classification of AML, can be performed as initial diagnostic step before WHO classification.

Noteworthy morphological aspects with regard to the sub-entities

AML-M0: Differential diagnostically, biphenotypic acute leukemia (EGIL classification), and also ALL, in particular with t(9;22), as well as AML-M5a, come into question. Immunophenotyping is required for the definitive establishment of AML-M0.

AML-M1: Because of the limited volume of cytoplasm and the low degree of maturity one often sees, particularly in the peripheral blood, so-called pseudo-nucleoli, corresponding to the Golgi apparatus that appears in the nucleus. They should not be confused with giant nucleoli. There exists a certain correlation between these morphological findings and a mutation in the NPM1 gene. The maturation of granulopoiesis to the stage of the promyelocyte or beyond is below 10%.

AML-M2: The maturation of granulopoiesis to the stage of the promyelocyte or beyond is above 10%. One finds a certain correlation between this FAB subtype with AML with translocation t(8;21). Of these cases of genetically defined AML, 90% manifest an M2 FAB subtype, and 10% an M1 subtype. The AML-M2 cases with t(8;21) often have type II blasts, or cells that are close to the promyelocyte (previously so-called type III blasts). According to WHO, however, AML is always present, even if the fraction of unambiguous blasts in the strict sense were below 20% (Tab. 15.15-3 – WHO classification of AML 2008)). This AML subtype often manifests long needle-like Auer rods, dysgranulopoiesis, and mild eosinophilia.

AML-M3: Most prominent are abnormal promyelocytes, often with bundles of Auer rods, so-called faggot cells. The MPO is always more than 90% strongly positive. Frequent leukopenia in the peripheral blood.

AML-M3 variant(v): the nuclei are bi-lobular, and can easily be confused with monocytes. Granules are hardly distinguishable and Auer rods occur less commonly than in M3. The MPO is strongly positive here as well, while the NSE is negative. Frequent leucocytosis in the peripheral blood.

AML-M4: Composite of myeloid and monocytic blasts, with bone marrow MPO above 3% and NSE above 20%. The monocytic blast fraction lies, thereby, between 20% and 80%.

AML-M4Eo: Blasts as in M4 and similar cytochemistry but, apart from that, unambiguous so-called pathological eosinophils with distinct dark granules. In contrast to normal eosinophils, these eosinophils are positive with chloracetate esterase. Proof for this sub-type is found in the demonstration of an inversion in chromosome 16 or the molecular correlate CBFB-MYH11.

AML-M5a: Mostly a monomorphic blast population with relatively blue cytoplasm with a net-like internal structure. Very marked NSE in over 80% of the blasts. Differential diagnostically, M0 ALL (also Burkitt type) and dedifferentiated multiple myeloma must be taken into consideration.

AML-M5b: With regard to the maturation of the mono blastic cells, this mature sub-type of mono blastic AML is easier to diagnose in the blood than in the bone marrrow. Here as well, the NSE is moderately to strongly positive in over 80% of the cases. This is more pronounced in the bone marrow than in the blood.

In AML-M5a and AML-M5b, gingiva hyperplasia or skin infiltrates, caused by the blasts, are frequently present at the time of diagnosis.

AML-M6: More than 50% of all nucleated cells belong to the erythrocyte series. Of the other, non-erythropoietic cells, at least 20–30% are blasts (according to FAB). Of more historical interest in the relevance of the strong PAS positivity in the immature cells erythrocytes. This can provide support for the M6 diagnosis, since normal erythropoiesis does not manifest this reaction, and PAS is not longer absolutely necessary in the diagnostic investigation of AML, particularly of AML-M6.

AML-M7: With this sub-type, which is observed far more frequently in children than in adults, bone marrow fibrosis with punctio sicca often occurs. Even if the blasts have a certain specific morphology, with cytoplasmic evaginations, and look, to a certain extent, like megakaryocytes, the diagnosis cannot be made with certitude based solely on cytomorphological criteria. In suspected AML-M7, immunophenotyping or APAAP with at least CD41 or CD61 must be performed.

WHO classification of acute myeloid leukemia (AML)

Within the framework of a purely cytomorphological differentiation between AML and MDS, as well as with respect to bilineal acute leukemia and ALL, it has again initially been specified in the 2008 WHO classification of AML (Tab. 15.15-3 – WHO classification of AML 2008) that with greater than 20% blasts in the bone marrow, one is dealing with AML and thereby, the category of RAEB-T, which belongs to myelodysplasia, is omitted /1/.

As the most important first step towards a biological classification of clinical symptomatology, the WHO has consolidated four genetically defined sub-types of AML with specific balanced trans locations into as a single group; this as a first stage in the sense of a hierarchy. For the four genetically defined groups named in Tab. 15.15-3 – WHO classification of AML (2008), the term AML is also valid, to a certain extent, if the number of bone marrow blasts is below 20%. The integration into MDS is, thereby, ruled out.

As the second step in the WHO classification, a sub-group that was defined, inter alia, on the basis of morphological criteria was exposed – namely, AML with multilineal dysplasia. Thereby, the dysplastic changes are classified according to the criteria of Goasguen and Bennett /8/.

The WHO considers multilineal to refer to the demonstration of dysplasia in 2 or 3 cell lineages in at least 50% of the analyzed bone marrow cells. In so doing, a far higher limiting value for AML than for MDS as evidence for dysplasia is established; for the latter, only 10% of the cells must display dysplastic properties. This group also includes patients who, at the same time, have only cytogenic changes, such as those found in MDS, as well as those who, in their previous medical history, had MDS or MDS/MPN overlap. This AML category, thereby defined in a miscellaneous manner according to WHO, must be further evaluated with regard to its autonomy in prospective clinical studies. Various analyses suggest that although, admittedly, the morphological differences can be captured and that they partially correlate with certain genetic sub-groups, an independent prognostic relevance remains to be demonstrated.

At the third stage of the WHO hierarchy, subgroups are identified that can only be defined while taking into account the patient’s medical history; here, therapy-associated MDS and AML in particular are included. The further sub-classification of these AML groups according to the type of previous therapy (according to alkylating agents, topoisomerase II inhibitors, other medication or radiotherapy) is absolutely necessary for the description of the biology and the clinical course. Not only clinical differences but also, and in particular, cytogenetic and molecular genetic differences stand out in these sub-groups, the further identification and biological description of which, via this WHO proposal, is made possible in a meaningful manner. AML following previous MDS or MPS is also captured in this sub-group according to WHO.

Only at the fourth stage of the WHO AML classification are the old, purely morphological or immunophenotypic sub-groups according to the FAB criteria shown referred to. Thereby, further sub-entities are added: Acute basophilic leukemia, acute pan myelosis with fibrosis, and myeloid sarcoma or chloroma. It remains to be seen whether these very rare AML sub-groups emerge more clearly with their compilation according to the WHO criteria. To a certain extent, they can be diagnosed only when the histological examination is taken into consideration.

Biphenotypic leukemia, which is also now classified according to WHO, is dealt with in Section 15.15 – Acute leukemias. They should be defined on the basis of the immunophenotype, according to the criteria of the EGIL group /6/, and they are classified between acute lymphatic and acute myeloid leukemia.

In this group, undifferentiated acute leukemia with bilineal properties and the genetic changes t(9;22) or t(4;11) is accounted for. An algorithm for the primary diagnosis of AML and follow up investigations is presented in Fig. 15.15-2 – Standard diagnostic investigation of acute lymphatic leukemia.

Cytogenetics, FISH and molecular genetics in leukemia.

In leukemia, characteristic chromosomal aberrations, which define autonomous entities with typical morphology and characteristic clinical course, are recognized. They are also increasingly linked with direct therapeutic consequences. Thus, in the new WHO classification of acute myeloid leukemia, specific chromosomal changes have been incorporated as decisive classification criteria. Apart from classical cytogenetics – the analysis of chromosomal changes – fluorescence in situ hybridization (FISH) and, particularly, molecular techniques such as the polymerase chain reaction (PCR), increasingly and independently play an additional very important role. This applies to the diagnostic investigation and classification of leukemia, as well as to the determination of the therapeutic response (minimal residual disease, MRD). With regard to cytogenetics and molecular genetics in leukemia, see the review literature /79/.

Refer to

15.15.1.2 Acute lymphocytic leukemia (ALL)

Cytomorphology, cytochemistry (Pappenheim, myeloperoxidase, previously also PAS) and especially immunophenotyping, chromosome analysis and molecular genetics are responsible for the primary diagnostic investigation of ALL (Fig. 15.15-2 – Standard diagnostic investigation of acute lymphocytic leukemia). The therapy is essentially influenced by the findings from immunophenotyping and cytogenetics/ molecular genetics. Apart from the distinction between B and T cell lineages, many additional aspects are to be considered. Above and beyond that numerous chromosomal aberrations, which are of prognostic and therapeutic relevance, have been described. On the one hand ALL is classified based upon karyotype into so-called ploidy groups (i.e., according to the number of chromosomes) and on the other hand the classification is made according to structural aberrations. The most common translocation in adult ALL is that of t(9;22)(q34;q11). It is associated with an unfavorable prognosis and necessitates targeted therapy.

With the introduction of (e.g., Imatinib) a specific tyrosine kinase inhibitor, the detection of the Philadelphia chromosome and its molecular correlate, the BCR-ABL rearrangement, has taken on a high level of therapeutic relevance. In a small proportion of ALL, so-called crytic BCR-ABL rearrangements, which are not detectable with chromosome analysis, are present. Therefore, screening with FISH or RT-PCR should be performed in all ALL B-lineage cases, in view of the important therapeutic consequences /710/. Within the framework of scientific analyses, assessment of the therapeutic response is provided by real time PCR, either with leukemia-specific fusion transcripts or with patient-specific immunoglobulin or T-cell receptor rearrangements. Crucial progress in therapy control may be expected to emerge from these data. Initial results in children are very promising /11/.

The classification of ALL according to purely morphological criteria, as was the case with the FAB classification (L1, L2, L3), no longer plays a role. If need be, a certain reminiscence is rendered by the fact that the subtype of mature B-ALL with t(8;14) usually corresponds to the so-called (FAB) L3 sub-type. The cells then usually manifest a very dark blue cytoplasm and a many vacuoles. However, in this case as well, the morphology alone cannot be considered to be sufficient, due to the immense therapeutic consequences. It absolutely must be supplemented by immunophenotyping and cytogenetics or molecular genetics.

15.15.2 Significance of the bone marrow histology in acute leukemia

In contrast to the significance of the bone marrow histology in chronic leukemia and in the staging of lymphoma, its relevance in acute leukemia is, rather, secondary. Individual aspects speak, apart from the previously described methods of smear cytomorphology and cytochemistry, immunophenotyping and cytogenetics and molecular genetics, for the supplementary implementation of a bone marrow punch in:

  • Punctio sicca and leukopenia or a lack of blasts in the peripheral blood
  • Marked fibrosis (e.g., AML-M7) or packed marrow, which then similarly leads to aspiration punctio sicca
  • Determination of angiogenesis is corresponding therapeutic studies with angiogenesis inhibitors
  • Differential diagnosis of severe aplastic anemia and hypo cellular MDS, or in suspicion of bone marrow tumor cell infiltration
  • Staging and suspected diagnosis of lymphoma, in order to be able to capture the bone marrow involvement with certitude. In this case the bone marrow punch, including immunohistology, and immunophenotyping from the bone marrow aspirate, are complementary but not always concordant, so that the most correct assertion can only be made with the use of both methods.

Therefore, in the diagnosis of acute leukemia, it is imperative to consider a bone marrow biopsy in every individual case. Admittedly, this can certainly usually be omitted but in certain cases, where necessary, it should be performed at the same time as the other samplings, in order to avoid a second procedure. For further current literature on the diagnostic investigation of leukemia, see Ref. /1213/.

References

1. Swerdlow SH, Campo E, Poleri SA, Harris NL, Stein H, Siebert L, et al., The 2016 revision of the World health Organization classification of lymphoid neoplasms. Blood 2016; 127: 2375–90.

2. Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, Le Beau M, et al. The 2016 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia. Blood 2016. doi: 10.1182/blood2016-03-643544.

3. Bennett JM, Catovsky D, Daniel MT, et al. Proposed evised criteria for the classification of acute myeloid leukemia. A report of the French-American-British Cooperative Group. Ann Intern Med 1985; 103: 620–5.

4. Bennett JM, Catovsky D, Daniel MT, et al. Criteria for the diagnosis of acute leukemia of megakaryocyte lineage (M7). Ann Intern Med 1985; 103: 460–2.

5. Bennett JM, Catovsky D, Daniel MT, et al. Proposal for the recognition of minimally differentiated acute myeloid leukemia (AML-M0). Br J Haematol 1991; 78: 325–9.

6. Béné M-C, Castoldi G, Knapp W, et al. Proposals for the immunological classification of acute leukemias. European Group for the Immu-nological Characterization of Leukemias (EGIL). Leukemia 1995; 9: 1783–6.

7. Löffler H, Haferlach T, Schoch C. WHO-Klassifikation der akuten myeloischen Leukämien (AML) und der myelodysplastischen Syndrome (MDS) – Hämatologische Erkrankungen – Ein diagnostisches Handbuch. DMW 2002; 127: 447–5. Heidelberg; Springer; 2010.

8. Goasguen JE, Matsuo T, Cox C, Bennett JM. Evaluation of the dysmyelopoiesis in 336 patients with de novo acute myeloid leukemia: Major importance of dysgranulopoiesis for remission and survival. Leukemia 1992; 6: 520–5.

9. Haferlach T. (Hrsg.), Ludwig W-D, Haferlach T, Schoch C. Labordiagnostik bei Leukämien und Lymphomen. Classification of acute leukemias. Bremen, Unimed 2007.

10. Schoch C, Schnittger S, Bursch S, et al. Comparison of chromosome banding analysis, interphase- and hypermetaphase-FISH, qualitative and quantitative PCR for diagnosis and for follow-up in chronic myeloid leukemia: A study on 350 cases. Leukemia 2002; 16: 53–9.

11. van Dongen JJM, Seriu T, Panzer-Grünmayer ER, et al. Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood. Lancet 1998; 352: 1731–8.

12. Löffler H, Rastetter J, Haferlach T. Atlas der klinischen Hämatologie. Heidelberg; Springer; 2004.

13. Theml H, Diem H, Haferlach T. Taschenatlas der Hämatologiel. Stuttgart; Thieme, 2002.

14. Journal article. A patient with hypokalemia and hypoxemia – what is the culprit? Clin Chem 2023; 69 (11): 1220–5.

15.16 Myelodysplastic syndrome

Torsten Haferlach

The myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by clonal proliferation of hematopoietic stem cells, recurrent genetic abnormalities, myelodysplasia, ineffective hematopoiesis, peripheral-blood cytopenia, and high risk of evolution to acute myeloid leukemia /1/. The MDS arises within the context of genetic changes in hematopoietic stem cells. These induce ineffective hematopoiesis, often with cytopenia in the peripheral blood. Clinically, symptoms of anemia (ineffective erythropoiesis), increased susceptibility to infection (granulocytopenia), and hemorrhagic diathesis (thrombocytopenia) emerge. MDS are a group of diseases that develop in advanced age; the median age at the time of occurrence is 69 years. The annual incidence at over 70 years of age is 30 : 100,000.

15.16.1 Diagnostic investigation of MDS

The diagnosis of MDS is currently made based upon cytomorphological test of bone marrow and peripheral blood.

The objective of the diagnosis is the differentiation of MDS from other clonal myeloid diseases, such as AML, and also from paroxysmal nocturnal hemoglobinuria (PNH), from severe aplastic anemia, and from reactive and other benign changes which may be associated with dysplastic hematopoiesis. Apart from cytomorphology, cytogenetics, which not only confirms the presence of a clonal disease in the case of an aberrant karyotype, but also is of considerable prognostic value and is acknowledged in the new WHO classification, is also of central diagnostic value /1/.

Up until now, however, it has been necessary to apply various morphological classifications to MDS. Thus, up until recently, the FAB classification, which is now being extended and, in a stepwise manner, replaced by the WHO classification, was considered to be valid /12/.

The WHO classification of MDS is based, in the first place, mainly upon cytomorphological criteria: (i)dysplasia of granulopoiesis and/or erythropoiesis and/or megakaryopoiesis, (ii) the demonstration of ring sideroblasts, (iii) the relative and absolute number of monocytes, and (iv) the fraction of myeloid blasts in the blood and/or the bone marrow. For proof of dysplasia in MDS, only 10% of the cells have to manifest the dysplasia criteria /3/ mentioned in:

In the current WHO proposal for the classification of MDS the RAEB-T category, with a bone marrow blast fraction of ≥ 20–30%, is assigned to AML (Tab. 15.16-2 – International Prognostic Scoring System: grouping). The RA and RARS entities are maintained, while the RAEB and CMML entities are further subdivided according to their bone marrow blast fraction. Furthermore, the category of refractory cytopenia with multi lineage dysplasia (RCDM), and a subgroup of unclassifiable MDS, have been introduced. It remains to be seen whether this further subdivision of MDS – which is, again, based upon purely morphological criteria – is improved and changed very predictably with molecular markers, and whether new clinical relevance will have to be defined and tested /2/. It is, however, certainly meaningful to list the 5q-syndrome entity as a distinct category, since it can be clearly differentiated, clinically and genetically, from all other MDS subgroups, and has a better prognosis as well as a more specific therapeutic option (e.g., lenalidomide). Here, however, additional insights from molecular genetics (e.g., TP53 mutations in patients with 5q- syndrome) increasingly play a role /3/.

Within the framework of the cytomorphological diagnostic investigation, at least 200 (500, according to WHO) bone marrow cells and 20 megakaryocytes should be evaluated; in MDS, signs of dysplasia should be demonstrable in at least 10% of the cells. Pseudo- Pelger neutrophils, peroxidase defects of segmented neutrophils, ring sideroblasts and micro megakaryocytes, and the proliferation of bone marrow blasts to 5–19%. These morphological changes partially correlate with the presence of clonal cytogenetic and molecular genetic markers; they also exhibit, however, considerable examiner dependence. It remains to be seen whether this further division of MDS is clinically relevant. It is, however, certainly meaningful to list the 5q- syndrome entity as a distinct category, since it can be clearly differentiated, clinically and genetically, from all other MDS subgroups, and is associated with a more favorable prognosis.

This does not hold true for the 5q- syndrome which, with a clear genetic marker, is characterized by bone marrow blasts of below 5% and, often, a normal or even elevated peripheral blood thrombocyte count.

In the morphological area, therefore, the evaluation of neutrophil hypo granulation should not remain the sole diagnostic criterion. In general terms, it is often difficult to diagnose early stage refractory anemia (RA) with cytopenia and dysplasia with only one lineage, and the monitoring of disease progression at intervals of 2–3 months prior to making a final diagnosis of MDS. Similarly, the recently introduced entities of RN for pure neutropenia and RT for pure thrombocytopenia which, along with RA are low listed as RCUD (refractory cytopenia with one lineage dysplasia), should be assessed. With reference to the differentiation of hypoplastic MDS and aplastic anemia, it must be taken into consideration that signs of dysplasia in erythropoiesis also occur in the latter, and, therefore, in contrast to dysplasia signs in other lines and bone marrow blast cell proliferation, are of no diagnostic value. The demonstration of cytogenetic changes certainly speaks rather for the presence of MDS, than for aplastic anemia, but proves neither the one nor the other. PNH should also be included in the differential diagnosis. Histology, including immunohistology, is strongly recommended for the differentiation of these entities.

Apart from cytomorphology and cytogenetics, multi parametric flow cytometry is increasingly finding its way into the diagnostic work-up in suspected MDS. Thus, for the different lineages of granulopoiesis, monocytopoiesis and erythropoiesis, this method features the possibility of qualitatively assessing signs of dysplasia in the form of aberrant antigen expression patterns, and of quantifying the blasts. The correlation with the cytomorphological findings is, consequently, high. In addition, valuable diagnostic information, of prognostic relevance, can be gathered in cases that are difficult to classify cytomorphologically.

15.16.2 Recognition of prognostic factors in the diagnosis of MDS

In can generally be said that the prognosis in patients with MDS is reduced in comparison with the general population, particularly in younger patients and in cases of high-risk MDS. The currently most meaningful and widely used system for the estimation of prognosis is the International Prognostic Scoring System (IPSS) /4/, which is based on the biomarker of bone marrow blast cells, karyotype changes and number of cytopenias. Refer to:

The scoring system is based upon 816 patients with MDS, the large majority of which was not previously treated, so that the spontaneous course could be estimated. The biological insights into the fundamental principles and the clinical course of MDS, and the parallels to acute myeloid leukemia (AML) that are present with regard to many aspects have, at least in young and high-risk patients, lead to the use of AML-typical therapies and partially limit, thereby, the applicability of the IPSS score. New insights from cytogenetics have to be incorporated /56/. The consideration of the requirement for transfusion for the prognosis estimation is proposed – WPSS /7/. The closeness of the correlation between the two diseases of MDS and AML is shown by the genetic aberrations (Tab. 15.16-4 – Characteristic cytogenetic alterations in MDS, in comparison with AML). The table shows that in the diagnostic investigation of MDS, while cytomorphology is of substantial and fundamental relevance, the question of prognosis and the distinct biological entity can certainly be answered with other parameters, particularly cytogenetic parameters /8/. In this context, the IPSS score has already pointed out the right direction.

Thus, it holds true for MDS as well as for acute leukemia and chronic myeloproliferative syndromes, that only by means of a combination of cytomorphology /59/ and cytogenetics, possibly informative at present, supplemented with immunophenotyping and molecular genetics, can the diagnosis be made. The WHO classification of 2008 /1/ will, therefore, have to be extended promptly in order to show, beyond the classification, its clinical and prognostic relevance in prospective studies. The therapy that is recommended following diagnosis is more clearly focused, away from an often only supportive, and at times even nihilistic approach, with substances having biologically different modes of action, such as lenalidomide or azacitidine; it can only be employed, in individual patients and in a targeted manner, along with the use of the laboratory methods mentioned in the foregoing.

References

1. Cazzola M. Myelodysplastic syndromes. N Engl J. Med 2020; 383: 1358–74.

2. Bejar R, Stevenson K, Abdel-Wahab O, et al. Clinical effect of point mutations in myelodysplastic syndromes. N Engl.J Med 2011; 364: 2496–506.

3. Jadersten M, Saft L, Smith A et al. TP53 mutations in low-risk myelodysplastic syndromes with del(5q) predict disease progression. J Clin Oncol 2011; 29: 1971–9.

4. Greenberg P, Cox C, Le Beau MM et al. International Scoring System for evaluating prognosis in myelodysplastic syndromes. Blood 1997; 89: 2079–88.

5. Haase D, Germing U, Schanz J et al. New insights into the prognostic impact of the karyotype in MDS and correlation with subtypes: evidence from a core dataset of 2124 patients. Blood 2007; 110: 4385–95.

6. Schanz J, Steidl C, Fonatsch C et al. Coalesced multicentric analysis of 2,351 patients with myelodysplastic syndromes indicates an under-estimation of poor-risk cytogenetics of myelodysplastic syndromes in the international prognostic scoring system. J Clin Oncol 2011; 29: 1963–70.

7. Malcovati L, Germing U, Kuendgen A et al. Time-dependent prognostic scoring system for predicting survival and leukemic evolution in myelodysplastic syndromes. J Clin. Oncol. 2007; 25 (23): 3503–3510.

8. Haferlach C, Bacher U, Haferlach T et al. The inv(3)(q21q26)/t(3;3)(q21;q26) is frequently accompanied by alterations of the RUNX1, KRAS and NRAS and NF1 genes and mediates adverse prognosis both in MDS and in AML: a study in 39 cases of MDS or AML. Leu-kemia 2011; 25: 874–7.

9. Haferlach T. The molecular pathology of myelodysplastic syndrome. Pathobiology 2018; 23: 1-6.

15.17 Myoproliferative neoplasms

Torsten Haferlach

Under the umbrella term of chronic myeloproliferative neoplasms (MNP), the following entities have, in the narrower sense, been consolidated up to the present time:

  • Polycythemia vera (PV)
  • Essential thrombocythemia (ET)
  • Primary myelofibrosis (PMF)
  • Chronic myeloid leukemia (CML).

The new WHO classification of 2008 has added the following entities /1/:

  • Chronic eosinophilic leukemia (not specified more precisely)
  • Chronic neutrophilic leukemia and mastocytosis.

MPN’s occur sporadically; they are thereby traced back to acquired and not congenital clonal genetic changes. In all cases, it is presumed that the starting cells are pluripotent hematopoietic stem cells.

By means of the stepwise elucidation of a clinically, morphologically, cytogenetically and molecular genetically well-defined clinical picture, new possibilities have now become available, especially in the diagnostic investigation and therapy of CML and, since 2005, for JAK2-mutated MPN as well /23/.

All the more important, first of all, is, therefore, the differentiation within MPN and the definition of BCR-ABL1-positive CML, based upon morphological, clinical and, particularly, diagnostic laboratory findings. Refer to:

However, overlapping traits, and phenotypes of the other MPN’s that to blend into one another, are also seen clinically since, in all of these, JAK2 mutations are observed. Furthermore, in the diagnosis and differential diagnosis of MPN, bone marrow histology is, in addition to the cytogenetic and molecular genetic diagnostic investigation of major importance (Fig. 15.17-1 – Algorithm for molecular genetic testing in myeloproliferative neoplasia).

With the exception of ET, MPN manifests an almost complete displacement of the fatty bone marrow. Specifically, in the diagnostic investigation of MPN, the points listed in the Tables are to be taken into consideration:

15.17.1 Chronic myeloid leukemia

Within the MPN group, chronic myeloid leukemia (CML) is, cytogenetically, the best-studied form. The presence of a Philadephia translocation distinguishes CML unambiguously from all other myoproliferative diseases. As early as 1960 Nowell and Hungerford demonstrated, for the very first time, a tumor-specific chromosomal change in patients with CML /4/. At that time, they discovered a small marker chromosome which was later named Philadelphia chromosome. Following the introduction of banding techniques in cytogenetics, it was possible to identify Philadelphia chromosome as a shortened chromosome 22. It was shown that shortening of chromosome 22 occurs due to a translocation between the long arms of chromosomes 9 and 22: t(9;22)(q34; q11) /5/.

The development of molecular genetic procedures permitted the identification of the gene involved: ABL1 on the long arm of chromosome 9 in chromosome band 9q34, and the so-called breakpoint cluster region (BCR) on the long arm of chromosome 22 in chromosome band 22q11. In this way, translocation t(9;22)(q34;q11) leads, at the molecular level, to the formation of two leukemia-specific hybrid genes: BCR-ABL1 on derivative chromosome 22, and ABL1-BCR, on derivative chromosome 9. The BCR-ABL1 gene codes for a chimeric protein, which manifests elevated tyrosine kinase activity in comparison with normal ABL ; this plays a determining role in the pathogenesis of CML /6/. Consequently, the diagnosis of CML is dependent upon the clinical picture and the blood and bone marrow findings, but also absolutely requires the demonstration of the pathognomonic BCR-ABL1 fusion transcript. This applies particularly since the introduction of therapy with tyrosine kinase inhibitors /7/.

Patients with CML and a cytogenetically normal karyotype (ca. 5%) manifest, both with fluorescence in situ hybridization (FISH) as well as with RT-PCR, a BCR-ABL1 rearrangement. Using metaphase-FISH, the BCR-ABL1 fusion gene can be demonstrated either on chromosome 22 or less often on chromosome 9. This form of CML is designated Philadelphia-negative, BCR-ABL1-positive CML, but this is irrelevant clinically. However, it leads to the suggestion that chromosome analysis alone is not sufficient in the diagnosis of CML.

Cytomorphology and histology

Of all of the MPN forms, the most severe leukocytosis (up to 700 × 109/L) occurs in CML. There occurs, thereby, a left shift, through to the blast cells (usually below 5%), in both peripheral blood and bone marrow aspirate samples. The bone marrow is hyper cellular, and exhibits a massive increase in granulopoiesis in comparison with erythropoiesis (up to a ratio of 20: 1; normal is 3 : 1). In addition, eosinophilia and, in particular and relatively pathognomonically, basophilia are found. In all MPN, but in particular in CML, glycolipid-storing cells, so-called pseudo-Gaucher cells, and sea blue histiocytes, are found in many cases, due to increased cell turnover in the bone marrow. In CML, fibrosis of the bone marrow is seldom seen at the time of diagnosis.

The diagnostic investigation of CML, at first diagnosis and during the disease course, should follow certain algorithms which can then govern treatment /8/. Cytomorphological testing of the blood and bone marrow, as well as bone marrow histology, are generally considered to be obligatory with regard to the primary diagnosis. In addition, chromosome analysis (optimally on bone marrow), and FISH and PCR analyses for BCR-ABL1. PCR can be performed quantitatively and serves, in cases of a favorable therapeutic response, as a sensitive marker of disease progression. It is recommended that, in the further course, evaluations take place at intervals of three months. Classical cytogenetic testing should also be performed again later on an annual basis. The reason is that under tyrosine kinase inhibitor treatment as well as, occasionally, under interferon therapy, previously observed Philadelphia-independent cytogenetic changes such as trisomy 8 or monosomy 7 occur /9/. Their relevance and influence on disease progression is unclear, but is still being further validated.

Furthermore, TKI resistance due to mutations at the site of action of the substances has been observed. In suspected cases, mutation analysis should be performed, as this can make possible a modification of treatment.

Following allogenic bone marrow transplantation, the renewed demonstration of the BCR-ABL1 fusion transcript by means of quantitative PCR, which is predictive of a relapse, is clinically relevant.

CML phases in relation to the clinical course

With the use of various therapeutic modalities (e.g., TKI, interferon and allogenic transplantation) the CML clinical course scenarios have changed in comparison with what they were previously. Patient survival has been substantially prolonged. Nonetheless, it remains meaningful, from the clinical-morphological point of view, to use the 3 phases of CML for orientation (Tab. 15.17-3 – WHO classification of CML based upon the morphological findings/1/.

This division has also been retained in the new WHO classification, and allows for clear patient assignment. It can never, however, be permitted to take on therapeutic consequences on its own. Rather, classical cytogenetics, FISH, PCR and quantitative PCR, as well as mutation analysis, are to be performed in order to make the correct diagnosis and, in particular, to guide treatment in CML patients /8/.

15.17.2 Polycythemia vera

Cytomorphology and histology

In Polycythemia vera (PV), the blood count shows a substantial elevation of the hematocrit to over 50%, the hemoglobin level is 16–22 g/dL, and moderate leukocytosis and thrombocytosis are observed. Bone marrow cytology in PV shows hyper cellularity as well as uniform proliferation of all three cell lineages. On iron staining, iron reserve, which is incorporated into the numerous erythrocytes, is lacking.

Histologically, one sees an elevated megakaryocyte number with giant forms and cluster formation, as well as augmented granulopoiesis and erythropoiesis with deficient storage iron, hyperplasia of the sinus system, and varying degrees of fibrosis as well as osteopenia. In PV, the bone marrow findings are normal in only 10% of the cases. The diagnostic criteria are listed in Tab. 15.17-4 – Diagnostic criteria of polycythemia vera.

The diagnosis of PV may not, however, be made without molecular analyses of JAK2 V617F mutations (in 95% of PV cases); reactive polyglobulia can, thereby, be ruled out in almost all cases /123/ (Tab. 15.17-4 and Fig. 15.17-1 – Algorithm for molecular genetic testing in myeloproliferative neoplasia). Patients with pure polyglobulia occasionally manifest a JAK2 exon 12 mutation /10/. Differential diagnostically, however, hypoxia caused by cardiac or pulmonary factors, erythropoietin-producing tumors, elevated androgens or hyperplasia of the red cells due to nicotine abuse must still be ruled out in patients without JAK2 mutations. However, a bone marrow puncture is no longer obligatory for the diagnosis of PV, since the biomarkers can usually also be tested using peripheral blood.

The implementation of cytogenetic testing in PV is recommended, according to the WHO classification. The incidence of chromosome aberrations increases with the persistence of the disease. It is higher in patients who are receiving myelosuppressive therapy. In these cases it is impossible to say, however, whether this reflects an influence of the therapy or, rather, is simply related to the fact that patients with progressive disease are more often treated with myelosuppresive agents. The transformation of the disease into MDS or AML is, likewise, associated with a higher rate of karyotype changes. Overall, it therefore seems that the demonstration of chromosome aberrations is associated with poor prognosis.

15.17.3 Primary myelofibrosis

Cytomorphology and histology

In primary myelofibrosis (PMF), the peripheral blood findings are uncharacteristic, usually anemia with reticulocytosis as well as functional splenectomy, Howell-Jolly bodies, and teardrop erythrocytes. In severe bone marrow fibrosis with extramedullary hematopoiesis, normoblasts also appear in the peripheral blood. The leukocyte and thrombocyte counts do not manifest unequivocal changes and, not uncommonly, numbers that are rather below normal or even increased are present at the time of diagnosis. Due to disturbed thrombocyte function, bleeding time is sometimes prolonged.

In PMF, the bone marrow cytology is often not evaluable on account of the severe fibrosis and the consequent punctio sicca. If this is, nonetheless, required and imposed in addition to the histology, it can be attempted to make touch imprints of a biopsy punch.

The histological picture shows fibrosis of varying severity (MF 0–3) with the optional occurrence of woven bone formation, inflammatory marrow changes with lymphocyte infiltrates, erythrocyte extravasates, plasmacytosis and inflammatory vascular changes with enlarged sinusoides, vessel wall sclerosis, and intrasinusoidal hematopoiesis. In addition, clusters of atypical megakaryocytes and megaloblastoid erythropoiesis are found. At diagnosis, blast cells are usually not increased. During the course of the disease, cytopenia increases, the extent of the splenomegaly may become very great, transformation to acute leukemia is observed.

Cytogenetically, a deletion in the long arm of chromosome 20(20q-) is the most common chromosome aberration in patients with PMF. It is observed, like the deletion in the long arm of chromosome 13, in 6–7% of the patients. Further karyotype alterations that have been described include numerical changes in chromosomes 7, 8 and 9, as well as structural aberrations of 1q and 5q /1/. It is recommended that chromosome analysis be performed; in punctio sicca, a biopsy punch can also be processed in physiological saline containing heparin for further analysis.

Apart from that, molecular markers should definitely be determined: in the first place, JAK2 and MPLW515 (Tab. 15.17-5 – Diagnosis criteria for primary myelofibrosis). Newer, targeted therapies have recently been made available /11/.

15.17.4 Essential thrombocythemia

Cytomorphology and histology

The cardinal symptom in essential thrombocythemia (ET) is marked thrombocytosis, which can reach as high as 5 × 1010/L. According to WHO, ≥ 450 × 109/L are required (Tab. 15.17-6 – Diagnostic criteria of essential thrombocythemia). In the smear, giant thrombocytes and thrombocyte aggregates are observed. Bleeding time can be normal or shortened but, due to disturbances of thrombocyte function, may also be prolonged. Potassium is released from the thrombocytes, leading to hyperkalemia. In the bone marrow, in the presence of normal granulopoiesis and erythropoiesis, ET manifests diagnosis-determining megakaryocyte clusters, which are often located around the central-intermediate sinus. The diagnosis of ET is, at times, a diagnosis of exclusion. It, as well, necessitates a step-wise algorithm of molecular diagnostic investigation (Fig. 15.17-1 – Algorithm for molecular genetic testing in myeloproliferative neoplasia).

Only some 5% of ET patients have clonal karyotype abnormalities. In these cases, a gain of chromosome 9 is most frequently observed. The performance of a complete chromosome analysis seems, therefore, not to be a requirement; it is, nonetheless, meaningful within the framework of an initial diagnosis in suspected MPN.

15.17.5 Chronic eosinophilic leukemia, not otherwise specified

According to the WHO classification of 2008, chronic eosinophilic leukemia (not otherwise specified) is also listed in the chapter on MPN. It is classified here because, in contrast, diseases with eosinophilia and evidence of molecular changes in the FIP1L1-PDGFRA, FGFR1, or PDGFRA and PDGFRB genes are allocated to a separate chapter /112/. Thus, following exclusion of the molecular changes or even specific cytogenetic findings, this CEL, which cannot be classified in a more precise manner, remains as part of the MPN chapter. It is defined as eosinophilia in the blood (1.5 × 109/L), less than 20% blast cells, and no chromosome alterations.

15.17.6 Mastocytosis

Mastocytosis is also listed in the MPN chapter of the WHO classification of 2008. The following are distinguished: cutaneous mastocytosis (CM), indolent systemic mastocytosis (ISM), systemic mastocytosis with an associated clonal hematologic non-mast cell lineage disease (SM-AHNMD), aggressive systemic mastocytosis (ASM), mast cell leukemia (MCL), mast cell sarcoma (MSC), and extra cutaneous mastocytoma.

Further detail with regard to the complex diagnostic criteria for the individual subgroups is not addressed /13/. Among others, various dermatological investigations are required, as are laboratory biomarkers (e.g., measurement of serum tryptase), immunophenotyping, or histology and immunohistology. In addition, gene mutations are to be investigated such as the analysis of KIT (usually D816V) mutations.

References

1. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th. 2008. Lyon, International Agency for Research on Cancer (IARC).

2. James C, Ugo V, Le Couedic JP et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005; 434 (7037): 1144–1148.

3. Kralovics R, Passamonti F, Buser AS et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352 (17): 1779–1790.

4. Nowell PC, Hungerford DA. A minute chromosome in human granulocytic leukemia. Science 1960; 132: 1497.

5. Rowley JD. A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature 1973; 243: 290–3.

6. Heisterkamp N, Stephenson JR, Groffen J, Hansen PF, de Klein A, Bartram CR u. Grosveld G. Localization of the c-abl oncogene adjacent to a translocation breakpoint in chronic myelocytic leukaemia. Nature 1983; 306: 239–42.

7. Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001; 344: 1031–7.

8. Baccarani M, Cortes J, Pane F et al. Chronic myeloid leukemia: an update of concepts and management recommendations of European Leukemia Net. J Clin Oncol 2009; 27: 6041–51.

9. Schoch C, Haferlach T, Kern W, et al. Occurrence of additional chromosome aberrations in chronic myeloid leukemia patients treated with imatinib mesylate. Leukemia 2003; 17: 461–3.

10. Scott LM, Tong W, Levine RL et al. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. N Engl J Med 2007; 356: 459–68.

11. Verstovsek S, Kantarjian H, Mesa RA et al. Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med 2010; 363: 1117–27.

12. Cools J, de Angelo DJ, Gotlib J, et al. A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome. N Engl J Med 2003; 348: 1201–14.

13. Horny HP, Sotlar K, Valent P. Mastocytosis: state of the art. Pathobiology 2007; 74 (2): 121–132.

15.18 Immunophenotyping of acute leukemia and non-Hodgkin lymphoma

Richard Schabath, Wolf-Dieter Ludwig

The diagnosis and classification of acute leukemia (AL) and non-Hodgkin lymphoma (NHL) is oriented to the classification of the World Health Organization (WHO) for tumors of the hematopoietic and lymphatic tissue /1/. In this document, acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) of precursor B and precursor T cells are differentiated from mature B, T and NK cell neoplasms. In the differential diagnostic differentiation of the subtypes of AL and NHL, morphological and histological features, immunophenotype, and genotype are taken into consideration. The analysis of these cell biological features of tumor cells, commonly implemented in parallel, has made the identification of new subtypes or entities, whose clinical picture and therapeutic influenceability are different, substantially easier. In addition, understanding of the mechanisms that are pathogenetically relevant to leukemia genesis and lymphoma genesis could be improved by means of the characterization of genetic changes in leukemia and lymphoma cells /23, 45/.

Immunophenotyping in bone marrow and/or peripheral blood is an essential component in the initial diagnostic investigation and the course of AL and leukemic NHL. It is based upon the demonstration of different antigens using monoclonal antibodies (MAB), which are expressed by precursor and/or mature myelopoietic and lymphopoietic cells, often as a function of their stage of maturation /14, 6, 78/. The binding of MAB’s to membrane-bound or intracellular antigens can be analyzed using different methods. Thereby, the labeling of cell suspensions with immunofluorescence techniques and their analysis using multi parametric flow cytometry, as well as immunoenzymatic staining, are considered to be routine methods /89, 10, 11, 1240/.

The advantages of flow cytometric immunophenotyping are:

  • Rapid analysis of the samples in spite of high cell counts (over 106 cells in a single sample)
  • High sensitivity of detection
  • Simultaneous analysis of various biomarkers such as 2–10 fluorescent and light scattering properties of the cells
  • Exact quantification of the results
  • Statistical data analysis.

For recommendations, indications, standardization and quality assurance of immunophenotyping in AL and NHL by multiparametric flow cytometry and immunoenzymatic procedures, see Section 52.2 and references /47, 9, 10, 1140/.

15.18.1 Immunological classification of acute leukemia

The essential objectives of the immunophenotyping of acute leukemia are:

  • To classify morphologically and cytochemically undifferentiated leukemia of B cell, T cell, NK cell and myeloid cell lineage, as well as to determine the stage of maturity of the leukemia cells
  • To identify biologically and/or prognostically relevant subtypes and to diagnose them in a standardized, study-conform manner
  • To identify the expression of proteins that are involved in the regulation of important cell biology functions, (e.g.; adhesion, proliferation, differentiation, and apoptosis)
  • Detection of target structures for targeted therapy (e.g., with MAB’s against CD20, CD33 or CD52)
  • To make possible the treatment of non-eliminated residual leukemia cells (minimal residual disease) by means of its identification.

Fig. 15.18-1 – Flow diagram for immunophenotyping in acute leukemia illustrates the stepwise approach in the immunological classification of AL /420/.

Starting from the morphological diagnosis of AL, which is always based upon the light microscopic evaluation of pan-optically stained smears, an unequivocal diagnosis and definition of the subtype is made by means of lineage assignment of the leukemia blast cells and the determination of the immunological subtype and stage of maturity of the AL.

Lineage assignment of the leukemia blast cells

The lineage assignment of the leukemia blast cells occurs based upon the detection of membrane-bound or cytoplasmic antigens that are expressed by immature lymphoid or myeloid progenitor cells. Particularly relevant to the diagnostic investigation of AL are the cytoplasmic antigens that are already expressed in very immature cells in a lineage specific manner /1314/:

  • CD13, CD33, myeloperoxidase (MPO) and lysozyme in myeloid cells
  • CD19, cyCD22 and cyCD79a in B lymphoid progenitor cells
  • cyCD3 in T lymphoid progenitor cells.

These antigens, which can be detected immunoenzymatically or following fixation and permeabilization of leukemia cell suspensions as well as with flow cytometry /15/, are expressed to a certain extent, including membrane-bound, by mature leukemia or lymphoma cells (CD3, CD22, CD79a).

Determination of the immunological subtype and stage of maturity

The determination of the immunological subtype and stage of maturity of AL using MAB’s against antigens whose expression is limited to immature lympho-hematopoietic progenitor cells, or is associated with different stages of differentiation of lymphopoiesis or myelopoiesis.

Based upon their expression pattern, the antigens that are important for immunophenotyping of AL are subdivided into:

  • Lineage-specific traits: (i) MPO for the myeloid cell lineage, (ii) cy/m for immunoglobulins, (iii) CD22, CD79a for B and T cell receptors α/β and γ/δ, (iv) CD3 for the T cell line
  • Panmyeloid antigens (e.g., CD13, CD33, CD65)
  • Pan-B (e.g., CD19, CD22, CD79a)
  • Pan-T antigens (e.g., CD3, CD2, CD5, CD7)
  • Lineage-associated antigens (e.g., CD14 for monocytic, CD15 for granulocytic, CD235a (Glycophorin A) for erythrocytic and CD41 or CD61 for megakaryocytic cells)
  • Progenitor cell-associated antigens, such as CD10, CD34, CD117, HLA-DR, terminal deoxynucleotidyl transferase (TdT).

The analysis of the antigens listed in Fig. 15.18-1 – Flow diagram for immunophenotyping in acute leukemia allows for unambiguous lineage assignment and subtype definition in almost all cases /20/.

Tab. 15.18-1 – Basic panel for the initial diagnosis in acute leukemia shows the basic panel for the initial diagnostic investigation of AL, recommended by the German competence network for acute and chronic leukemia.

15.18.1.1 Acute undifferentiated leukemia

Morpholgical or cytochemical acute undifferentiated leukemia (AUL) in which, with immunophenotyping, not more than one antigen of myeloid or B or T lymphoid cell lineage is detectable, is only diagnosed very infrequently (less than 1% of all AL) /11617/. AUL, also designated morphologically as stem cell leukemia, expresses progenitor cell-associated antigens (CD34, CD38, CD117, HLA-DR, TdT) exclusively.

The definitive assignment of AUL, important for the therapeutic course of action, necessitates additional analyses such as, ultrastructural demonstration of MPO or platelet peroxidase /1819/ or of cytogenetic or molecular genetic abnormalities /14/ which are characteristic of AML or ALL. These complex analyses are not, however, routinely performed within the framework of the initial leukemia diagnostic investigation.

15.18.1.2 Immunophenotyping of ALL

The identification and assignment of ALL to B or T cell lineage is easily possible, based upon the cyCD3, cyCD22, cyCD79a antigens that are first expressed intra-cytoplasmatically in lymphoid progenitor cells /142021/ as well as the membrane antigens CD7 and CD19. These antigens can be detected in immature precursor B or T cells, and in over 99% of the corresponding immunological subtypes of ALL.

For the recognition of immature T-ALL subtypes, the simultaneous analysis of CD7 and cyCD3 is important, since some 10–20% of immature AML cells express CD7 /4/, while cyCD3 is considered to be a specific marker of the T cell series /13/.

The precise characterization of the immunological subtype, or the determination of the stage of maturity of ALL, is accomplished by the analysis of additional B cell and T cell-associated antigens (Fig. 15.18-1 – Flow diagram for immunophenotyping in acute leukemia). The characterization allows differentiation of the following subtypes /421/:

  • B progenitor cell ALL
  • B cell ALL/Burkitt’s lymphoma
  • T progenitor cell ALL

The characteristics of these subtypes are shown in Tab. 15.18-2 – Terminology, frequency and phenotype of the immunological ALL subtypes.

In 5–40% of ALL patients, the blast cells co express myeloid antigens (my + ALL). This is associated particularly with immature subtypes (pro-B ALL, pro-T ALL or pre-T ALL) /26/.

The differentiation of the subtypes is important for the definition of risk groups and assignment to the different therapy forms in the German multi-center ALL therapeutic trials in children (ALL-BFM) and adults (GMALL):

15.18.1.3 Immunophenotyping of AML

In AML, immunophenotyping has not achieved the status that it has attained in ALL.

Within the framework of classification, immunophentyping is necessary mainly for the identification of:

  • Minimally differentiated AML, subtype M0: MPO cytochemically negative, expression of pan myeloid antigens and/or demonstration of MPO using MAB’s in the absence of lineage-specific B cell or T cell characteristics /19/.
  • Acute megakaryoblastic leukemia, subtype M7: membrane-bound, infrequently solely intracytoplasmic expression of CD41 and/or CD61 /18/.

In 75–90% of all AML patients, leukemia cells express the antigens CD13, CD33 and CD65; however, all three pan myeloid markers are detectable in only approximately 55% of the patients, while at least one of these antigens is found in more than 98% of the cases /22/. MPO can be detected using MAB’s in just 90% of AML patients. In addition, CD117, the receptor for the stem cell factor (c-kit protooncogene), which is expressed by 1–4% of normal bone marrow cells and some 60–70% of AML cells, but only rarely by immature ALL cells, has turned out to be a valuable marker for the immunological characterization of AML /23/.

By means of the simultaneous analysis of these antigens and the use of MAB’s against erythroids (e.g., glycophorin A) platelet-associated antigens such as CD41 and CD61, as well as MPO, close to 100% of AML cases can be identified with immunological cell markers, and differentiated from ALL. For the differentiation of AML and ALL, the demonstration of TdT is hardly relevant, since 10–40% of AML cases, depending upon the method used (immunofluorescence or immunocytochemistry), express the enzyme TdT, which was previously considered to be a lymphoid marker.

It is possible to distinguish between immature myeloid and granulocytic differentiated leukemia cells by means of combined intracytoplasmic demonstration of MPO and lactoferrin (LF), where undifferentiated myeloid cells are MPO-positive and LF-negative, while granulocytic differentiated leukemia cells express both MPO and LF /14/.

Due to the lack of availability of monoblast-specific MAB’s it is, as a rule, not possible to immunologically differentiate immature monoblastic leukemia (FAB M5a) from other subtypes of immature AML (e.g., FAB M0/M1).

In AML, as in ALL, aberrant expression of blast cell lymphoid markers occurs. In 10–25% of the patients, co-expression of T-lymphoid antigens, especially CD4, CD7 and/or CD2, can be demonstrated, while B-lymphoid antigens including CD10 (below 10%) are only expressed infrequently /25/.

Due to the heterogeneity of antigen expression in AML, with the exception of the FAB-M0 and FAB-M7 subtypes, it is not possible to establish, with certainty, a correlation of the immunophenotype with morphologically or cytochemically defined FAB subtypes, or with cytogenetically and molecular biologically defined WHO subgroups of AML /2224/. A correlation can only provide an initial clue with regard to the AML subtype, until the cytogenetic and molecular biology results are made available (Tab. 15.18-3 – Correlation of the FAB classification, cytogenetics and immunophenotype).

15.18.1.4 Mixed phenotype acute leukemia

AL subtypes in which the pathological blast cell population simultaneously expresses myeloid and lymphoid antigens (aberrant antigen expression) are diagnosed with increasing frequency (up to 5% of all AL). These subtypes were initially classified as biphenotypic acute leukemia (BAL) /2036/.

With the publication of the WHO classification of 2008, an internationally accepted nomenclature for this heterogeneous subgroup of AL now exists /1/. The mixed phenotype acute leukemia (MPAL) immunological classification score is based upon this classification /20/ and has replaced the EGIL recommendations (Tab. 15.18-4 – Score for the definition of mixed phenotype acute leukemia). However, with regard to MPAL prognosis and therapy, it is mainly the underlying cytogenetic or molecular biological aberration and not the immunophenotype that is critical /3637/.

15.18.1.5 Detection of residual leukemia cells in AL with immunophenotyping

In AL, the demonstration of residual leukemia cells during and following the initial course of chemotherapy has acquired increasing relevance with regard to subsequent therapeutic planning and prognostic assessment of the disease. Up to now, the assessment of remission has been based upon morphological evaluation of the bone marrow. Due to the low sensitivity of morphology (detection limit 10–2 i.e., 1 leukemia cell per 100 normal cells) more sensitive methods for the detection of minimal residual disease, such as immunophenotyping (detection limit 10–3–10–5) and molecular genetic analyses (detection limit of the polymerase chain reaction 10–4–10–5) have been employed /727, 2838/.

Detection of residual leukemia cells with immunophenotyping is based on the expression of leukemia-associated immunophenotypes, which can be depicted using multiparametric flow cytometry in the great majority of AL cases. The following are considered to be important biomarkers with regard to the distinction between leukemic and normal progenitor cells:

  • Aberrant or asynchronous antigen expression
  • Lack of expression of differentiation antigens
  • Altered antigen density on leukemia cells.

Apart from the rapidity of the test, the possibility of quantifying the number of residual leukemia cells and of determining their vitality are considered to be advantages of immunophenotyping by multiparametric flow cytometry for the demonstration of minimal residual disease.

An important prerequisite for the unambiguous identification of residual leukemia cells with immunophenotyping is the precise characterization of the leukemic blasts at the time of diagnosis by multiparametric flow cytometry, with which the expression of 3–10 antigens per cell can also be simultaneously assessed, along with light scattering properties. Single-color or dual-color analyses of leukemia cells are not sufficient for the characterization of leukemia-specific traits and should, therefore, no longer be used for the diagnosis of minimal residual disease.

Examples of suitable antigen combinations for the diagnosis of minimal residual disease in AL patients, and data on the frequency of occurrence of various aberrant or asynchronous phenotypes in leukemia subtypes, are shown in Tab. 15.18-5 – Antigen combinations for the diagnosis of minimal residual disease in patients with acute lymphatic leukemia. Based upon evidence for these combinations of antigens, it is possible to identify residual leukemia cells in a sample containing 10,000 normal hematopoietic progenitor cells (detection limit 10–4).

In ALL, the clinical significance of immunophenotyping and molecular biological demonstration of residual leukemia cells at different time points during the course of treatment could be demonstrated in numerous studies and in some cases in prospective, multicenter studies /2738/. In AML as well, sufficient clinical data now exist to justify the routine use of immunophenotyping for the diagnosis of MRD /28/. However, for the detection of minimal residual disease there is considerable competition between flow cytometry and longer established molecular biology methods (e.g.,quantitative PCR).

15.18.1.6 Immunophenotyping of NHL

Immunophenotyping also plays a decisive role in the diagnostic investigation of leukemic NHL. Its objectives, apart from the differentiation of mature lymphoid neoplasms from AL, are:

  • Assignment of the malignant cells to B, T or NK cell lineages
  • Proof or exclusion of clonality of malignant B cells using kappa or lambda light chain restriction
  • Differentiation of mature lymphoid neoplasms, especially mature T cell neoplasms, from reactive conditions (e.g., EBV or Cytomegalovirus infections or toxoplasmosis)
  • Monitoring of responsiveness to therapy (chemotherapy, monoclonal antibodies) by means of the early identification of residual leukemia or lymphoma cells (minimal residual disease).

The assignment of mature B, T and NK cell neoplasms to the corresponding cell lineage or to a certain maturity stage, based upon a panel of different MAB’s, the composition of which is influenced to a considerable extent by the question under consideration (e.g., initial diagnostic investigation or demonstration of minimal residual disease) /7/. The immunophenotype expression profiles of normal B and T cells and of mature B, T and NK cell neoplasms that are depicted in:

Scores have been proposed which, based upon a characteristic expression profile of membrane-bound antigens, significantly improve the precision of the differentiation between B-CLL and other mature B cell neoplasms, particularly for the differentiation of chronic lymphocytic leukemia of B cell lineage from other mature B cell neoplasms. In 90–95% of the cases, typical B-CLL can be distinguished from other mature B cell neoplasms by means of analysis of five antigens which are expressed on leukemia cells in B-CLL either not at all (CD 79b, FMC7), weakly (membrane-bound immunoglobulins), or strongly (CD5, CD23) /31/. In this regard, an additional useful marker is CD200, which is usually expressed by B-CLL cells, while mantel cell lymphoma which, as CD5-positive NHL can be problematic with regard to differentiation from B-CLL, is CD200 negative /39/.

Studies of mutations in the variable portions of the immunoglobulin heavy chain gene (IgVH) have led to the recognition of two different cell biological subtypes of B-CLL which, with regard to clinical course, immunophenotype, and cytogenetic and molecular genetic findings, are clearly different /32/. Membrane-bound (CD38) and intracytoplasmic (ZAP-70) antigens are expressed differently by these two subtypes and are therefore increasingly used as diagnostic and prognostic biomarkers. CD38 or ZAP-70 positivity in B-CLL cells usually correlates with lack of mutation of the IgVH gene and an unfavorable clinical course /3334/.

In mature B cell neoplasms as well, immunophenotyping by multiparametric flow cytometry is extremely sensitive of residual leukemia cells (detection limit 10–4/35/.

In site of increasingly precise information regarding the immunophenotype of mature B, T and NK cell neoplasms, as well as further methodical development, unambiguous assignment to the entities defined in the WHO classification is, based upon bone marrow or peripheral blood immunophenotyping, often not possible in leukemic mature lymphoid neoplasia /1/. Further diagnostic measures (e.g., bone marrow or lymph node histology, cytogenetic or molecular genetic analyses) are, therefore, usually required for the definitive classification and prognostic assessment of the disease.

References

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3. Arber DA, Orazi A, Hasserjian, Thiele J, Borowitz MJ, Le Beau MM, et al. The 2016 revision of the World health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016; 127: 2391- 405.

4. Ludwig WD, Haferlach T, Schoch C. Classification of Acute Leukemias: Perspective 1. In: Pui CH, ed. Treatment of Acute Leukemias: New Directions for Clinical Research. Current Clinical Oncology. Totowa, NJ; Humana Press 2003: 3–41.

5. Kuppers R, Klein U, Hansmann ML, Rajewsky K. Cellular origin of human B-cell lymphomas. N Engl J Med 1999; 341: 1520–9.

6. Leucocyte Typing VII. White cell differentiation antigens: Proceedings of the Seventh International Workshop and Conference held in Harrogate, United Kingdom. Oxford; University Press, 2002.

7. Jennings CD, Foon KA. Recent advances in flow cytometry: application to the diagnosis of hematologic malignancy. Blood 1997; 90: 2863–92.

8. Orfao A, Schmitz G, Brando B, Ruiz-Arguelles A, Basso G, Braylan R, et al. Clinically useful information provided by the flow cytometric immunophenotyping of hematological malignancies: current status and future directions. Clin Chem 1999; 45: 1708–17.

9. Ratei R, Nebe T, Schabath R, Kleine HD, Karawajew L, Ludwig WD, et al. Flow-Cytometric Immunophenotyping of Acute Leukemias and Flow Cytometry in the Diagnosis of Non-Hodgkin’s Lymphomas. In: Sack U, Tarnok A, Rothe G, eds. Cellular Diagnostics: Basic Principles, Methods and Clinical Applications of Flow Cytometry. Basel, Karger, 2009: 612–667.

10. Rothe G, Schmitz G, Adorf D, Barlage S, Gramatzki M, Hanenberg, et al. Consensus protocol for the flow cytometric immunophenotyping of hematopoietic malignancies. Leukemia 1996; 10: 877–95.

11. Davis BH, Foucar K, Szczarkowski W, Ball E, Witzig T, Foon KA, et al. U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: medical indications. Cytometry 1997; 30: 249–63.

12. Owens MA, Vall HG, Hurley AA, Wormsley SB. Validation and quality control of immunophenotyping in clinical flow cytometry. J Immunol Methods 2000; 243: 33–50.

13. Janossy G, Coustan-Smith E, Campana D. The reliability of cytoplasmatic CD3 and CD22 antigen expression in the immunodiagnosis of acute leukaemia: a study of 500 cases. Leukemia 1989; 3: 170–81.

14. Knapp W, Strobl H, Majdic O. Flow cytometric analysis of cell surface and intracellular antigens in leukemia diagnosis. Cytometry 1994; 18: 187–98.

15. Ludwig WD, Rhagavachar A, Thie E. Immunophenotypic classification of acute lymphoblastic leukemia. Baillière’s Clin Haematol 1994; 7: 235–62.

16. Campana D, Hansen-Hagge TE, Matutes E, Coustan-Smith E, Yokota S, Shetty V,et al. Phenotypic, genotypic, cytochemical, and ultrastructural characterization of acute undifferentiated leukemia. Leukemia 1990; 4: 620–4.

17. Matutes E, Bucceri V, Morilla R, Shetty V, Dyer M, Catovsky D. Immunological, ultrastructural and molecular features of unclassificable acute leukaemia. In: Ludwig WD, Thiel E, eds. Recent advances in cell biology of acute leukaemia – impact on clinical diagnosis and therapy. Recent results in cancer re-search. Vol 131. Berlin; Springer 1993: 41–52.

18. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C. Criteria for the diagnosis of acute leukemia of megakaryocytic lineage (M7). Ann Intern Med 1985; 103: 460–2.

19. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR, Sultan C. Proposals for the recognition of minimally differentiated acute myeloid leukemias (AML-M0). Br. J. Haematol 1991; 78: 325–9.

20. Béné MC, Castoldi G, Knapp W, Ludwig WD, Matutes E, Orfao A, van’t Veer MB. Proposals for the immunological classification of acute leukemias. Leukemia 1995; 9: 1783–6.

21. Ludwig WD, Reiter A, Löffler H, Gökbuget N, Hoelzer D, Riehm H, Thiel E. Immunophenotypic features of childhood and adult acute lymphoblastic leukaemia (ALL): Experience of the German multicentre trials ALL-BFM amd GMALL. Leuk Lymphoma 1994; 13: Suppl 1: 71–6.

22. Creutzig U, Harbott J, Sperling C, Ritter J, Zimmermann M, Löffler H, et al. Clinical significance of surface antigen expression in children with acute myeloid leukaemia: results of study AML-BFM-87. Blood 1995; 86: 3097–108.

23. Béné MC, Bernier M, Casanovas RO, Castoldi G, Knapp W, Lanza F,et al. The reliability and specificity of c-kit for the diagnosis of acute myeloid leukemias and undifferentiated leukemias. Blood 1998; 92: 596–99.

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25. Drexler HG, Thiel E, Ludwig WD. Review of the incidence and clinical relevance of myeloid antigen-positive acute lymphoblastic leukemia. Leukemia 1991; 5: 637–45.

26. Drexler HG, Thiel E, Ludwig WD. Acute myeloid leukemias expressing lymphoid-associated antigens: Diagnostic incidence and prognostic significance. Leukemia 1993; 7: 489–98.

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29. Cro L, Guffanti A, Colombi M, Cesana B, Grimoldi MG, Patriarca C, et al. Diagnostic role and prognostic significance of a simplified immunophenotypic classification of mature B cell chronic lymphoid leukemias. Leukemia 2003; 17: 125–32.

30. Ahmad E, Garcia D, Davis BH. Clinical utility of CD23 and FMC7 antigen coexistent expression in B-cell lymphoproliferative disorder subclassification. Cytometry 2002; 50: 1–7.

31. Moreau EJ, Matutes E, A’Hern RP, Morilla AM, Morilla RM, Owusu-Ankomah KA, et al. Improvement of the chronic lymphocytic leukemia scoring system with the monoclonal antibody SN8 (CD79b). Am J Clin Pathol 1997; 108: 378–82.

32. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999; 94: 1848–54.

33. Kröber A, Seiler T, Benner A, Bullinger L, Bruckle E, Lichter P, et al. V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood 2002; 100: 1410–6.

34. Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M, et al. ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 2003; 348: 1764–75.

35. Sánchez ML, Almeida J, Vidriales B, López-Berges MC, Garcia-Marcos MA, Moro MJ, et al. Incidence of phenotypic aberrations in a series of 467 patients with B chronic lymphoproliferative disorders: basis for the design of specific four-color stainings to be used for minimal residual disease investigation. Leukemia 2002; 16: 1460–9.

36. Matutes E, Pickl WF, Van’t Veer M, Morilla R, Swansbury J, Strobl H, et al. Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification. Blood 2011; 117: 3163–71.

37. Gerr H, Zimmermann M, Schrappe M, Dworzak M, Ludwig WD, Bradtke J, et al. Acute leukaemias of ambiguous lineage in children: characterization, prognosis and therapy recommendations. Br J Haematol 2010; 149: 84–92.

38. Ratei R, Basso G, Dworzak M, Gaipa G, Veltroni M, Rhein P, et al. AIEOP-BFM-FCM-MRD-Study Group. Monitoring treatment response of childhood precursor B-cell acute lymphoblastic leukemia in the AIEOP-BFM-ALL 2000 protocol with multiparameter flow cytometry: predictive impact of early blast reduction on the remission status after induction. Leukemia. 2009; 23: 528–34.

39. Palumbo GA, Parrinello N, Fargione G, Cardillo K, Chiarenza A, Berretta S, et al. CD200 expression may help in differential diagnosis between mantle cell lymphoma and B-cell chronic lymphocytic leukemia. Leukemia Research 2009; 33: 1212–6.

40. Béné MC, Nebe T, Bettelheim P, Buldini B, Bumbea H, Kern W, et al. Immunophenotyping of acute leukemia and lymphoproliferative disorders: a consensus proposal of the European Leukemia Net Work Package 10. Leukemia 2011; 25: 567–74.

Table 15.1-1 Life span and daily turnover of blood cells in the circulation /1/

Blood cell

Life span

Turnover/
24 hours

Erythrocyte

120 days

2.0 × 1011

Reticulocyte

24 hours

2.0 × 1011

PMN1)

21 hours

1.0 × 1011

Eosinophil

6–18 hours

Basophil

8 hours

Monocyte

14 hours

8.4 × 109

Thrombocyte

10 days

1.0 × 1011

1) Life span in tissue 4-5 days, PMN polymorphonuclear neutrophil granulocyte

Table 15.1-2 Hematological tests for assessment of the hematopoieses /2728/

Comment

Hemoglobin concentration (Hb value)

The measurement of the hemoglobin concentration is the most reliable (precision, validity) and best standardized hematology test. The Hb value is an effective parameter for assessing the organism’s erythrocyte mass and thus also of the oxygen-transporting capacity of the blood, although it is only an indirect measure of red blood cell mass.

Erythrocyte count

The erythrocyte count, also known as the red blood cell count (RBC), is an indicator of the red blood cell mass of the organism. It is not, however, a good diagnostic parameter for the identification of a decrease in cell mass in anemia or an increase caused by polycythemia, since changes in the erythrocyte volume are not taken into consideration.

MCV

The mean corpuscular volume (MCV) is a red cell index of the size of the peripheral red blood cells. Hematology analyzers measure the MCV directly. However, since all red blood cells (including reticulocytes and, possibly, normoblasts) are measured, the MCV cannot automatically be equated with normocytosis, microcytosis or macrocytosis, without knowledge of the erythrocyte distribution width.

MCH

The mean corpuscular Hb content of the red blood cells (mean corpuscular hemoglobin, MCH) is a red cell index that expresses, in pg, the mean Hb content of all of the red blood cells. MCH is a measure that remains constant over the life span of the erythrocyte. The MCH determines the volume of the erythrocyte during maturation of the progenitor cells.

%HYPO

The Hb concentration in individual red blood cells is measured, and the proportion with a concentration of below 280 g/L is expressed as percentage of the total number. The %HYPO is a direct measure of the iron demand of erythropoiesis and can only be determined with certain hematology analyzers.

MCHC

The mean cellular hemoglobin concentration (MCHC) is a red cell index which expresses the Hb concentration of the circulating red blood cells in g/L. Changes in the MCHC are indicative of a disturbance in the relationship between the Hb content of the erythrocytes and their volume. Normochromic and hypochromic anemia are differentiated based upon the MCHC value.

Hematocrit (HCT)

The HCT, also termed packed red blood cell volume (PCV), is the product of the erythrocyte number × MCV. It is a redundant parameter in the diagnostic investigation of anemia.

RDW

The red blood cell distribution width (RDW) is calculated from the distribution histogram as the standard deviation or coefficient of variation of the MCV. In microcytosis, an elevated RDW is indicative of iron deficiency, and a normal RDW suggests the presence of heterozygous β-thalassemia.

Reticulocyte count

The reticulocyte count is an indicator of the erythropoietic effectiveness of the bone marrow. Erythropoiesis is differentiated into normo-, hypo- and hyper regenerative forms.

Reticulocyte RNA

Reticulocyte RNA content is an indicator of the extent of the stimulation of erythropoiesis. A large fraction of reticulocytes with high RNA content indicates intensive stimulation (e.g., following presence in high altitude after two days or after administration of erythropoiesis-stimulating agents).

Reticulocyte hemoglobin content

Mean reticulocyte Hb content (CHr, RetHe) is an early and dynamic indicator for the iron demand of erythropoiesis. In combination with the determination of %HYPO and MCH, a diagnostic pattern is available which differentiates short term iron demand of erythropoiesis (CHr decrease within 72 hours), middle term iron demand (%HYPO increase within 2 to 4 weeks) and longer term iron demand (MCH decrease within 3 to 6 months). The CHr and RetHe are effective markers for the diagnosis of iron-deficient erythropoiesis, a state where the iron demand of erythropoiesis is greater than its supply, independent of the stored iron content.

Differentiated leukocyte count

The differentiated leukocyte count, determined with a hematology analyzer in combination with the blood count, has gained acceptance based upon its acceptable precision and short analysis time. There are, nonetheless, limitations, and automated differentiation should be supplemented with the blood smear examination in symptomatic patients and if one or more blood cell count parameters are flagged by the hematology analyzer.

Blood smear

There are two relevant indications for a microscopic blood smear investigation:

  • Verification of the hematology analyzer blood cell count, if this seems to be implausible
  • The obtaining of further diagnostic information from blood cell morphology, particularly evidence for blood cells that are normally only detectable in the bone marrow.

Thrombocyte count

Indicator of the organism’s thrombocyte mass.

MPV

The mean platelet volume (MPV) is an indicator of stimulation of thrombopoiesis. With a low platelet count, an elevated MPV is indicative of peripheral platelet consumption, for (e.g., due to disseminated intravascular coagulation) while a reduced MPV is, rather, suggestive of an intrinsic disorder of megakaryopoiesis.

Table 15.1-3 Differential diagnosis of pancytopenia /33/

Clinical and laboratory findings

Aplastic Anemia

Aplastic anemia is characterized by hypocellular bone marrow with an absence of myeloid forms. The term refers to an empty bone marrow. Congenital and acquired forms of aplastic anemia exist. Acquired forms are caused by toxins, infections or autoimmune diseases.

Myelodysplastic Syndrome (MDS)

MDS is a primary myeloid neoplasm with a risk of transformation to acute leukemia. Clonal proliferation of hematopoietic stem cells causes dysplasia of the bone marrow and peripheral blood cytopenias.

For further information refer to Section 15.16 – Myelodysplastic syndrome.

Systemic Diseases

Increased concentration of tumor necrosis factor and interleukin-6 in infections and inflammations can cause suppression of the bone marrow that would cause reduced production of blood cells.

Toxins, Medications

Toxins and medications with myelosuppressive properties can cause low blood counts. Examples are illicit drugs like levamisole in cocaine, and excessive alcohol consumption.

Nutritional Disorders

Vitamin B12 and folic acid are required for DNA synthesis. In deficiencies arrest in the synthesis phase (s phase) of blood cell cycle occurs. Patients present with pancytopenia, magaloblastosis and elevated concentration of serum latate dehydrogenase.

Sequestration and peripheral Destruction of Blood cells

Peripheral destruction or sequestration of blood cells (sequestration in splenomegalia) can lead to pancytopenia. In most cases destruction of erythrocytes is predominant, immune thrombocytopenia and autoimmune thrombocytopenia can cause low thrombocyte and neutrophil counts, respectively.

Myelophthisis

Myelophthisis is the infiltration of bone marrow by non hematopoietic cells, resulting in disruption of normal hematopoiesis or the development of bone marrow fibrosis. Causes include solid tumors with metastasis to the bone marrow, granulomatous and lipid storage disorders.

Acute MyeLoid Leukemia

Patients with acute myeloid leukemia present with a pathologic cellular blood count: normochromic anemia, platelet count greatly diminished, and leukocyte count below 4,000/μl in 20–30% of cases.

For further information refer to Section 15.15 – Acute leukemias.

Table 15.2-1 Erythrocyte reference intervals

Adults /3/

4.1–5.4

4.4–5.9

Children /45/

Fetus /6/*

1. day*

4.3–6.3

15. gestation wk

1.9–3.0

0.5 month*

3.9–5.9

16. wk

2.2–3.2

1 months*

3.3–5.3

17. wk

2.3–3.2

2 months*

3.1–4.3

18.–21. wk

2.6–3.6

4 months*

3.5–5.1

22.–25. wk

2.4–3.8

6 months*

3.9–5.5

26.–29. wk

2.7–4.3

9–12 months*

4.0–5.3

> 30. gestation wk

2.5–5.1

1.5–3.0 yrs

3.7–5.3

4–9 yrs

3.9–5.1

10–12 yrs

4.1–5.2

13–16 yrs

4.0–5.0

4.3–5.6

Values expressed in 106/μL or 1012/L, * Values are 2.5th and 97.5th percentiles

Table 15.2-2 Reference intervals of erythrocyte indices

MCV
(fL)

MCH
(pg)

MCHC
(g/L)

RDW
(%)***

Adults* /2/

80–96

28–33

330–360

< 15

%HYPO /13/*

1–5%

Age

MCV
(fL)

MCH
(pg/cell)

MCHC
(g/L)

Children /514/**

Umbilical cord

101–125

33–41

310–350

1 day

98–122

33–41

310–350

2–6 days

94–135

29–41

240–360

14–23 days

84–128

26–38

260–340

24–37 days

82–126

26–38

250–340

40–50 days

81–125

25–37

260–340

2.0–2.5 months

81–121

24–36

260–340

3.0–3.5 months

77–113

23–36

260–340

5–7 months

73–109

21–33

260–340

8–10 months

74–106

21–33

280–320

11–13.5 months

74–102

23–31

280–320

1.5–3.0 yrs

73–101

23–31

260–340

4–12 yrs

77–89

25–31

320–360

13–16 yrs

79–92

26–32

320–360

Children /3/

MCV
(fL)**

Fetus

Week 15

127–159

Week 16

119–167

Week 17

121–153

Weeks 18–21

119–143

Weeks 22–25

109–141

Weeks 26–29

103–134

Week ≥ 30

97–132

* Values are 2.5 th and 97.5 th% confidence interval with non parametric distribution

** Values expressed as x ± 2s

*** Values are instrument-dependent

Conversion: 1 × 10–15 L = 1 fL; pg × 0.062 = fmol; g/L × 0.062 = mmol/L

Table 15.2-3 Classification of anemia based on MCV and RDW /18/

Microcytic isocytic

Microcytic anisocytic

Normocytic isocytic

Normocytic anisocytic

Macrocytic isocytic

Macrocytic anisocytic

MCV

RDW

MCV

RDW

MCV

RDW

MCV

RDW

MCV

RDW

MCV

RDW

Decrease

Normal

Decrease

Elevated

Normal

Normal

Normal

Elevated

Elevated

Normal

Elevated

Elevated

β-thalassemia minor

Iron deficiency anemia

Anemia of chronic disease

Osteomyelofibrosis

Aplastic anemia

Pernicious anemia

Table 15.2-4 Classification of anemia based on MCV, MCH and MCHC /15/

Erythrocyte
indices

Clinical and laboratory findings

MCV normal

MCH normal

MCHC normal

Normochromic normocytic anemia:

  • Non regenerative anemia (e.g., chronic renal disease, chronic inflammatory disease, systemic infection, chronic liver disease, malignant tumor, endocrine disease, maldigestion, malabsorption)

MCV normal

MCH elevated

MCHC elevated

Apparently hyperchromic anemia due to pre-analytic or analytic interference:

  • Intravascular hemolysis, in vitro hemolysis
  • Hyperlipidemia, causes erroneously high hemoglobin concentration
  • Heinz bodies in toxic hemolytic anemia, unstable hemoglobin, enzymopathy
  • Laboratory errors due to erroneously low hematocrit or erroneously high hemoglobin measurement.

MCV normal

MCH reduced

MCHC normal

Incipient iron deficiency anemia. The RDW is usually above 15%, the %HYPO above 5% and the reticulocyte Hb content (Ret-He, CHr) is below 28 pg. See Section 15.6 – Reticulocyte count and reticulocyte indices.

MCV reduced

MCH reduced

MCHC reduced

Most common form of anemia. In 80–90% of the cases in Northern and Central Europe, classical iron deficiency anemia or anemia of chronic disease with iron-restricted erythropoiesis is present; in some 5% of the cases, β-thalassemia is the cause. Hereditary sideroblastic anemia is a rare cause.

MCV reduced

MCH normal

MCHC elevated

Severe hereditary spherocytosis, a hemolytic disease with an increase of the erythrocyte number, the hemoglobin concentration, and the hematocrit. Hereditary spherocytosis is not a hyperchromic anemia.

MCV elevated

MCH reduced

MCHC reduced

Regenerative anemia (e.g., observed during the first few days of adequate supplementation in anemias due to an underlying deficiency of iron, copper or vitamin B6).

MCV elevated

MCH normal

MCHC normal/

low

  • Folic acid or vitamin B12 deficiency anemia, liver cirrhosis, alcoholism
  • Cancer patients under cytostatic therapy
  • Myelodysplastic syndrome, hereditary stomatocytosis.

MCV elevated

MCH elevated

MCHC elevated

Presence of high titer cold agglutinins, that cause erythrocyte agglutination. The erythrocyte number is underestimated, and the MCV is overestimated. In consequence, the erythrocyte number and the hematocrit are erroneously low, and the MCH and MCHC calculations are erroneously high.

Table 15.3-1 Formation of hemiglobincyanide from hemoglobin /2/

Hb (Fe2+) + [FeIII(CN)6]3– Hi (Fe3+) + [FeII (CN)6]4–

Hi (Fe3+) + CN Hi [FeCN]2+

Table 15.3-2 Hemoglobin reference intervals

Fetus /3/*

Week 15

109 ± 7

Week 16

125 ± 8

Week 17

124 ± 9

Weeks 18–21

117 ± 13

Weeks 22–25

122 ± 16

Weeks 26–29

129 ± 14

Weeks > 30

136 ± 22

Children /56/***

Umbilical cord

135–207

1. day

152–235

2–6 days

150–240

14–23 days

127–187

24–37 days

103–179

40–50 days

90–166

2.0–2.5 months

92–150

3.0–3.5 months

96–128

5–7 months

101–129

8–10 months

105–129

11–13.5 months

107–113

1.5–3 years

108–128

5 years

111–143

10 years

119–147

12 years

118–150

15 years

128–168

Adults /4/**

115–160

135–178

Data expressed in g/L.

* Values are x ± 1 s

** Values are 2.5th and 97.5th percentiles

*** Values are x ± 2 s.

Conversion:

– mmol/L = g/L × 0.0621; g/L = mmol/L × 16.1

– mmol/L = g/dL × 0.621; g/dL = mmol/L × 1.61

Table 15.3-3 Proposed low hemoglobin thresholds for white and black adults /9/

Group

Age (years)

Hb (g/L)

White men

20–59

137

≥ 60

132

White women

20–49

122

≥ 50

122

Black men

20–59

129

≥ 60

127

Black women

20–49

115

≥ 50

115

Based on the Scripps-Kaiser data for the 5th percentile. Ref. 7 Tab. 2. NHANES data are considered to be confirmatory.

Table 15.3-4 Proposed low hemoglobin thresholds for children in the USA /10/

Age (years)

Hb (g/L)

HCT (fraction)

1–1.9

110

0.330

2–4.9

112

0.340

5–7.9

114

0.345

8–11.9

116

0.350

12–14.9

118

0.355

123

0.370

15–17.9

120

0.360

126

0.380

≥ 18

120

0.360

136

0.410

Table 15.3-5 Correction for anemia during an extended stay at high altitude /10/

Altitude (feet)

Hb (g/L)

HCT (fraction)

< 3,000

0.0

0.0

3,000–3,999

+ 2.0

+ 0.005

4,000–4,999

+ 3.0

+ 0.010

5,000–5,999

+ 5.0

+ 0.015

6,000–6,999

+ 7.0

+ 0.020

7,000–7,999

+ 10

+ 0.030

8,000–8,999

+ 13

+ 0.040

9,000–9,999

+ 16

+ 0.050

≥ 10,000

+ 20

+ 0.060

The Hb or HCT value must be added to the reference interval value.

Table 15.3-6 Correction for anemia in smokers /10/

Smoker status

Hb (g/L)

HCT (fraction)

Non-smokers

0.0

0.0

Smokers (all)

+ 3.0

+ 0.0010

0.5–1 packs/day

+ 3.0

+ 0.0010

1–2 packs/day

+ 5.0

+ 0.0015

> 2 packs/day

+ 7.0

+ 0.0020

The Hb or HCT value must be added to the reference interval value.

Table 15.3-7 Classification of anemia according to erythropoietic activity of the bone marrow

Clinical and laboratory findings in hyporegenerative anemia

Ineffective erythropoiesis may be present due to inadequately low erythropoietin synthesis or to intrinsic hypo proliferation of erythropoiesis; however, a combination of both causes usually occurs. Causes are:

  • Deficiency states; iron, vitamin B12, folate, erythropoietin (chronic kidney disease), hormones (thyroid, hypophysis or adrenal gland)
  • Disorder of proliferation and/or stem cell differentiation; chronic disease (inflammation, autoimmune disease, tumor, liver disease), toxic (alcohol, cytostatics), radiation, aplastic anemia
  • Displacement of erythropoiesis; acute leukemia, myelodysplastic syndrome, myeloproliferative syndrome, malignant lymphoma, metastases of solid tumors in the bone marrow, storage diseases.

Clinical and laboratory findings in hyperregenerative anemia

In these anemias the erythropoietic response corresponds to the extent of the anemia. The increase in erythropoietin secretion is adequate with respect to the fall in Hb and the erythropoietic tissue is healthy. The following may be the underlying cause:

  • Therapy-associated regeneration of erythropoiesis (therapy with iron, folate or Vitamin B12)
  • Subacute bleeding (maglinant tumor)
  • Enhanced erythrocyte degradation; hypersplenism, hemolytic anemia ,direct toxic (e.g., malaria, Wilson’s disease), mechanical (e.g., artificial heart valves, vascular prostheses, disseminated intravascular coagulation)
  • Erythrocyte membrane defects (e.g., hereditary spherocytosis, elliptocytosis, paroxysmal nocturnal hemoglobinuria)
  • Hemoglobinopathies, enzyme defects

Table 15.3-8 Blood cell status in certain groups of individuals and patients

Clinical and laboratory findings

Anemia in children

In the first weeks of life, newborns undergo a decline in erythrocyte mass, which leads to a decrease in Hb value and in the hematocrit. In healthy infants aged 10–12 weeks, the Hb nadir seldom falls below 90 g/L. The fall is more marked in premature infants, in whom the nadir may be as low as 80 g/L or 70 g/L with birth weights of 1.0–1.5 kg and of below 1.0 kg, respectively. While full-term newborns can tolerate the fall in Hb and, therefore, one speaks of physiological anemia, premature infants may manifest symptoms that require blood transfusion or therapy with erythropoiesis-stimulating agents (ESA) /20/. The cause of the marked decline in Hb in premature newborns is iron deficiency, due to insufficient iron storage caused by the pre term birth, earlier postnatal activation of erythropoiesis, faster postnatal growth, and frequent blood sampling for diagnostic purposes. The reference intervals for the blood count and iron metabolism parameters in full term neonates are also valid for premature infants during the first year of life /21/.

Hb concentration and hematocrit increase continuously with age. In the USA, the Centers for Disease Control defined the lower limits for Hb shown in Tab. 15.3-4 – Proposed low hemoglobin thresholds for children in the USA.

Sports anemia /16/

Endurance athletes often show reduced levels of Hb and HCT. Sports anemia is caused mainly by an increase in plasma volume relative to the red blood cell mass causing hemodilution although erythropoiesis is stimulated. Acute heavy exercise decreases the arterial O2 saturation and changes renal hemodynamics; both are triggers for the erythropoietin production.

Anemia in the elderly /22/

The mean prevalence of anemia in individuals over the age of 65 is 17%. Approximately 60% of individuals over the age of 65 have diseases that lead to diminished Hb levels. In the Cardiovascular Health Study cohort, individuals older than 65 years had a poorer clinical outcome with Hb values of below 137 g/L (men) or 126 g/L (women). Only 1% of elderly individuals who are not hospitalized have Hb concentrations of less than 100 g/L. The anemia may possibly be masked in some cases due to diuretics-associated hemoconcentration. According to the Chianti study, the prevalence of anemia is considerably higher in individuals with a creatinine clearance of below 30 [mL × min–1 × (1.73 m2)–1/23/. Elderly individuals with low Hb values are at elevated risk of hospitalization and mortality, in comparison with those whose values are within the reference interval. This also holds for elderly individuals with mild anemia (women Hb 100–119 g/L, men 100–129 g/L). The three-year risks for hospitalization and mortality were associated with hazard ratios of 1.32 and 1.86, respectively /24/. One fourth of anemia cases in the elderly are not explained by nutrient deficiency (iron, vitamin B12, folic acid), renal insufficiency, chronic inflammation or myelodysplastic syndrome /25/.

Gestational anemia

Erythropoiesis undergoes substantial changes during pregnancy /26/. Starting from the 10th week of gestation, increases in blood volume by 30–40%, in plasma volume by 40–50%, and in erythrocyte mass by 20–30% occur. As of the 10th week of gestation this increase can be observed, with a peak between the 32nd and 34th week of gestation, thereafter it remains rather constant. WHO recommends that the Hb concentration should not fall below 110 g/L at any time during pregnancy and not below 100 g/L in the puerperium /8/. In the puerperium, Hb decreases by 8 g/L and the HCT by 0.10 relative to the levels before delivery, while the reticulocytes already increase during delivery. The Hb concentration and the hematocrit increase continuously from the 4th day postpartum onwards /27/.

Vegetarian adults

A variety of different definitions exist /76/:

  • Vegetarians are individuals who consume red meat no more than once a month, consume fish and chicken no more than once a week.
  • Someone who never consumes red meat and consumes chicken or fish no more than once a week.
  • Lacto-ovo vegetarians and lacto-vegetarians eat no meat products, fish or poultry but consume both diary products and eggs or dairy products, respectively.

Vegans exclude meat, fish, poultry, dairy products and eggs.

Adult vegetarians, especially females in the reproductive age, have a high prevalence of depleted iron stores (ferritin < 15 ug/L) and iron deficiency anemia (Hb < 120 g/L) compared to nonvegetarians. In children on a vegetarian diet serum ferritin concentration was significantly lower in nonvegetarians, but the concentration of the soluble transferrin receptor (sTfR) was significantly higher and the serum hepcidin concentration significantly lower than in nonvegetarians /77/.

Anemia in heart failure /16/

A high proportion of patients with chronic cardiac insufficiency have anemia and Hb values of 100–120 g/L. Anemia in this order of magnitude causes an increase in exercise cardiac output. This results in an increased preload wall stress and left ventricular work, which in turn increases oxygen consumption and accelerates myocyte loss. The hypoxia at the tissue level and decreased blood viscosity cause arterial vasodilatation, which decreases after load. A chronic volume overload state induced by anemia causes the addition and lengthening of myofibrils causing ventricular dilatation and an increase in wall tension (see Section 2.7 – Chronic heart failure).

Anemia predicts mortality in severe heart failure . In the Prospective Randomized Amlodipine Survival Evaluation (PRAISE) the relationships between baseline hemoglobin/hematocrit and mortality in patients with left ventricular ejection fraction of less than 30% and a NYHA classification functional class IIIb and IV were evaluated over the course of 15 months. Patients in the lowest quintile (Hb 116 ± 9 g/L) had a 52% higher mortality risk (hazard ratio 1.52) than the patients in the highest quintile (Hb 162 ± 9 g/L) /28/.

Anemia in the critically ill /29/

Critically ill patients in intensive care units often have anemia and Hb value ≤ 100 g/L. This is based upon the fact that these patients may have substantial changes in plasma volume (hyper hydration, dehydration), which render the assessment of the Hb concentration more difficult. About 62% of patients admitted to intensive care units have an Hb value of less than 120 g/L, and 29% below 100 g/L. Generally, normocytic, normochromic anemia is found, associated with hemorrhage (elevated reticulocytes), reduced erythrocyte formation due to inflammation (decreased reticulocytes), or hemolysis or sequestration of erythrocytes (increased reticulocytes). Reactive hemophagocytic syndrome may also be present as a result of an infectious trigger or secondary to malignant disease. The blood cells are phagocytized by histiocytes, resulting in pancytopenia.

Postoperative anemia /31/

For the postoperative management and diagnosis of anemia in surgical patients the following diagnostic management is recommended: whenever assess the iron status within 24 h postoperatively, if it has not been already performed in the pre-operative assessment. Monitor hemoglobin (Hb) 3-4 days postoperatively. Iron treatment should be considered if postoperative ferritin is below 100 μg/L or below 300 μg/L and transferrin saturation below 20%, or reticulocyte hemoglobin content is below 28 pg. The calculation of total iron deficiency is as follows: total iron deficiency (mg) = (target Hb – actual Hb) × weight (kg) × 0.24. Hb value is measured in g/L. Add another 10 mg/kg for replenishing iron stores.

Anemia of chronic kidney disease (ACD)

Renal anemia is normocytic, normochromic, and hypo regenerative. See also Tab. 15.10-2 – Diseases and conditions associated with an adequate rise in EPO concentration. The reasons are diminished erythropoietin (EPO) synthesis in relation to the Hb level and the cumulation of toxic substances which are excreted normally in the urine. Both factors cause intrinsic hypo proliferation of erythropoiesis and are possibly also responsible for the slight reduction in the erythrocyte life span. The National Kidney Foundation Disease Outcomes Quality Initiative (KDOQI) recommends that all patients with a GFR of below 60 [mL × min–1 × (1.73 m2)–1] be evaluated for anemia. According to the results of the Third National Health and Nutrition Examination Survey (NHANES III) /30/, a Hb value of below 110 g/L was diagnosed in 42.2% of patients with an eGFR of less than 30 [mL × min–1 × (1.73 m2)–1], and in 3.5% of those with a GFR of 30–59 [mL × min–1 × (1.73 m2)–1]. A relevant cause is an inadequate response of EPO to the Hb decrease. This is not the case in polycystic kidney disease, in which the synthesis of EPO is maintained. In these patients it is important to evaluate iron metabolism. See Section 7.3 – Ferritin, Section 7.4 – Soluble transferrin receptor (sTfR), and Section 7.5 – Transferrin saturation (TfS). Treatment for patients undergoing hemodialysis consists of administration of ESA . Functioning iron metabolism is important in order to assure effective erythropoiesis. Therefore, prior to the start of treatment, ferritin level should be at least 100 μg/L (better 200–500 μg/L), TfS should be ≥ 20% (better 30–40%), %HYPO should be below 10% (better below 2.5%), and for monitoring of iron demand of erythropoiesis under ESA therapy, CHr (Ret-HE) is determined. An increase to ≥ 29 pg indicates no iron demand of erythropoiesis /32/. In a study a high-dose intravenous iron regimen administered pro actively (400 mg monthly unless the ferritin concentration was > 700 μg/L or the transferrin saturation was 40% or higher), resulted in lower dose of ESA being administered /33/.

Anemia in diabetes

Approximately one fifth of non-hospitalized diabetics has anemia, defined by Hb levels of below 120 g/L (women) and 130 g/L (men). According to one study /34/:

  • Patients with a GFR above 60 [mL × min–1 × (1.73 m2)–1] and without albuminuria have higher Hb values than those with albuminuria
  • Patients with macro albuminuria have lower Hb values than those with micro albuminuria
  • Patients with a GFR below 60 [mL × min–1 × (1.73 m2)–1] and normo albuminuria have similar Hb values to those with a GFR of greater than 60 [mL × min–1 × (1.73 m2)–1] and macro albuminuria.

In diabetics, therefore, the evaluation of anemia should be performed not only in impaired GFR, but also in the presence of albuminuria in patients with a normal GFR.

Clinically, the anemia can emerge earlier than the renal insufficiency.

The pathophysiology of anemia in diabetic nephropathy is multifactorial. Relevant factors are damage to the interstitial cells and the renal vascular architecture, with consequent interstitial fibrosis. The more marked this is, the less erythropoietin is produced. There is limited evidence to suggest that correction of anemia, specifically in patients with complications of diabetes, might be beneficial. However, while correction of anemia in patients with advanced kidney disease may stabilize left ventricular hypertrophy, earlier intervention may lead to regression.

Alcoholism

Alcoholism induced anemia is multifactorial. Important pathophysiological factors are:

  • Toxic suppression of erythropoiesis due to inhibition of ribosomal and mitochondrial protein synthesis
  • Megaloblastic changes due to deficient folic acid metabolism caused by alcohol
  • Deficient vitamin B6 metabolism and, consequently, mitochondrial accumulation of iron in erythroblasts and formation of ring sideroblasts
  • Changes in the erythrocyte membrane due to alcohol, resulting in hemolysis and reduced erythrocyte life span.

Laboratory findings: the Hb level is 80–120 g/L, the mean MCV is 5–10% higher than in healthy controls, and the RDW manifests dimorphism. In the blood smear, acanthocytosis and stomatocytosis and ring sideroblasts can be present.

Cancer

Patients receiving ongoing chemotherapy who present with anemia (Hb below 110 g/L or Hb decrease more than 20 g/L from a baseline level below 120 g/L) and absolute iron deficiency (serum ferritin below 100 ug/L) should receive iron treatment with an i.v. iron preparation to correct iron deficiency. If ESA (erythropoietin stimulating agent) treatment is considered, iron treatment should be given before the initiation of ESA therapy in the case of functional iron deficiency (transferrin saturation < 20% and serum ferritin > 100 ug/l) /61/.

Red blood cell (RBC) transfusion should be considered in patients with Hb < 70–80 g/L or severe anemia related symptoms (even at higher Hb levels) and the need for immediate and Hb symptom improvement, the administration of RBC transfusion without delay is justified /61/.

Studies reporting the outcome of patients receiving transfusions during radical surgery for non-metastatic cancer report that RBC transfusions were associated with an increased risk of death (hazard ratio 1.36; 95% CI 1.26–1.46). The survival was reduced even in cancer at survival and increased the risk of relapse /74/.

Table 15.3-9 Classification and differentiation of microcytic anemia

Clinical and laboratory findings

Microcytic anemia

Depending on the hematology analyzer, an MCV below 80–83 fL is characteristic. It is always necessary to assess the MCV, in association with the erythrocyte distribution width, in order to rule out erythrocyte dimorphism or to assess erythrocyte morphology in the blood smear. If a low MCV has been confirmed it is differentiated, based upon the MCHC, whether hypochromia (MCHC below 320 g/L) or normochromia (MCHC ≥ 320 g/L) are present.

Microcytic, hypochromic anemia can result from a defect in the globin genes (hemoglobinopathy or thalassemia), a defect in heme synthesis, or erythroid precursor cell demand for iron. Usually iron restricted erythropoiesis, which is due to nutritive iron deficiency or chronic or acute bleeding is the main cause /35/.

Iron deficiency anemia

In women up to the age of 50, menstrual bleeding, in men of the same age group nutritional iron deficiency, and in both genders above the age of 50 gastrointestinal bleeding due to tumors are most common causes of iron deficiency /36/. In children, reduced iron intake is the main cause of iron deficiency. Depending upon the extent and duration of iron deficiency, Hb, MCV, MCH and MCHC are reduced. The erythrocyte count is usually low-normal or only moderate decreased. The hypochromia usually predominates relative to the microcytosis /7/. RDW is increased to ≥ 15%. It takes some 8 weeks from the time of empty iron stores, with transition to iron-restricted erythropoiesis, until the occurrence of an elevated percentage of hypochromic erythrocytes. If the proportion of hypochromic erythrocytes (%HYPO) is determined, this time is reduced to 2–3 weeks, and with the determination of reticulocyte Hb content (CHr, RetHE), it is reduced still further, to some 4 days. Erythrocyte poikilocytosis (irregular shape) and anisocytosis (unequal size) are seen in the blood smear. In iron-restricted erythropoiesis, the %HYPO increases to ≥ 5%, and the CHr decreases to below 28 pg. Under effective oral iron therapy, the Hb value increases within 4 weeks to over 10 g/L, as do the CHr after one week. For further diagnostic investigation, see Chapter 7 – Iron metabolism and Section 15.2 – Erythrocytes (cell count and indices). In iron deficiency anemia, erythropoiesis is hypo regenerative to normo regenerative.

β-thalassemia

In β-thalassemia, the biochemical defect is the lack of synthesis of β-globin chains of the erythroid precursor cells (see Section 15.7 – Hemoglobinopathies). In heterozygous trait carriers (thalassemia minor), this leads to deficient erythrocyte hemoglobinization. In homozygous patients (thalassemia major), not only is there a lack of β-globin chains but also, erythropoiesis is impaired due to a relative excess of α-globin chains. These excessive chains, which cannot bind to HbA, precipitate in the erythroid precursor cell and leads to cell death and dyserythropoiesis /37/. Thalassemia major is associated with severe hypochromic anemia in childhood. In uncomplicated cases of thalassemia minor, Hb concentrations are 110–120 g/L. This is maintained by means of a high erythrocyte count of (5–7) × 1012/L. MCV is below 80 fL, usually even below 75 fL, and it can even decrease to 55 fL. MCH is significantly decreased, MCHC is greater than 310 g/L, RDW is normal since the microcytosis is uniform. Microcytosis predominates in relation to hypochromia and a %MICRO/%HYPO ratio above 0.9 with a hypochromic erythrocyte fraction ≥ 19% is indicative of heterozygous β-thalassemia /38/. This can be associated with mild reticulocytosis and elevated concentrations of soluble transferrin receptor. Hematological values in heterozygous and homozygous β-thalassemia are shown in Tab. 15.3-10 – Hematological data in patients with β-thalassemia.

HbE syndrome

After HbS, HbE is the second most common Hb variant. The gene occurs with a prevalence of some 10% in Southeast Asia. In the region of Cambodia, Laos and Thailand, the prevalence of HbE is 20–40%. HbE is a mutation that is due to the substitution of glutamine with lysine at position 26 of the β-globin chain. The cause is the exchange of guanine with adenine at codon 26 of the β-globin gene. Since, in addition, this mutation leads to a change in the splicing site, a decrease in functional β-globin mRNA occurs with, in consequence, reduced formation of β-chains, resulting in a milder thalassemia phenotype. There exist three HbE syndromes; common to all of them is microcytosis and a varying HbE proportion /39/:

  • HbEE is the homozygous phenotype. This is a benign condition with no, or only minimal, anemia. The MCV is 20–25 fL lower than normal, and the life span of the erythrocytes is normal. The erythrocyte count is elevated, while the reticulocyte count is usually normal. In the blood smear, up to 75% target cells can be detected, on Hb electrophoresis no HbA is found, and HbE is detected only in combination with a low percentage of HbF.
  • The heterozygous phenotype is HbAE. Individuals of this type are, for the most part, asymptomatic; anemia is not present. The erythrocyte count is slightly elevated, the MCV is 10–15 fL below normal. A few target cells are visible in the blood smear. Hb electrophoresis shows an HbE fraction of some 30%; the rest is HbA.
  • HbE/β-thalassemia phenotype. Heterozygous HbE and β-thalassemia is present, with a clinical picture that is similar to that of β-thalassemia. The clinical symptomatology is variable; it can be associated with mild microcytic anemia and can, also, require transfusion.

Hereditary spherocytosis (HS) /40/

HS is a hemolytic anemia in Caucasians that is often genetically determined, with an incidence of 1 in 2000–5000 births. Some 75% of the cases are inherited as an autosomal dominant disease, the rest are inherited in a recessive manner or a novel mutation may be involved. The molecular defects are very heterogeneous, and include genes that code for the erythrocyte proteins spectrin, ankyrin, and band 3. All of these proteins are involved in the structure of the red blood cell cytoskeleton, and a deficiency or dysfunction leads to the formation of spheroid, fragile and osmotically labile erythrocytes which are captured and destroyed by the spleen. The defective protein can be detected with SDS polyacrylamide gel electrophoresis. The spectrin deficiency phenotype is usually associated with severe anemia and more spherocytes in the smear than the band 3 deficiency phenotype. In one study /41/, HS manifested as mild, moderate and severe forms in 38%, 11%, and 9% of the cases, respectively. The membrane defect (spectrin, ankyrin, band 3) was not clearly associated with the severity of the HS. The diagnosis is usually already made in children, based upon anemia, icterus and splenomegaly; in adults, the disease is diagnosed on the basis of splenomegaly and bile stones. The severity of the anemia correlates with the extent of hemolysis and the splenomegaly. Splenectomy leads to reversal of the anemia.

Laboratory findings: Hb low or normal, MCV low (particularly good to determine with hematology analyzers that use flow cytometric techniques), MCHC elevated, RDW increased, percentage of hyper dense red blood cells increased, reticulocytosis, elevated erythropoietin. Following splenectomy, normalization of Hb, reticulocytes and erythropoietin. HbS typically comprises 35% to 45% of the total hemoglobin, unless concurrent iron deficiency or alpha thalassemia is present Iron deficiency and alpha thalassemia reduce the percentage of hemoglobin comprised by HbS in sickle trait.

Sideroblastic anemia /42/

Sideroblastic anemia is characterized by anemia with the emergence of ring sideroblasts in the bone marrow. Ring sideroblasts are erythroblasts characterized by iron accumulation in perinuclear mitochondria due to impaired iron utilization. There are two forms of sideroblastic anemia:

  • The inherited sideroblastic anemia is a rare and heterogenous disease caused by mutations of genes involved in heme biosynthesis, iron-sulfur (Fe-S) cluster biogenesis, Fe-S cluster transport, and mitochondrial metabolism.
  • The acquired sideroblastic anemia is relatively common and caused by drugs or alcohol; however, the best known acquired sideroblastic anemia is refractory anemia with ring sideroblasts (RARS), a subtype of myelodysplastic syndrome.

X-linked sideroblastic anemia (XLSA) /42/

XLSA is the most common inherited sideroblastic anemia. A mutation in the gene ALAS2 of the hematopoiesis-specific aminolevulinate synthase (ALAS) occurs. The enzyme ALAS2 catalyzes the first step in heme synthesis, the formation of aminolevulinic acid from glycine and succinyl-CoA (see also Fig. 7.1-6 – First step of heme synthesis). Many mutations occur, most of which are missense mutations. Hemizygous men manifest microcytic anemia and iron overload. The disease can already be diagnosed in newborns, but it often only becomes clinically apparent in middle age.

Sideroblastic anemia due to SLC25A38 gene mutation is the next most common inherited sideroblastic anemia. The iron-transferrin containing endosomes of the cell deliver iron directly to mitochondria. The SLC25A38 gene regulates the iron importer protein mitoferrin 1 (Mfrn1; SLC25A38), which is a member of the solute carrier family localized in the inner mitochondrial membrane, and is used for heme synthesis and iron-sulfur cluster biogenesis.

Laboratory findings: the most important characteristics are microcytic, hypochromic anemia, often with Pappenheim bodies (iron-positive inclusions), erythrocyte dimorphism (one microcytic and one normocytic population), ring sideroblasts in bone marrow, in particular iron accumulation in orthochromatic erythroblasts, secondary iron overload due to ineffective erythropoiesis. The disease is manifested in all age groups; it is more common in men than in women, but the anemia is more severe in women.

Iron refractory iron deficiency anemia (IRIDA)

A new cause of hereditary anemia called IRIDA (OMIM 206200) has recently been described. IRIDA is due to a mutation in the TMPRSS6 gene encoding the membrane-bound serine protease matriptase-2. Matriptase-2 cleaves hemojuvelin, a major regulator of hepcidin expression and that this function is altered in this genetic form of anemia /71/. In contrast to low hepcidin levels, observed in acquired iron deficiency, in patients with matriptase-2 deficiency, serum hepcidin is inappropriately high for the low iron status and accounts for the absent/delayed response to oral iron treatment. Refer to Fig. 7.6-4 – Signals and pathways for the regulation of hepcidin expression by stimulating factors. The prevalence of this condition is not known, but it has certainly been under-diagnosed up to now. Clinically, IRIDA patients are chacterized by a hypochromic microcytic anemia. The clinical phenotype develops only after the neonatal period.

Laboratory findings: hypochromic microcytic anemia, very low serum and transferrin saturation levels, ferritin concentrations are mostly within the reference range. Normal to high hepcidin levels characterize IRIDA. In inflammatory conditions a laboratory diagnostic pattern is analyzed similar to what is observed in IRIDA. However, the anemia is normo- or moderately microcytic as opposed to the marked microcytosis in IRIDA.

Post-transplantation anemia (PTA) /43/

PTA (Hb in women below 120 g/L, in men below 130 g/L) is a common problem that may hinder patient’s quality of life. It occurs in 12–94% of patients, and is most common in the immediate post transplant period. Thus, in one study of 240 kidney transplant recipients, the prevalence of PTA was 76%; one year later it was 21%, and after 4 years it was 36% /44/. During the first 6 months, predominantly microcytic anemia was present. One cause of microcytosis is therapy with sirolimus. The extent of the anemia is dependent upon the chronic kidney disease (CKD) stage following transplantation. The anemia prevalence in CKD stages 1, 2, 3, 4 and 5 was, in each case, 0%, 3%, 7%, 27% and 33%.

Patients who are disease-free after two or five years after hematopoietic cell transplantation (HCT) have a greater than 80% subsequent 10-year survival rate. Many studies show that HCT survivors suffer from significant late effects that adversely affect morbidity, mortality, working status, and quality of live. Laboratory tests for detection of late effects are TSH (hypothyroidism), glucose and HbA1c (diabetes), lipids (dyslipidemia), Cortisol-stimulation test (adrenal insufficiency), testosterone (gonadal dysfunction), FSH, LH (gonadal dysfunction), ferritin (iron overload), ALT (hepatitis) /69/.

Post-transplantation lymphoproliferative disorders (PTLD) account for 21% of all cancers in recipients of solid organ transplants. In the adult population the incidences of recipients are: kidney transplants 0.8–2,5%, pancreatic transplants 0.5–5%, Liver transplants 1–5.5%, heart transplants 2–8%, lung transplants 3–10%, and multi organ and intestinal transplants ≤ 20%. PLTD is a consequence of therapeutic immunosuppression /70/

Hemolytic anemia

Hemolysis occurring in hematologic diseases is often associated with an iron loading anemia. The iron overload is the result of entrance of free hemoglobin in the circulation. Massive hemolysis is confirmed by a complete decrease of haptoglobin and hemopexin, increased lactate dehydrogenase, an increase in red cell distribution width, a reduced half-life of red blood cells, an increase in ferroportin, and a decrease in hepcidin. Tissue iron overload derived from heme or hemoglobin is primarily localized in the liver and spleen macrophages rather than hepatocytes. Because of depressed hepcidin formation of the hepatocytes enteral iron absorption is increased. Serum ferritin is increased but transferrin saturation remains in the normal range. The absence of hepatocyte iron overload is a consequence of both the huge increase in erythroblast production and urinary iron losses /72/.

Table 15.3-10 Hematological data in patients with β-thalassemia /37/

Parameter

Normal

Hetero-
zygous

Homo-
zygous

Hb (g/L)

110–150

80–110

40–70

MCH (pg)

27–33

16–24

16–24

MCV (fL)

75–90

60–75

60–75

Reticulocytes (%)

1–2

1–2

2–10

HbA (%)

96–98

90–95

0–20

HbF (%)

0–2

1–5

60–90

HbA2 (%)

1–3

4–6

0–10

Erythroblasts

0

0

Many

Table 15.3-11 Classification and differentiation of normocytic anemia

Clinical and laboratory findings

Normocytic normochromic anemia – Generally

In normocytic normochromic anemia, MCV, MCH, and MCHC values are normal. These anemias are subdivided into hypo regenerative and hyper regenerative forms, corresponding to their regenerative erythropoietic activity. Major hyper regenerative anemias are posthemorrhagic anemia and hemolytic anemia. Corresponding to the regenerative erythropoietic activity, an increase in reticulocytes and in sTfR is to be expected. The pathophysiology of hypo regenerative normocytic normochromic anemias is different and is associated mainly with inflammation, malignancies, or diminished erythropoietin synthesis /7/.

Hyper regenerative normocytic anemia

Hyper regenerative anemias are due to:

  • Erythrocyte destruction (hemolysis; erythrocyte life span is decreased)
  • External or internal bleeding (e.g., hemorrhage or gastrointestinal bleeding)
  • Treatment of deficiency anemia (e.g., during the regeneration phase following iron substitution or substitution with vitamin B12 or folate).

Laboratory findings: characteristic findings are reticulocytosis and, in the blood smear, polychromasia of the erythrocytes. The regenerative responses (i.e., reticulocytosis and polychromasia) are more marked in hemolytic anemia than in anemia due to bleeding. In hemolytic anemia, this is due to recirculation of iron stored in macrophages and of factors exerting stimulatory effects on erythropoiesis /45/.

Hemolytic anemia (HA) – Generally

HA comprises some 5% of anemias and can have the causes listed in Tab. 15.3-12 – Causes of hemolytic anemia. Hemolysis can be associated with normocytic or microcytic anemia. The hemolysis can take place in the vascular system (intravascular hemolysis) or in the spleen (extravascular hemolysis) in the presence of splenomegaly. This pathological process is termed hypersplenism. Both types of hemolysis can cause hyperbilirubinemia. While reticulocytosis is an indicator of both extravascular and intravascular hemolysis, decreased haptoglobin (Hp) mainly occurs with intravascular hemolysis. The half-life time of Hp is 4 days, but the Hp-Hb complex survives for only a few minutes. Since, in hemolysis, the Hp catabolic rate is much greater than that of its synthesis, the Hp concentration is reduced. Intravascular and extravascular hemolysis often occur together, but one form predominates. Plasma total protein concentration does not change in HA /46/. HA is associated with hypercoagulability /47/.

HA is hyperregenerative; on days 2–3 reticulocytosis of 15% is reached, and this can be as high as 50% after one week, particularly in immune mediated hemolysis. For further information on hemolytic anemia, see Sections LD, reticulocytes and haptoglobin as well.

Hereditary HA is essentially caused by an erythrocyte membrane disorder (hereditary spherocytosis, hereditary ellipocytosis), by erythrocyte enzyme defects, by diminished formation of normal Hb (thalassemia syndrome) or by the synthesis of qualitatively abnormal Hb (HbE syndrome, sickle cell anemia). With the Hb disorders, those with α-globin chain abnormality are already manifested clinically at birth, those with β-globin chain disorders first become obvious at the age of 4–6 months /39/. Refer also to Tab. 7.6-2 – Disorders of hepcidin and ferroportin regulation.

– Hereditary elliptocytosis

This disease is occasionally discovered during the assessment of a blood smear. These patients have an elliptocyte proportion of 15–70% relative to the total erythrocyte population. They are, normally, not anemic, and their automated blood count is usually normal. Approximately 5–20% of the patients have compensated or mild anemia and reticulocytosis of about 20%.

– Enzyme defects

Enzyme defect anemia makes up some 18% of non-spherocytic hemolytic anemia. Pyruvate kinase and glucose-6-phosphate dehydrogenase deficiencies affect, respectively, 54% and 30% of these anemias (see Section 15.8 – Erythrocyte enzymes).

– Sickling disease (SCD) /48/

SCD comprise a group of autosomal co-dominant hemoglobinopathies characterized by mutations in the gene encoding the hemoglobin ß-subunit on chromosome 11. The gene codes the assembly of two β-globin chains to HbA. The mutant β-allel (βS) codes for the production of the variant hemoglobin S(HbS). In the mutated sickle cell gene (βS), adenine is substituted with thymine and thus, in amino acid position 6 of the β-globin chain, valine is incorporated instead of glutamic acid. The heterozygous carrier state, known as sickle cell trait, results in the production of both hemoglobin A and S and has predominately a benign clinical picture. Five haplotypes of the βS-allele specific to different regions of the world exist. The haplotypes differ from one another in the severity of the clinical picture to which they give rise, and in the frequency and the pattern of the clinical symptoms. SCD patients are virtually resistant to malaria, since the intraerythrocytic parasites are degraded during hemolysis.

SCD is the most common hemoglobinopathy in the world, with a high incidence in Mediterranean countries, the near east, central Africa, and certain parts of India. In peoples of African origin, there are three major beta globin haplotypes (Benin, Bantu, and Senegal) which dominates in North America and the Caribbean. The Asian haplotype accounts for the disease in India and the Eastern Province of Saudi Arabia /79/.

Polymerization of deoxygenated HbS (PO2 < 40 mmHg) is the primary event in the pathogenesis of SCD. HbS is transferred from solid single molecules into a gel by binding of valine in position 6 to phenylalanine in position 85 in the adjacent HbS resulting in a typical fibre structure. The sickle cell gets its typical shape by polymerization of the fibre structures inside the erythrocyte /49/. If the HbS fraction is greater than 50%, the erythrocytes loose their plasticity and clog the capillaries of almost all organs, whereupon trophic disturbances and necrosis occur. Pain crises, which can last for minutes or days, result from the shift in the micro flow pathways. Four sickle cell syndromes are distinguished; their hematological findings are summarized in Tab. 15.3-13 – Laboratory findings in sickle cell anemia /39/:

  • HbSS is the homozygous phenotype. In Africa, some 120,000 children of this type are born annually; in the USA 3000–4000. Patients with this phenotype have normocytic normochromic anemia with Hb values of 60–100 g/L and reticulocytosis of 10–25%. Erythrocyte survival time is shortened to 5–20 days. While normally the sickle shape reverts to the discoid shape with oxygenation of the cells, with the homozygous phenotype up to 30% of the sickle cells are irreversibly altered and hemolyzed. The membrane rigidity of sickle cells is associated with the increased activity of the Ca2+ sensitive K+-channel and potassium-chloride co transporters cause dehydration and generate the dense cells (MCHC ≥ 460 g/L) /49/. The cells are visible in the blood smear and contain Howell-Jolly bodies. Patients with this phenotype manifest the clinical symptomatology outlined in the foregoing.
  • HbAS is a heterozygous phenotype consisting of 35–45% HbS, and the remainder HbA. In certain regions of West Africa, 20–25% of the population are carriers of the sickle cell gene; in the USA, this is the case for 10% of the black population. HbAS phenotypes do not manifest anemia; they have normal blood cell morphology.
  • HbSC phenotype: heterozygous inheritance of HbS and HbC, in the proportion of 45–55%, gives rise to symptoms milder than the HbSS disease. The anemia is mild, the MCV is reduced, and reticulocytosis of 3–6% is measured. In Africa, its prevalence is one fifth that of HbSS disease. The HbSC phenotype, like the HbSS disease, also manifests pain crises, and necrosis of the renal papillae, necrosis of the femoral head, and retinopathy are frequently observed. In contrast to HbSS disease, in which splenic atrophy occurs as of the age of 5 years, the HbSC phenotype manifests lifelong splenomegaly.
  • HbS/β-thalassemia phenotype: heterozygosity for HbS and β-thalassemia is present. This phenotype occurs preferentially in Mediterranean countries. The HbS fraction is 60–90%, and the remainder is HbF or, in a few cases, HbA. Anemia is mild (Hb approximately 100 g/L) and MCV and MCH are decreased. The clinical course is milder than in phenotypes HbSS and HbSC, since the increased erythrocyte HbF content prevents the formation of sickle cells.

Clinical findings: SCD is characterized by the painful recurrence of vaso-occlusive events, multi-organ morbidity and an increased risk of early death. There is strong evidence that elevated HbF level is associated with relatively mild clinical manifestations of SCD /66/. Young SCD patients have a diminished urinary concentrating ability, electrolyte regulation, and enhanced proximal tubular function. Because of increased renal plasma flow, the glomeruli are hypertrophic and the GFR is elevated. Proteinuria develops in the second decade of life; 10–20% of young patients often already manifest macroalbuminuria and, later, focal glomerular sclerosis. In the Baby Hug trial, it was shown that an incipient disorder of kidney function can be diagnosed early with DPTA-GFR, and that organ damage can be prevented with early hydroxyurea treatment /50/. Chronic administration of blood transfusion is frequent in SCD patients, and this leads to iron overload. Ferritin values of < 1,500 μg/L signal acceptable iron overload; values > 3,000 μg/L are associated with liver. The spleen is one of the first organs to be damaged in SCD. The damage is often silent and progressive, resulting in functional asplenia with a high risk of infections. Some children with SCD experience acute splenic sequestration and chronic hypersplenism.

Laboratory findings: RBC, hemoglobin, and hematocrit are low in hemolytic anemia patients with heterozygous S/0 and homozygous SS mutations White blood cell and platelets are increased and fluctuating. However reticulocyte counts vary and are affected by a variety of variables. In patients taking hydroxyurea, mean corpuscular volume (MCV) is increased. Because of the various RBC subpopulations, higher red cell distribution width is observed /51/.

Gene therapy with LentiGlobin consists of autologous transplantation of hematopoietic stem and progenitor cells transduced with the BB305 lentiviral vector a modified β-globin gene, which produces the antisickling HbAT87. One-time treatment with LentiGlobin resulted in sustained production of HbAT87Q in most red cells, leading to reduced hemolysis and severe vaso-occlusive events /80/.

– Paroxysmal nocturnal hemoglobinuria (PNH)

PNH is a rare, acquired non-malignant clonal stem cell disease, caused by a mutation of the Phosphatidyl inositol glycan-class (PIG)-A gene. PNH results from hematpoietic stem cells and its progeny that are unable to synthesize the glycosylphosphatidylinositol (GPI) anchors, which serves to tether the cell surface complement inhibitory proteins CD55 and CD59. Both of the proteins are are key regulators of the complement pathway and protect host cells from complement-mediated removal. The defect, or deficient GPI anchor protein, does not support the transfer of N-acetylglucosamine to glycosylphosphatidylinositol for the synthesis of the intact GPI anchor. The proteins that are to be anchored by GPI are synthesized, but they are not integrated into the cell membrane and are detectable in high concentrations in the plasma. The GPI anchor also permits the influx of GPI-specific phospholipase C for the separation of the anchored proteins from the cell membrane. The prevalence of PNH is 1 : 100,000 to 1 : 500,000. The complement dysregulation leads to chronic hemolysis and thrombosis characteristic for PNH /78/.

Clinical symptoms: PNH is characterized by a unique clinical triad: intravascular hemolysis, a high risk of thrombosis, and bone marrow impairment. By frequency: thrombosis 40%, anemia 35%, hemoglobinuria 26%, hemorrhage 18%, aplastic anemia 13%, gastrointestinal symptoms 10%, icterus 9%, iron deficiency anemia 6%.

Hemolysis: the somatic mutation in the X-linked gene PIGA makes the mutant hematopoietic stem cell and its progeny unable to synthesize the glycosylphosphatidyl inositol anchor. Therefore CD55 and CD59 do not protect hematopoetic cells from activated complement, the consequences are hemolytic anemia and hemoglobinuria due to chronic intravascular hemolysis. Other complaints are stomach pain, esophageal spasm, difficulties in swallowing.

Thromboembolism (TE) /52/: thromboembolism in PNH is associated with a 7-fold higher mortality risk. In 46% of patients, TE develops in spite of anticoagulation. The TE risk is 7-fold higher in patients with lactate dehydrogenase (LD) ≥ 1.5-fold the upper reference interval value in comparison to patients with lower levels. The TE risk is 3-fold increased in the presence of abdominal or thoracic pain, or in dyspnea. With LD ≥ 1.5-fold and thoracic pain, abdominal pain, dyspnea and hemoglobinuria, TE risks are increased 19-fold, 18-fold, 10-fold and 10-fold, respectively.

Bone marrow impairment: aplastic anemia and myelodysplatic syndrome may result.

Laboratory findings: increased LD, increased bilirubin, decreased hemoglobin, decreased haptoglobin, hemoglobinuria. Confirmation of the diagnosis with flow cytometry. The GPI anchored surface proteins are missing in all of the hematopoietic cell lines (CD55/CD59 deficiency). Cells with normal expression or with partial or complete loss of expression are termed, respectively, type I, type II and type III cells. Whereas in untreated patients the direct Coombs’ test is negative (because red cells with bound C3 are rapidly lysed) patients treated suboptimal by the C5 inhibitor eculizumab can have a positive Coombs’ test /81/.

– Hemolytic uremic syndrome (HUS) /53/

HUS is a thrombotic micro angiopathy, characterized by hemolytic anemia, thrombocytopenia and acute kidney failure. A distinction is made between the following types:

  • The D(+)HUS type that is associated with diarrhea, triggered by intestinal infection with Shiga toxin-producing E. coli O157:H7 but also other subtypes like O103; O111, O26. These bacteria/toxins are responsible for approximately 95% of all HUS cases. The source of these enterohemorrhagic E. coli (EHEC) is infected cattle. The prevalence in Germany and Austria is 0.7/100,000 children below the age of 15 years, and 1.5–1.9/100,000 children below the age of 5. The infective dose is some 100 germs. Orally ingested germs colonize the intestine, permeate the intestinal barrier, and reach the blood.
  • An extremely rare type of HUS (SPA-HUS), associated with Streptococcus pneumonia, which can be accompanied by sepsis, meningitis, and pneumonia with pleural empyema.
  • Atypical HUS D(–). It is based upon excessive complement activation in renal glomeruli and arteriolar cells. The cause is a mutation in the factor B gene.

The risk of HUS following infection with Shiga toxin-producing E. coli is 10–15%; it is often self-limiting and improves within 1–3 weeks of disease onset. However, a permanent reduction in GFR occurs in 10–20% of the cases.

Laboratory findingss:’ hemolytic anemia with Hb ≤ 100 g/L, fragmented erythrocytes, reticulocytosis, thrombocytopenia < 150 × 109/L, creatinine above the 97th percentile for the corresponding age, increased bilirubin and LD, which is markedly elevated in atypical HUS.

– Malaria /54/

The disease is triggered by an anopheles mosquito bite; sporozoites are released into the blood of the affected individual, from where they take hold in the liver. Schizonts mature in the liver, and merozoites from disintegrating schizonts pass into the blood and colonize in the erythrocytes. In the erythrocytic phase, they can take two reproductive pathways:

  • The sexual pathway which, following a repeated mosquito bite, makes transmission to other individuals possible
  • The asexual pathway. Accordingly, the merozoite develops to a ring form, which matures to the trophozoite and then to the schizont. This disintegrates and releases numerous merozoites, which again affect additional erythrocytes. In P. falciparum infection, this is a synchronous process that lasts 48 hours in each case.

Laboratory findings: anemia, haptoglobin decrease, LD elevation. Decrease in serum iron and inappropriately low reticulocytosis relative to the anemia, rise in ferritin. In chronic forms, the erythropoietic response is impaired, possibly due to a diminished response to erythropoietin and enhanced erythrophagocytosis. See Chapter 44 – Parasitic infections for further malaria findings.

– Warm autoimmune hemolytic anemia (wAIHA)

The wAIHA is a rare autoantibody mediated immune disorder. The pathology of the disease is caused by IgG-, IgM- or IgA-type autoantibodies associated, or not, with molecules of the complement system, and directed against self-erythrocytes. Red blood cell (RBC) destruction is induced by activation of FcγR-bearing effector cells, mainly in the spleen after sequestration and trapping by splenic macrophages. The most common RBC antigen targets are membrane proteins. Data of a study /68/ demonstrate that the ability of anti-RBC autoantibodies to induce phagocytosis, trogocytosis and antigen dependent cytotoxicity is not related to their antigen specificity, but the clinical severity may be dependent on the functional activity of the anti-RBC antibodies.

Acute hemorrhage

In acute hemorrhage erythrocytes and plasma proteins are lost, but not to the same proportion /45/. Immediately following acute hemorrhage, the erythrocytes are redistributed from the small vessels, and the spleen, particularly from the vessels in the region of the nervus splanchnicus. Therefore, blood levels of Hb and erythrocytes in the large vessels are normal or show a moderate decrease. Only after 12–24 hours a stronger decline occurs, and the nadir is reached 48–72 hours after the acute hemorrhage. Following 2–4 weeks, the values recover to pre-bleeding levels. Total protein concentration in serum drops after 4–6 hours, particularly if the patients drink following the volume loss. The nadir of total protein is reached within 12–24 hours, and the concentrations recover to pre-bleeding levels 1–3 weeks later. Based upon total protein, hemolytic anemia can be differentiated from acute hemorrhagic anemia within the first 24 hours. Total protein is normal and/or remains constant in the former case, while in the latter, a decrease occurs.

Laboratory findings: in acute hemorrhage, Hb level, erythrocyte count and the Hct fall proportionately. Reticulocytosis can be detected on the second or the third day and reaches 15% following 1–2 weeks. With comparable bleeding, regeneration of anemia following internal blood loss occurs more rapidly than with external bleeding, since iron and proteins are conserved in internal blood loss.

– Postpartum hemorrhage

Wordwide postpartum hemorrhage accounts for 8% of maternal deaths in developed regions of the world and 20% of maternal deaths in developing regions. The traditional definition of postpartum hemorrhage is blood loss of more than 500 mL after a vaginal delivery or more than 1.000 mL after cesarean delivery. Typical clinical signs and symptoms of hypovolemia e.g., tachycardia and hypovolemia due to postpartum hemorrhage may not appear until blood loss exceeds 25% of total blood volume (> 1,500 mL during pregnancy). Postpartum hemorrhage is considered to be primary when it occurs within the first 24 hours after delivery and secondary when it occurs between 24 hours and up to 12 weeks after delivery. The cause of postpartum hemorrhage is summarized by the four “Ts” tone (utrine atony), trauma (lacerations or uterine rupture), tissue (retained placenta or clots), and thrombin (clotting factor deficiency) /75/. The most common cause is uterine atony (70% of cases).Transfusion is typically begun when the blood loss exceeds 1,500 mL. The goals are to maintain a hemoglobin level more than 80 g/L (tranfusion protocol 450 mL red cells, fresh frozen plasma 1 Unit, cryoprecipitate 1 Unit (factor VIII, fibrinogen), perhaps 1 U apheresis platelet concentrate.

Hypo regenerative normocytic anemia – Generally

Hypo regenerative normocytic anemia is usually normochromic, with minimal variability in erythrocyte morphology. The reticulocyte count is usually below 40 × 109/L.

– Anemia of chronic disease (ACD)

ACD occurs in infection, chronic inflammation, chronic liver disease and malignant tumors /55/. The trigger of the anemia is IL-6 stimulated hepcidin expression within the framework of an acute phase response. The effects are: inhibition of proliferation of erythropoiesis, reduction in iron availability due to inhibition of the cellular iron exporter ferroportin in macrophages and enterocytes. Since the erythroblast takes up less iron via the transferrin receptor (TfR) and releases none due to the ferroportin blockade, normocytic normochromic anemia is common during the first years of ACD. The Hb level is 100–120 g/L, transferrin saturation is > 16%, ferritin is > 100 μg/L, sTfR is normal, CRP is often > 5 mg/L. Among internal medicine patients, ACD comprises 40–70% of all cases of anemia. Approximately 10–20% of ACD patients develop a mild, hypochromic anemia, due to iron-restricted erythropoiesis (ACD/IRE).

In ACD/IRE either bleeding or hepcidin induced long term reduced intestinal iron absorption are the causes of decreased iron supply for erythropoiesis (see also Section 7.6 – Hepcidin). Sensitive indicators for the identification of IRE in ACD are a decrease of the reticulocyte Hb content (CHr, Ret-He) below 28 pg or a %HYPO of > 5% /56/. Hb can be normalized by means of treatment of the underlying disease or therapy with erythroid-stimulating agents. ACD includes forms of anemia that are due to renal insufficiency, disorders of liver function, and endocrine disorders.

– Anemia of cancer /57/

The prevalence of anemia in patients with different cancer types (39% at enrollment and 68% becoming anemic at least once during a 6-month period) has been shown in the European Cancer Anemia Survey /58/. Mean Hb values are 100–120 g/L, and they seldom decline to below 80 g/L. In cancer patients, ACD and chemotherapy are the main causes of anemia, which becomes more severe due to chronic loss of blood and nutritional iron deficiency. The mortality risk in cancer patients with anemia is increased by 65% /59/, and average annual health care cost per patient is 4-fold increased. Of the patients with solid tumors, those with colorectal, lung and ovarian carcinoma have the highest prevalence of anemia and the greatest requirement for transfusion. In patients undergoing chemotherapy, there is a direct correlation between the Hb value and quality of life. The following factors can cause anemia of cancer /60/:

Directly tumor-related (e.g., loss of blood, hemolysis, hypersplenism, bone marrow infiltration)

Indirectly, due to chemotherapy, radiation therapy, or due to the ACD, which is particularly the case with solid metastatic tumors. Approximately one third of tumor patients have elevated CRP levels, defined as values greater than 8 mg/L.

Management of anemia and iron deficiency /61/:

  • Red blood cell transfusion for patients with Hb-levels below 70–80 g/l
  • Intravenous iron therapy if ferritin below 100 μg/L and transferrin saturation below 20% for patients with Hb levels 80–100 g/L
  • Intravenous iron therapy if ferritin below 100 μg/L or transferrin saturation below 20% for patients with Hb levels 100–110 g/L

Aplastic anemia /62/

Aplastic anemia is featured by bone marrow hypo cellularity and peripheral pancytopenia. Aplastic anemia can be due to congenital (20%) or acquired causes (80%). All hematopoietic cells are diminished. Symptoms are fatigue, tachycardia, dyspnea, bruising, mucocutaneous bleeding, and nose bleeds. The incidence is 2–6 cases per million per year. The age distribution is biphasic, with peaks at 15–25 as well as at ≥ 60 years of age. The bone marrow is hypo cellular, and hematopoietic cells are replaced by fat cells. Causes are: Idiopathic (70%), SLE, Sjögren syndrome, rheumatoid arthritis, myasthenia gravis, medications (phenytoin, azathioprine, isoniazid, thyreostatics), Parvovirus B19, B cell lymphoproliferative diseases.

Laboratory findings: marrow cellularity of less than 25% of normal or less than 50% with hematopoietic cells representing less than 30% of the residual cells and at least two of the following blood counts: leukocyte count ≤ 3.5 × 109/L (neutrophil count ≤ 0.5 × 109/L), platelets < 20 × 109/L, reticulocytes < 1%.

AIDS

Cytopenia is infrequent in the early stages of HIV infection; it occurs only in clinically manifest AIDS disease.

Laboratory findings: more than 70% of patients have predominantly normochromic normocytic anemia. Leukopenia and thrombocytopenia are present in greater than 70% and 40% of the cases. Hb concentrations are, as a rule, 90–100 g/L. Disorders of iron metabolism are present, as in ACD, with reduced iron, elevated serum ferritin and diminished transferrin saturation /63/.

Dock 11 deficiency /83/

Dedicator of cytokinesis (DOCK 11) participates in actin cytoskeleton dynamics through its guanine nucleotide exchange factor (GEF) activity, resulting in activation of the small Rho guanosine triphosphatases (GTPases) RAC and cell division cycle 42 (CDC42). Clinically DOCK 11 deficiency includes phenotypes associated with CD42 mutations, such as recurrent infections, immune dysregulation, a normocytic anemia, thrombocyte anomalies and neurodevelopmental abnormalities. Unlike patients carrying CD42 mutations patients with DOCK 11 deficiency have no detectable facial abnormalities, neutropenia, monocytopenia, or hemophagocytic lymphohistiocytosis. The erythroid hypoplasia of the patients is consistent with a bone marrow defect and the observed erythroid-differentiation defect in DOCK11 knockdown CD34+ cord blood cells. DOCK 11-deficient T-cells show reduced transendothelial migration in vitro, yet also increased migration speed under confinement in vivo and in vitro, which suggests that DOCK 11 is involved in leukocyte diapedesis and interstitial migation.Autoinflammation has been observed in several actin-related deficiencies and has been linked to increased inflammasome-mediated secretion of interleukin 1β and interleukin 18.

Table 15.3-12 Causes of hemolytic anemia

  • Antibody-mediated: idiopathic, medications, infections, isoimmunization, transfusion reaction, lymphoproliferative disease, rheumatic disease
  • Mechanical: prosthetic heart valves, implants, vasculitis (micro angiopathic hemolysis), arteriovenous malformations
  • Disorders of hemoglobin synthesis or structure
  • Erythrocyte enzyme defects, erythrocyte membrane defects
  • Miscellaneous: march hemoglobinuria, parasitic infection, snake poison, thermic damage.

Table 15.3-13 Laboratory findings in sickle cell anemia, courtesy of Ref. /39/

Hb
variants

Hb
(g/L)

HCT

MCV
(fL)

Retic.
(%)

ISC

Fractions of
Hb variants

HbSS

60–100

0.20–0.30

80–90

10–

15

4+

80–95% HbS
2–20% HbF
2–4% HbA2

HbS/β0 thal.

60–100

0.20–0.30

60–70

10–

15

3+

75–95% HbS
2–20% HbF
3–6% HbA2

HbS/β+ thal.

80–120

0.30–0.36

65–75

3–

6

1+

50–85% HbS
10–30% HbA
2–20% HbF
3% HbA2

HbSC

100–120

0.30–
0.36

70–80

5–

10

1+

50% HbS
50% HbC

ISC, irreversibly sickled cells, microscopically detectable

Table 15.3-14 Classification and differentiation of macrocytic anemia

Clinical and laboratory findings

Folic acid deficiency, vitamin B12 deficiency, antimetabolites (folic acid, purine and pyrimidine analogs) in cancer therapy /7/

The vitamin deficiency and medication with antimetabolites results in retarded purine and pyrimidine (notably thymidilate) synthesis. Therefore DNA does not replicate, but RNA and hemoglobin synthesis continues leading to typical changes, especially in rapidly dividing tissues such as the bone marrow. With constant cytoplasm formation, the diminished cell division leads to the development of larger cells. Typical changes in the peripheral blood are pancytopenia, elevated MCV, MCH and red cell diameter indexes, MCHC is reduced or low-normal. The granulocytes have polylobulated nuclei. Since the Hb content has not increased in proportion with the cell volume, the MCH is high-normal but the MCHC is low-normal. Therefore, macrocytic erythrocytes are hyperchromic relative to the MCH, and hypochromic relative to the MCHC. LD is elevated due to the ineffective erythropoiesis. The reticulocyte count is reduced, but it can be elevated in bleeding. The combination of iron, vitamin B12 or folate deficiency simulates a normal MCV value, if the erythrocyte distribution width is not evaluated.

Chronic alcoholism, liver disease

In chronic alcoholism and chronic active liver disease, the impairment of DNA synthesis is also due to insufficient availability of folic acid and vitamin B12.

Congenital dyserythropoietic anemia (CDA) /6465/

CDA belongs to a group of inherited conditions characterized by maturation arrest during erythropoiesis with a reduced reticulocyte production in contrast with erythroid hyperplasia in bone marrow. Three classical types have defined on the basis of bone marrow morphology. CDA I and II are autosomal recessive diseases. Whereas CDA I displays abnormalities in chromatin structure, CDA II patients have a marked increase in bi- and multi-nucleated erythroblasts in their bone marrow. CDA type III is an autosomal dominant disease with giant multi-nucleated erythroblasts in bone marrow.

CDA I: the Hb value is 90–110 g/L, and the MCV is 100–120 fL. Hemolytic anemia with an inadequately low reticulocyte count, mild icterus, and low haptoglobin concentration are present. The bone marrow is hyper cellular, containing 30–60% early and late polychromatic erythroblasts with nuclei of abnormal size and shape. The proerythroblasts and the early basophil erythroblasts are normal. Typical are incompletely divided cells with chromatin bridges between the erythroblasts.

CDA II: these patients have anemia, icterus (90%), splenomegaly (70%), and hepatomegaly (45%). The anemia is normocytic with anisocytosis and poikilocytosis, in children there is a slight elevation in the MCV, the reticulocyte count is normal to slightly elevated. The bone marrow is hyper cellular but normoblastic, and contains 10–15% binuclear erythroblasts.

Diagnosis of a CDA relies on examinations of the erythroblasts in a bone marrow biopsy which demonstrates the presence of chromatin bridges between erythroblast nuclei and a spongy Swiss cheese appearance of the chromatin in CDA I, binucleated cells and endoplasmic reticulum remnants in CDA II, and medullary hyperplasia with the presence of giant erythroblasts containing up to 10 nuclei in CDA III.

Laboratory findings: hypo regenerative anemia, macrocytosis, intermittent hyperbilirubinemia, decrease in haptoglobin, increase in LD, atypic electrophoresis of erythrocyte membrane protein.

Table 15.4-1 Equations for HCT calculation

Calculation of relative centrifugal force (RCF):

RCF (gn) = 0.00001118 × r × N2

gn, gravity; r, rotor diameter in cm; N, number of revolutions in rpm

Calculation of the HCT:

HCT = Length of RBC column (mm) Length of total blood column (mm)

Some hematology analyzer determine the sum of electrical impulses and divide them by the number of impulses. The calculation is performed according to the equation:

HCT = MCV (fl) × RBC (L) 10 15

Table 15.4-2 Hematocrit reference intervals

Adults

Fraction

Relative %

Caucasians /2/

0.42 (0.36–0.48)

42 (36–48)

0.46 (0.40–0.53)

46 (40–53)

Blacks /1/

0.38 (0.34–0.43)

38 (34–43)

0.42 (0.34–0.48)

41 (34–48)

Athletes /3/

0.41 (0.37–0.45)

41 (37–45)

0.45 (0.40–0.50)

45 (40–50)

Data expressed as fraction and in relative percent. Values are x ± 2s or the 2.5th and 97.5th percentiles.

Fetus (week of pregnancy) /4/

  • 15

0.28–0.42

(28–42)

  • 16

0.34–0.42

(34–42)

  • 17

0.31–0.43

(31–43)

  • 18–21

0.31–0.45

(31–45)

  • 22–25

0.31–0.47

(31–47)

  • 26–29

0.32–0.50

(32–50)

  • ≥ 30

0.30–0.58

(30–58)

Neonates /56/

  • Umbilical cord blood

0.48–0.56

(48–56)

  • Venous blood
    (2 h after delivery)

0.49–0.71

(49–71)

  • Venous blood
    (6 h after delivery)

0.44–0.68

(44–68)

Children /78/

  • 2–6 days

0.40–0.70

(40–70)

  • 1–2 weeks

0.38–0.70

(38–70)

  • 2–3 weeks

0.38–0.60

(38–60)

  • 3–7 weeks

0.36–0.46

(36–46)

  • 7–12 weeks

0.30–0.38

(30–38)

  • 10–12 mo

0.35–0.43

(35–43)

  • 4–5 yrs

0.32–0.40

(32–40)

  • 6–8 yrs

0.32–0.41

(32–41)

  • 10–13 yrs

0.34–0.44

(34–44)

  • 14–16 yrs

0.35–0.43

(35–43)

0.38–0.49

(38–49)

Data expressed as fraction and in brackets in relative percent. Values are x ± 2s or the 2.5th and 97.5th percentiles.

Table 15.4-3 Diseases and conditions with decreased HCT

Clinical and laboratory findings

Pregnancy

During the first two trimesters of pregnancy, hydremia and hemodilution develop with a disproportionate increase in plasma volume. The red cell mass and the plasma volume increase by 30% and 50%, respectively. The result is a relative fall in the HCT of 25–30%, and a fall in Hb of 20–40 g/L. In one study /26/, the HCT was 0.34–0.43 in non-pregnant women, 0.31–0.41 during the first trimester of pregnancy, 0.30–0.38 during the second trimester, and 0.28–0.39 during the third.

Hemorrhage

Immediately after acute blood loss, the HCT is normal with a decreased blood volume. Following 12–36 hours the blood volume is, once again, normal, the lost volume having been replaced by fluids from the interstitial and intracellular spaces. In consequence, the HCT decreases continuously and in parallel with the Hb value. A decrease in the HCT of 0.03 corresponds to an fall in Hb of 10 g/L. A single HCT determination provides only a limited information of the volume loss; the monitoring of the course is more valid /27/.

In hemorrhage with a decline in the HCT to below 0.25 to 0.30, a decision must be made with regard to the transfusion of erythrocytes particularly in the elderly and in patients with cardiac disease. Young patients with normal cardiac function often tolerate, without hypoxic organ damage, an isovolemic decrease in the HCT to 0.20 to 0.25, corresponding to a Hb value of 60–75 g/L. With the transfusion of 300 mL of packed red cells containing an erythrocyte fraction of 200 mL to a 70 kg adult, a rise in the HCT of 0.03 may be expected. The number units of packed cells that must be transfused, and the HCT that can be reached after transfusion, can be calculated by means of the equations that are shown in Tab. 15.4-4 – Calculation of desired packed red cells in dependence of the HCT /28/.

Following transfusions or infusions, varying extents of fluid displacement depending upon the type of blood product or infusion occur between the intra- and extracellular space. A corresponding equilibrium can be observed following a minimum of 24 hours, at which time the HCT permits the assessment of the composition of the blood.

Acute gastrointestinal bleeding is the second-most common indication for transfusion of blood products in the UK. Clinical evidence now favors restrictive transfusion of erythrocytes in the hemodynamically stable bleeding patient. It is specifically important to consider the specific case of the cirrhotic patient with gastrointestinal bleeding. Deranged laboratory measures of coagulation may not be associated with increased bleeding risks.

Increase in plasma volume

Conditions associated with an increase in plasma volume, for e.g., intraoperative hyperhydration with plasma expanders, or internal diseases that cause an expansion of the plasma volume, such as primary hyperaldosteronism, lead to a decrease in the HCT and anemia. In approximately half the cases anemia can be explained as a result of hemodilution, rather than by a reduction in red cell mas, especially in chronic heart and liver diseases /39/.

Pre-operative anemia (see also Section 19.2 – Oxidative stress)

The 30-day post-operative mortality in patients undergoing non-cardiac surgery was investigated as a function of the pre-operative HCT in comparison with operated patients with a normal HCT. In mild anemia (HCT in women < 0.36 to > 0.29; in men < 0.39 to > 0.29) as well as in moderate to severe anemia (HCT ≤ 0.29 in both men and women), the odds ratio for post-operative mortality was 1.41. If additional risk factors were present, the odds ratios for morbidity and mortality were higher than in cases of anemia or a risk factor alone /29/. Each perioperative transfusion, even of only one unit packed blood, increased morbidity and mortality /30/.

Table 15.4-4 Calculation of desired packed red cells (PC) in dependence of the HCT /28/

Number of PC (nPC) that has to be transfused at the actual HCT (HCTa), in order to reach the desired HCT (HCTd):

nPC = BV × (HCTd – HCTa) 20,000 – 300 × HCTd

Expected value of the HCT (%) [HCTd] following administration of a certain number of PC (nPC):

HCTd = BV × HCTa + nPC × 200 × 100 BV + nPC × 300

Explanation: the volume of one PC is 300 mL, and it contains 200 mL of red cells. HCTa, actual HCT; HCTd, desired HCT; HCT is expressed in %. BV is the estimated normal blood volume of an individual, and is calculated as follows:

BV (mL) = Body weight in kg × 67

BV (mL) = Body weight in kg × 77

Table 15.4-5 Diseases and conditions with increased hematocrit (HCT)

Clinical and laboratory findings

Polycythemia vera (PV) /31/

The myeloproliferative neoplasms (PV, essential thrombocytosis and primary myelofibrosis) are unique hematopoietic stem-cell disorders that share mutations that constitutively activate physiologic signal-transduction pathways responsible for hematopoiesis. In addition to phenotypic mimicry, each type of myeloproliferative neoplasm is capable of evolving into another type, making diagnosis, risk assessment, and therapeutic choices difficult /32/.

PV occurs in patients of all ethnic backgrounds, however it is much more common in those of European than Asian descent, having an annual incidence ranging from 2 to 10 cases per million population per year. PV is generally a disease of older individuals, occurring most frequently between 50 and 70 years of age; childhood and adolescent disease is exceptionally rare. The most common complaints are thrombosis (deep vein thrombosis, stroke, myocardial ischemia, Budd-Chiari syndrome, mesenteric ischemia) and bleeding complications (epistaxis, oral mucosal hemorrhage, gastrointestinal hemorrhage, or nonspecific ecchymosis). Hyper viscosity syndrome due to sluggish blood flow and micro thrombi may cause hypertension, headache, stupor, disturbances of vision, dizziness, tinnitus, claudication and erythromelalgia. Pruritus occurs following prolonged exposure to warm water, due to histamine release from activated basophil granulocytes. More than half of the patients have splenomegaly. Gouty arthritis is not uncommon. World Health Organization diagnostic criteria for PV are shown in Tab. 15.4-6 – World Health Organization diagnostic criteria for polycythemia vera.

The pathogenesis of PV is the result of different mutations:

  • Some 95% of patients with PV have the JAK2 V617F mutation in exon 14 of the JAK2 gene. The enzyme (Janus kinase) encoded by this gene is a cytoplasmic tyrosine kinase that is involved in signal transmission of different cytokine receptors, including the EPO receptor. The mutation enhances the Janus kinase activity and leads to an EPO-independent stimulation of erythropoiesis. The mutation is acquired, and can also be detectable in primary thrombocythemia and primary myelofibrosis. An abnormal clone in the bone marrow leads to the disease.
  • Other patients have mutations in exon 12 of the JAK2 gene (JAK2 F537-K539delinsL, JAK2 H538QK539L, JAK2N542-E543del, JAK2 K539L). These patients have a predominantly erythroid phenotype.

Laboratory findings /19/: suspicion of PV exists with HCT > 0.52 (Hb > 185 g/L) or 0.48 (Hb > 165 g/L) in men and women, respectively, along with normocytic normochromic erythrocytes. More than 40% of patients have leukocytosis > 12 × 109/L or neutrophil granulocytosis > 10 × 109/L. If oxygen saturation is normal, EPO is determined. Leukocytes are investigated for the JAK2 V617F mutation because erythrocytes lacks the nucleus. PV is present if a homozygous mutation is found. More than 60% of the patients have thrombocytosis > 400× 109/L and more than 70% have an elevation in vitamin B12 concentration of 25% above the upper reference interval value. EPO concentrations are low or subnormal. The erythrocyte sedimentation rate is markedly reduced. Bilirubin and uric acid can be increased. Bleeding time is normal, but the global coagulation tests can be pathological due to the high HCT, since the anticoagulant to plasma ratio is no longer correct.

Primary erythrocytosis /3334/

Primary erythrocytosis (also known as primary familial and congenital polycythemia, PFCP) is a pathology of erythroid progenitors, which display hypersensitivity to EPO. However, the erythropoiesis is polyclonal and PFCP patients do not show any risk of their disease transforming into myelofibrosis or leukemia /34/.

The erythropoietin receptor (EPOR) is expressed by erythroid progenitors. Transcription of the EPO gene is regulated by mechanisms that are dependent upon the cellular response to changes in oxygen supply. The binding of EPO to its receptor leads to the transient activation of preformed EPOR-JAK2 complexes and down streaming signaling pathways, including signal transducer and activators of transcription (STAT), phosphatidylinositol 3 kinase/akt and mitogen-activated protein kinase pathways. Activated STAT causes homodimerization of the EPOR in the presence of EPO and auto phosphorylation of JAK2 occurs. Once JAK2 is activated, specific EPOR tyro sines are phosphorylated and form docking sites for adaptor molecules (See Fig. 15.4-2 – Homodimer erythropoietin receptor showing phosphorylated tyro sines)

Around 20 germ line heterozygous nonsense and frame shift mutations located in exon 8 of the gene EPOR have been described so far in PFCP, all leading to the truncation of the C-terminal part of EPOR. The C-terminal part of the EPOR includes several conserved tyrosine residues that are docking sites for positive and negative regulators of EPOR signaling. The EPO hypersensitivity of PFCP progenitors is, therefore, usually explained by the disappearance of negative regulatory domains located in the truncated part of the receptor.

EPO hypersensitivity induced by the EPOR mutant p.Gln434Profs*11 is explained by the appearance of a new C-terminal tail /34/. The latter, by increasing EPOR dimerization, stability and cell-surface localization, causes pre activation of EPOR and JAK2, constitutive signaling and hypersensitivity to EPO.

Laboratory findings: Erythrocytosis with subnormal EPO concentration. Gene panel sequencing improves the diagnostic work-up of patients with erythrocytosis /38/.

High oxygen-affinity hemoglobins /19/

High oxygen affinity hemoglobins may be stable or unstable, and as they do not release oxygen readily to the tissues the resulting hypoxia drives EPO production and thus erythrocytosis. Mutations in the genes that code for the α- and β-chains can lead to hemoglobins with high-affinity binding of O2. The hemoglobin-oxygen dissociation curve is shifted to the left indicating abnormal Hb which has increased affinity for O2. As a rule, with oxygen partial pressure of 20 mm Hg, O2 saturation in the capillaries is 35%. With Hb variants with high oxygen affinity this can, however, reach 60%.

Laboratory findings: erythrocytosis, high or, relative to the Hb value, inappropriately high EPO concentrations. The oxygen partial pressure at which Hb saturation of 50% is reached is low. Example: control 27 mm Hg, patient 16.5 mm Hg.

VHL gene mutation

All affected patients are homozygous for a single mutation C598T. The amino acid 200 arginine changed to tryptophan in the von Hippel-Lindau (VHL) gene. The VHL gene plays an important role in hypoxia sensing where cells sense a decrease in O2 and allow the organism to adapt. In the renal peri tubular fibroblasts, the VHL protein regulates the degradation of hypoxia-inducible factor 1 (HIF-1), which is made up of one α- and one β subunit and exists in three isoforms (HIF-1α, HIF-1β and HIF-1γ). O2 activates prolyl hydrolase which then hydroxylates HIF-α. If the O2 saturation is normal, this leads to the binding of VHL protein and ubiquitination of HIF-α and the destruction of the proteins. EPO synthesis is reduced. In hypoxia HIF-α associates with its β-subunit, the complex binds to hypoxia responsive elements leading to increased production of EPO. With a homozygous mutation of the VHL gene, the normal mechanism is disrupted, HIF-α is driven down the hypoxia pathway resulting in increased EPO production and erythrocytosis (Fig. 15.4-3 – Increased synthesis of erythropoietin in von Hippel-Lindau gene mutation). The disease is also called Chuvash polycythemia.

Laboratory findings: the oxygen partial pressure at which Hb saturation of 50% is reached is low. Inappropriate elevated EPO concentrations, identification of the VHL gene mutation.

Bisphosphoglycerate mutase deficiency /19/

During glycolysis the 2,3-bisphosphoglycerate (2,3-BPG) shunt involves conversion of 1,3-BPG to 2,3-BPG catalyzed by the enzyme bisphosphoglycerate mutase. In the erythrocyte 2,3-BPG binds to the hemoglobin tetramer and converts the Hb molecule to a low oxygen affinity state (Fig. 15.3-2 – The O2 dissociation curves in dependence of PO2 and the red cell content of 2,3-DPG). The binding of 2,3-BPG therefore shifts the oxygen affinity curve to the right. Lack of 2,3-BPG shifts the oxygen affinity curve to the left as the Hb tetramer is kept in a high O2 affinity state. Due to a mutation in the gene that encodes for bisphosphoglycerate mutase, the activity of the enzyme is low or absent. Little or no 2,3-BPG is formed and results in a left shifted oxygen affinity curve (Fig. 15.4-4 – Effect of 2,3-diphosphoglycerate (2,3-DPG) on O2 saturation curve) a high affinity Hb and a compensatory erythrocytosis.

Laboratory findings: erythrocytosis, oxygen partial pressure at which Hb saturation of 50% is reached is low. Inappropriately elevated EPO concentrations, demonstration of bisphosphoglycerate mutase gene mutation.

Neonates

Immediately after delivery, neonates have a HCT ≤ 0.56 in umbilical cord blood and ≤ 0.70 in venous blood 2 hours later /56/. Higher values are indicative of abnormal flow kinetics (hyper viscosity). Other authors select a threshold HCT of 0.65. According to one study /35/, only 47% of neonates with an umbilical cord blood HCT above 0.65 have hyper viscosity, and only 23% of those with hyper viscosity have erythrocytosis. 2,3-BPG concentrations in fetal erythrocytes are lower than in adults, and this leads to a left shift of the oxygen affinity curve. In consequence, HbF can bind O2 with greater affinity, and can accept the O2 from the maternal blood in the placenta.

Smoker

Smokers’ polycythemia is caused by CO intoxication, which leads to proliferation of erythropoiesis due to hypoxia and in which, additionally and for unknown reasons, the plasma volume is diminished /21/. Smokers often have leukocytosis of above 10 × 109/L.

Acquired erythrocytosis – Generally

Acquired erythrocytoses are differentiated into central and local hypoxia driven processes. They are associated with elevated EPO concentrations, and do not lead like polycythemia vera to an increase in platelets and leukocytes.

– Hypoxia at altitude /21/

The HCT and the Hb values increase linearly to an altitude of approximately 4000 meters. Above that there is a disproportional increase up to about 6000 meters, from where on a decrease in erythropoiesis is found. The erythropoiesis is diminished dramatically when the arterial O2 saturation decreases to below 60%. An arterial O2 saturation of less than 92% is considered low. In blood with HCT values higher than 0.55 the blood viscosity is increased. This enhances the energy demand for the heart to pump blood and may impair the microcirculation.

– Local hypoxic causes

Inadequate O2 supply to the renal O2 sensors, due to impaired renal function, results in augmented EPO synthesis and erythrocytosis. This is the case in renal artery stenosis, end-stage renal disease, polycystic kidney disease, hydronephrosis, and following kidney transplantation.

– Post-transplantation erythrocytosis (PTE)

PTE is a complication of renal transplantation; the incidence is 5–17% /36/. The HCT rises to as high as above 0.60 many months following transplantation, making phlebotomy necessary. In such cases, thromboembolic complications can occur. The erythrocytosis is often transient in nature. The cause of the inappropriately high EPO secretion relative to the HCT is unknown.

– Autonomous EPO synthesis

Paraneoplastic polycythemia is found in cerebellar hemangioblastoma, meningioma, parathyroid gland hyperplasia, hepatocellular and renal cell carcinoma, pheochromocytoma, and leiomyosarcoma of the uterus /19/.

– ESA therapy

The administration of erythropoiesis-stimulating agents (ESA) for the purpose of EPO doping, leads to erythrocytosis. Androgen doping also leads to erythrocytosis.

Thrombotic risk

Certain disorders with elevated hematocrit, such as polycythemia, Chuvash erythrocytosis, primary familial and congenital polycythemia or erythrocytosis (EPOR mutation) and EPAS1 gain-of-function mutations, are associated with thrombotic complications. These conditions are characterized by diverse cellular and metabolic changes that could be directly associated with thrombotic risk, irrespective of hematocrit level /33/.

Table 15.4-6 World Health Organization diagnostic criteria for polycythemia vera /20/

 

Criteria

A1

Elevated RBC mass > 25% above mean normal predicted value, or Hb > 185 g/L in men, > 165 g/L in women

A2

No case of secondary erythrocytosis, including absence of familial erythrocytosis; no elevation of erythropoietin due to hypoxia (arterial PO2 ≤ 92%), high affinity hemoglobin, truncated erythropoietin receptor, inappropriate erythropoietin production by tumor

A3

Splenomegaly

A4

Clonal genetic abnormality other than Ph chromosome or BCR/ABL fusion gene in marrow cells

A5

Endogenous erythroid colony formation in vitro

B1

Thrombocytosis > 400 × 109/L

B2

WBC > 12 × 109/L

B3

Bone marrow biopsy showing pan myelosis with prominent erythroid and megakaryocytic proliferation

B4

Low serum erythropoietin level

Polycythemia vera is diagnosed when A1+A2 + any other A, or A1+A2 + any 2B. RBC, red blood cell; Hb, hemoglobin; Ph, Philadelphia; WBC, white blood cell

Table 15.5-1 Agents capable of inducing methemoglobinemia /5/

Local
anesthetics

Nitrites/
nitrates

Analgesics

Benzocaine

Lidocaine

Procaine

Sodium nitrite

Nitroglycerin

Amyl nitrite

Butyl nitrite

Isobutyl nitrite

Primaquine

Phenazo-
pyridine

Phenacetin

Anti-
microbials

Miscellaneous

Dapsone

Sulfanilamide

Sulfathiazole

Aniline dyes

Chlorates

Nitrobenzene

Aminophenol

Celecoxib
(Cox-2 inhibitor)

Table 15.5-2 Correlation of clinical symptoms with increasing proportion of metHb /7/

MetHb
fraction (%)

Symptomatology

< 15

Usually asymptomatic

15–20

Cyanosis, headache, drowsiness

20–45

Marked cyanosis, nausea

45–70

Severe cyanosis, vomiting, confusion, seizures

> 70

Usually lethal

Table 15.5-3 Clinical symptoms in dependence of the COHb concentration /8/

COHb (%)

Clinical and laboratory symptoms

0–10

No relevant symptoms (smokers)

> 10–15

No relevant symptoms, possibly shortness of breath on physical exertion (heavy smokers)

> 15–25

Usually no effect at rest, shortness of breath on physical exertion, possibly dizziness and headache; dilatation of cutaneous capillaries

> 25–35

Headache, dizziness, vomiting, pulse acceleration, irritability, disturbances of judgment, mild fatigability, vision disorders

> 35–45

Similar to 25–35%, but more severe, additionally state of confusion, signs of paralysis, fainting even on mild exertion

> 45–55

Marked impairment of consciousness or unconsciousness, increased respiratory and pulse rate, collapse, danger of death with prolonged exposure

> 55–65

Additionally seizures, respiratory paralysis

> 65

Immediate danger of death

Table 15.5-4 Hemolysis and plasma free hemoglobin

Clinical and laboratory findings

Venovenous and venoarterial extracorporal membrane oxigenation (ECMO) /20/

ECMO is a lifesaving support that is used in critically ill patients. Three main pathophysiologic aspects in the short-term ECMO setting are in patients with free hemoglobin:

  • Regulation vascular tone. Free Hb interacts with NO and catalyzes transformation of NO into nitrate if oxygen-Hb is involved or into a ferric-NO complex if deoxy-Hb is involved. Subsequent NO depletion is then responsible for a loss in a major negative regulator of the vascular smooth muscle tone, inducing a potent vasoconstriction. The low NO-dependent vasoconstriction is responsible for both an increase in systemic and vascular pulmonary vascular resistance /25/.
  • Abnormal coagulation and platelet activation. NO depletion is responsible for platelet function in endothelial behavior towards platelet aggregation and coagulation. Free Hb increases the procoagulant state in diseases such as paroxysmal nocturnal hemoglobinuria and sickle cell disease.
  • Renal tubular toxicity: Heme-containing proteins are known to be toxic to kidneys.

All the pathologic changes caused by free Hb in the bloodstream are associated with an increased morbidity and perhaps mortality.

Severe hemolysis may be present during venovenous ECMO. A peak in free Hb and creatinine can be a predictor of acute renal failure. Severe increase in free Hb (> 500 mg/L) in the first 24 hours of ECMO onset is an independent predictor of mortality. In a study /26/ patients with free Hb > 300 mg/L had a 10-fold increase in hospital mortality and seemed associated with renal impairment. Two factors are recognized to promote and generate hemolysis in ECMO: thrombosis (head pump and oxygenator) and strong mechanical stress of erythrocytes (head pump) /20/.

Hemolysis is a common phenomenon during ECMO support in most cases. Usually the intensity of hemolysis is low, generating tolerable free Hb concentration.

Severe Sepsis

Results of a study are /27/: In non-survivors of severe sepsis free Hb concentration was twice the value compared to survivors (57 mg/L vs. 147 mg/L, Harboe method). In postoperative patients the free Hb concentration was 45–78 mg/L (ELISA method). Thirty-day survival of patients was markedly lower in patients with high free Hb than in patients with low free Hb.

Massive Transfusion And/or TRansfusion of Stored Blood

Aged stored blood hemolyzes after massive transfusion. Free Hb led to vascular and kidney injury indicating that intravascular hemolysis is a pathological mechanism in several human diseases /28/.

Table 15.6-1 Hematocrit (HCT) correction and shift correction /9/

RI (%) = Observed count × HCT (%) 45 RPI = Observed count (%) × HCT (%) Days at a HCT 45

Table 15.6-2 Expected lower RPI thresholds and reticulocyte count as function of HCT /16/

HCT

Reticuloc. × 109

RPI

0.35

150

2–3

0.30

250

> 3

< 0.25

> 250

> 4

RPI, reticulocyte production index

Table 15.6-3 Reticulocyte reference intervals

 

Relative
(%)

Absolute
(109/L)

Children*

Pre-term delivery /11/

2.6 ± 1.0

72 ± 29

Full-term neonates /12/

2.4 ± 1.2

74 ± 26

0–14 days /12/

5.3 ± 2.0

318 ± 50

  • 14 days–1 yr /12/

2.0 ± 0.5

89 ± 23

2.0 ± 0.7

88 ± 20

2.2 ± 0.7

99 ± 28

2.1 ± 0.8

90 ± 27

  • 12 yrs to adult /12/

2.3 ± 1.2

92 ± 25

Adults /13/* manual

0.4–2.3

(19–111)

 

 

Count
(109/L)

Adults /14/** automated

Abbott CD 4000

30–117

31–115

Advia 120

25.9–97.5

36–101.1

Coulter LH 750

13.9–98.3

21.7–114.5

Horiba ABX, Pentra 12

27–91

39–113

Sysmex XE 2100

19.8–80.7

24.8–96.2

* Values expressed as x ± s; ** Values are 2.5th and 97.5th percentiles. The values provided in Ref. /12/ are valid for flow cytometry with the Becton Dickinson FACSCalibur. Since reference intervals depend on the hematology analyzer used, intervals for adults from 5 well-known manufacturers are shown.

Table 15.6-4 Diseases and conditions with reticulocytosis /17/

Clinical and laboratory findings

Acute hemorrhage

After an acute hemorrhage, the timing of the onset of reticulocytosis and its extent depend on the amount of blood loss. The rise usually occurs on the 2nd–3rd day and the reticulocytosis ranges from 5–15%.

Hemolytic anemia

Acute hemolytic anemias are associated with high reticulocyte counts but clinical signs may be absent. Reticulocytoses of > 50% are observed in immune hemolytic anemias. In efficient treatment with corticosteroids, the reticulocyte count declines within a few days. Acquired, mild hemolytic syndromes may be associated with a normal Hb concentration and a reticulocytosis of 2–5%.

In congenital hemolytic anemias, with the exception of G-6PD deficiency, the RBC volume is changed and slightly elevated reticulocyte counts are present.

In pyruvate kinase deficiency, a hereditary chronic hemolytic anemia with an autosomal mode of inheritance, the severity of the clinical manifestations is subject to great variability and reticulocytosis of up to 50%. After splenectomy, persistent reticulocytosis occurs which may range from 50–100% /22/.

Hypersplenism

A normocytic, slightly hemolytic anemia is present. The reticulocyte count is slightly elevated. In the peripheral blood smear, polychromasia and erythroblasts are found in some of the cases.

Treatment of deficiency anemia

In the case of deficiency of iron, folate, vitamin B6 or vitamin B12, the regeneration of the hypo regenerative erythropoiesis is indicated within one 1–2 weeks by a rise in the reticulocyte count, if there is efficient supplementation. After 2–4 weeks, a maximal reticulocytosis of 10–20% is measured.

Athletes, injection of erythropoiesis-stimulating agents (ESA) /23/

Top-level athletes have a normal reticulocyte count but often an elevated immature reticulocyte fraction (IRF), dependent upon activation of erythropoiesis due to the mild hemolysis caused by participation in sports. The IRF increases 36 hours following bolus injection of ESA, reaching a maximal value after 3 to 4 days and normalizing after 7 days. The reticulocyte life span in circulation is prolonged from 1.7 to 3.4 days. The overall result is a doubling of the reticulocyte count. Following repeated ESA administration, reticulocyte values are increased from days 7–24 and remain elevated for a further 7 days after the last ESA dose.

Aplasiogenic cytostatic therapy

An increase in the reticulocyte count is an early surrogate marker of a recovery of hematopoiesis following chemotherapy /24/.

Allogenic bone marrow transplantation

The reticulocyte maturity index (RMI) is a good indicator of transplant function and is as valuable a transplantation marker as the rise in neutrophile granulocytes. A rise in both of these parameters is seen during the early post transplantation phase, if the transplant functions. In clinical or subclinical infections, the rise in neutrophil granulocytes may be absent. The RMI, however, is not influenced by infectious processes and increases in the presence of a functioning transplant /25/.

Table 15.6-5 RMI and IRF reference intervals

 

LFR%

MFR%

HFR%

RMI adults /29/

Sysmex 9500

85–97

3–14

< 2,5

Advia 120

88–98

2–11

≤ 2,0

IRF adults /29/

  • Cell DYN 4000: proportion of reticulocytes with LFR, MFR and HFR in each case 0.14–0.35.

RMI, reticulocyte maturity index; IMR, immature reticiulocyte fraction; LFR, low size fraction reticulocytes; MFR, medium size fraction reticulocytes; HFR, high size fraction reticulocytes

Table 15.6-6 Diagnostic significance of reticulocyte count and reticulocyte maturity index (RMI) /32/

Anemia

Reticulocyte
count

RMI

Aplastic anemia

Aplastic crisis

/N

Intrinsic marrow
hypoplasia

N/

Bone marrow
regeneration

N/

Anemia of chronic
disease

/N

N

Iron deficiency

/N

Thalassemia

N/

N/

Myelodysplastic
syndrome

/N/

N/

Folate/vitamin B12
deficiency

/N

Hemolytic anemia

Hemorrhage

N/

Immature reticulocyte fraction: N, normal; low; high

Table 15.6-7 Reference interval for reticulocyte hemoglobin (CHr, Ret-He)

Adults

CHr

28–35 pg /37/

Ret-He

28–35 pg /36/

CHCMr

270–330 g/l /34/

RFHb

1.76 ± 0.59 g/L /38/

Neonates

CHr

33–38 pg /39/

Children

CHr

27.5–33 pg /40/

RFHb, Hb content of the reticulocyte fraction; CHCMr, Reticulocyte Hb content

Table 15.7-1 Normal human hemoglobins

Hb
type

Chain
formula

Occurrence

Quantitative
proportion

HbA

α2β2

Children > ½ year

Adults

97.0%

HbA

α2δ2

Children > 1 year

Adults

2.5–3.0%

HbF

α2γ2

Fetal/neonatal peroid

Children < 1 year

Children age-dependent, adults up to 0.5%

Hb
(embryo)

Normally no longer detectable after birth

Table 15.7-2 Prevalence of hemoglobinopathy gene carriers in the world’s population /4/

Country

Gene carrier

African nations

5–30%

Arab nations

5–40%

Up to 60% regionally

Central Asia and India

10–20%

Southeast Asia

5–40%

Up to 70% regionally

USA and Central America

5–20%

Italy

7–9%

Greece

6–7%

Turkey

7–10%

Germany, Great Britain, Spain, France, The Netherlands, Belgium, Scandinavian countries

Among total population 0.5 to 1%

Among immigrants 5%

Balkan countries

2–5%

Russia

Rare

Caucasus countries

Up to 5%

Table 15.7-3 The most important hemoglobinopathies in Germany

β-thalassemia syndromes

65.8%

HbS

21.2%

α-thalassemia syndromes

7.8%

HbE

2.1%

HbC

1.8%

Unstable Hb abnormalities

0.2%

Reference value: 35,831 laboratory diagnoses

100%

Table 15.7-4 Program for the laboratory diagnostic investigation of hemoglobinopathies

Hematology

Complete blood count and, possibly, reticulocyte count

Clinical
chemistry

Iron status: ferritin, transferrin saturation

Hemolysis markers: haptoglobin, LD, bilirubin

Hemoglobin
analysis

Electrophoresis and/or HPLC

HbF determination, HbF cell staining

Hb solubility test, sickle cell test for HbS

Molecular
genetics

DNA purification

PCR amplification

DNA sequencing

MLPA deletion diagnostics

Table 15.7-5 Basic information on molecular genetic methodology

DNA purification

Original material

Polymerase chain amplification

Technique for rapid amplification (duplication) of specific DNA sequences of different size.

DNA sequencing

Analysis of the nucleotide sequence of DNA molecules or fragments using enzymatic or chemical methods. The analysis is performed using automated DNA sequencers.

MLPA deletions

Multiplex ligation-dependent probe amplification, molecular genetic method with which, following large genomic deletions or duplications, individual gene regions or an entire gene can be investigated.

Table 15.7-7 Basic forms of hemoglobinopathies

Hemoglobinopathy

Basic defect

1. Thalassemia syndromes α-, β-, δβ-, γδβ-, γ and δ-thalassemia. Thalassemic hemoglobinopathies

2. Abnormalities of hemoglobin structure:

  • Structural defects of α- or β globin chains (e.g., HbS, HbC, HbE)

3. Types of combination and interaction (e.g.,

  • HbSC disease
  • HbS-β thalassemia
  • HbE-α thalassemia)

ad 1) Reduced or lack of synthesis of α-, β-, γ- or δ-globin chains

ad 2) Abnormal structure of α-chains or β-chains: Combination of different forms of thalassemia with one another or with abnormal hemoglobins

ad 3) Combinations of different abnormal hemoglobins with one another

Table 15.7-6 Reference intervals for hemoglobins /23/

 

Full-term
newborn
infants

Children
1–2 yrs

Children
> 2 yrs
and adults

HbA

17.7
(13.1–22.3)**

15–40**

96.0
(95.0–98.2)

97.5
(97–98.5)

HbA2

0.25
(0.05–0.45)

1.8
(1.5–3.0)

2.5
(1.5–3.2)

HbF

81.7
(77.5–86.9)*

60–85**

1.5 (≤ 2.0)*

0.8
(0.4–1.0)**

0.5
(≤ 0.5)**

Reference intervals for children < 1 year are age-dependent. Data expressed in percent. Values expressed as x ± s. Methods: if not otherwise specified: electrophoresis; * chromatography; ** alkali denaturation.

Table 15.7-8 Compilation of the most important criteria of α-thalassemias

Hereditary
status/
diagnosis

Arrangement of
α-globulin gene
cluster

Erythrogram,
hemoglobin
pattern (hp)

Cardinal symptoms

Normal
findings

▲▲/▲▲

αα/αα

Hb norm.

MCH norm.

hp norm.

No symptoms

Heterozygous
α+ thalassemia
(α thalassemia
minima)

▼▲/▼▼

–α/αα

Hb norm.

MCH < 27 pg

hp norm.

No symptoms, slight blood count changes

Homozygous
α+ thalassemia
(α thalassemia
minor)

▼▲/▲▼

–α/–α

Hb norm. or

MCH < 26 pg

hp norm.

Mild anemia, substantial blood count changes

Heterozygous
α0 thalassemia
(α thalassemia
minor)

▼▼/▲▲

––/αα

Hb norm. or

MCH < 24 pg

hp norm.

Mild anemia, substantial blood count changes

Mixed
heterozygosity
α+0 thalassemia
(HbH disease)

▼▼/▼▲

––/–α

Hb 80–100 g/L

MCH < 22 pg

hp: HbH
≈ 10–20%

Variable chronic hemolytic anemia

Homozygous
α0 thalassemia
(Hydrops fetalis
syndrome)

▼▼/▼▼

––/––

Hb < 60 g/L

MCH < 20 pg

hp: Hb Bart’s
80–90%

Hb Portland
≈ 10–20%

Life-threatening fetal anemia, generalized hydrops

Table 15.7-9 Compilation of the most important criteria of β-thalassemias

Hereditary status

Diagnosis

 

Erythrogram

Hb

pattern

Cardinal symptoms

Heterozygous β-thalassemia
(β thalassemia minor)

β++

β+

β0

Hb (g/L)

90–150

90–130

MCV 55–75 fL

MCH 19–25 pg

HbA2
> 3.2%

HbF
0.5–6%

Mild anemia without disease

Homozygous β-thalassemia*

Compound heterozygous β-thalassemia*

β++

β00

β+0

Hb < 70

MCV 50–60 fL

MCH 14–20 pg

HbA2
variable HbF
70–90%

Severe disease with permanently transfusion-dependent anemia

Mild homozygous or compound
heterozygous β-thalassemia
(β-thalassemia intermedia)

β++

I**

β+++

β+0

β00

Hb 60–100

MCV 55–70 fL

MCH 15–23 pg

HbA2
variable

HbF
to 100%

Moderately severe disease, variably transfusion-dependent

* β-thalassemia major; **, Influencing variables

Table 15.7-10 Clinical classification of the most important pathological hemoglobin variants

Variants associated with vascular occlusion
and hemolysis

e.g., HbS, HbC

Variants associated with the thalassemia

phenotype

e.g., HbE, Hb Lepore

Unstable hemoglobins associated with hemolysis

e.g., Hb Köln, Hb Leiden

Variants associated with disturbed oxygen transport function (familial erythrocytosis)

e.g., Hb Ohio

Variants associated with pathological methemoglobin (familial cyanosis)

e.g., HbM Iwate

Table 15.7-11 Diagnostic characteristics of sickle cell β-thalassemia

HbS-β0-thalassemia

HbS-β+-thalassemia

Appearance

Similar to severe
sickle cell disease

Appearance

Depending upon the residual β-globulin activity = mild to severe sickle cell disease

Laboratory diagnostic
investigation and findings

Hematology:

  • Hb 60–100 g/L
  • MCH < 22 pg

Laboratory diagnostic investigation and findings

Hematology:

  • Hb 90–120 g/L
  • MCH < 24 pg

Clinical chemistry

Hemolysis parameters
positive

Clinical chemistry

Hemolysis parameters positive

Hemoglobin analysis

  • HbA2 > 3.5%
  • HbS > 80%
  • HbF < 20%
  • HbA 0%

Hemoglobin analysis

  • HbA2 3.5%
  • HbA 3–30%
  • HbS 55–75%
  • HbF ≈ 20%

Molecular genetics

Demonstration of the
following mutations:

1. HbS = β codon
6 GAG GTG heterozygous

and

2. β0-thalassemia mutation heterozygous = compound heterozygosity

Molecular genetics

Demonstration of the following mutations:

1. HbS = β codon 6 GAG GTG heterozygous

and

2. β+-thalassemia mutation heterozygous = compound heterozygosity

Table 15.7-12 Characteristic features of the most important hemoglobin abnormalities

Sickness,
Genotype

Erythrogram

Hemoglobin
pattern

Cardinal symptoms

Sickle cell disease

HbSS

Hb 60–90 g/L

Normochromic
sickle cells

Positive hemolysis
markers

HbS 55–90%

HbA2 > 3.5%

HbF < 10 to

> 20%

Sickle cell crises/pain crises, acute organ syndromes, chronic hemolytic anemia

HbS heterozygosity

HbAS

Normal

HbS 35–40%

HbA2 ≥ 3.5%

No manifestation of disease

Sickle cell β+-thalassemia

HbSβ+-thalassemia

Hb 90–120 g/L
Hypochromia

Microcytosis

HbS > 55%

HbF > 20%

HbA2 > 3.5%

Variable, mild sickle cell disease

Sickle cell β0-thalassemia

HbSβ0-thalas-
semia

Hb 60–100 g/L

Hypochromia

Microcytosis

HbS > 80%

HbF < 20%

HbA2 > 3.5%

Severe sickle cell disease

HbSC disease

HbSC

Hb 100–130 g/L

Target cells

MCV < 75 fL

HbS 50%

HbC 50%

HbF < 5%

Attenuated symptoms of sickle cell disease, chronic hemolytic anemia

HbC disease

HbCC

Hb 100–120 g/L

Target cells

MCV < 75 fL

MCHC > 350 g/L

HbC > 95%

HbA2 2,5%

HbF 0.5%

Pain crises, organ dysfunction, chronic hemolytic anemia

HbC heterozygosity

HbAC

Normal

HbC 50%

HbA 47%

HbA2 3%

No manifestation of disease

HbE heterozygosity

HbAE

Hb normal or low

Hypochromia

HbE

25–35%

Mild hypochromic anemia

HbE disease

HbEE

Hb 100–140 g/L

High red cell count

MCH 20 pg

MCV 65 fL

Target cells

HbE > 95%

HbA2 2.5%

HbF < 3%

Mild anemia, hemolysis due to infection/medication

HbE-β+-thalassemia

HbE-β+-thalas-semia

Hb variable

Hypochromia

Microcytosis

HbE + HbA2

25–80%

HbF 6–50%

HbA 5–60%

Variable intermediate hypochromic anemia

HbE-β0-thalassemia

HbE-β0-thalas-semia

Hb < 80 g/L

MCV < 60 fL

MCH < 22 pg

HbE to 85%

HbA2 < 5%

HbF 15–25%

Same as β-thalassemia major

Hemoglobino-
pathy with
unstable Hb

HbX; some 150
different variants

HbX/HbA

Hb variable –
significantly
anemic;
Heinz bodies,
hemolysis due
to viral infection/
medication

HbX 20%

HbA2 3–4%

HbF < 5%

Variable, partially transfusion-dependent chronic hemolytic anemia

Abnormal
hemoglobin
with disturbed
O2 transport/
function

Different variants

Polyglobulia
Increase in metHb

Different, according to the type of abnormality

Congenital cyanosis associated with HbM abnormalities, congenital polyglobulia associated with Hb abnormalities with increased O2 affinity

Table 15.8-1 Reference intervals for erythrocyte enzymes

Enzymes /16/

Substrate
turnover*
(g Hb/min.)

Substrate turnover*
(1011 red cells/min.)

Glycolysis

  • Pyruvate kinase

20.2 ± 2.2

41 ± 10

  • Hexokinase

1.0 ± 0.1

2.3 ± 0.5

  • Glucose phosphate
    isomerase

44.7 ± 4.8

124 ± 13

  • Triosephosphate
    isomerase

2,180 ± 254

6,055 ± 705

Pentose phosphate cycle and glutathione metabolism

  • Glucose-6-phosphate
    dehydrogenase

11.0 ± 1.6

30.6 ± 4.5

  • 6-phosphogluconate
    dehydrogenase

9.5 ± 1.5

26.2 ± 4.1

  • Glutathione reductase

4.6 ± 0.8

25.7 ± 3.0

Values expressed as x ± s; *Substrate turnover data in μmol

Table 15.8-2 Findings and enzymatic characteristics associated with the most important erythrocyte enzymopathies

Disease

Findings

Enzyme
characteristics

Glucose-6-
phosphate
dehydrogenase
(G6PD)
deficiency

Types of presentation: acute, potentially life-threatening hemolytic crisis following oxidative stress; most severe anemia, formation of Heinz bodies, basket cells, punched out cells; hemoglobinemia, hemoglobinuria; in neonates hyperbilirubinemia

Heterozygous, hemizygous or homozygous G6PD deficiency of the Mediterranean, African or Asian type. Very different enzyme activities.

Chronic hemolytic anemia

Homozygosity or heterozygosity of various G6PD deficiency types. Occurring mostly in the Caucasian population of North America and Europe.

Pyruvate kinase
(PK) deficiency

Variable symptomatology from mild, compensated hemolytic anemia to moderate severity, and to severe forms of hemolysis with very pronounced anemia. Hepatosplenomegaly, macrocytosis, pyknocytosis, poikilocytosis, relatively few reticulocytes.

Homozygous enzyme deficiency.

Very variable residual enzyme activity in the different pathological enzyme types. Special types: double heterozygosity for two enzyme variants.

Heterozygous
PK deficiency

Not clinically and hematologically apparent, except possibly for neonatal hyperbilirubinemia.

Enzyme activity of heterozygous defect carriers approximately 50% of normal.

Table 15.8-3 Medication and chemicals that cause hemolysis in G6PD deficiency

  • Acetanilide
  • Methylene blue
  • Naphthalene
  • Niridazole
  • Nitrofurantoin
  • Phenylhydrazine
  • Pamaquine
  • Pentaquine
  • Primaquine
  • Sulfanilamide
  • Sulfacetamide
  • Sulfapyridine
  • Sulfamethoxazole
  • Thiazolsulfone
  • Toluidine blue
  • Trinitrotoluol

Table 15.10-1 Reference intervals for erythropoietin in dependence of the hemoglobin concentration

 

EPO
(U/L)

Hemoglobin
(g/L)

Adults /1/*

6–32

125–180

Children /4/**

  • 0–6 days

33.0 ± 31.4

156 ± 22

  • 7–50 days

11.7 ± 3.6

128 ± 11

  • 51–100 days

21.1 ± 5.5

114 ± 10

  • 101–150 days

15.1 ± 3.9

112 ± 12

  • 151–200 days

17.8 ± 6.3

125 (n = 2)

  • > 201 days

23.1 ± 9.7

118 ± 8

* Values are the 2.5th and 97.5th percentiles. ** Values expressed as x ± s. The children’s cohorts (normal weight at birth) include only 2–13 individuals.

Table 15.10-2 Diseases and conditions associated with an adequate rise in EPO concentration

Clinical and laboratory findings

Hypoxia

In healthy individuals, a rise in serum EPO concentration occurs following 2 hours in an environment containing 11% O2, and after 8 hours the basal values are approximately doubled. A similar rise in EPO occurs 6 h following the second of two ascents to an altitude of 4,300 meters with an overnight stay at 1,200 meters after the first ascent. Nonetheless, with repeated climbs the EPO rise is more moderate /25/. Hypoxic conditions, caused by severe cyanotic heart disease or hemoglobinopathy with a mutation resulting in high affinity to O2, such as Hb Köln or Hb York, also lead to a rise in EPO. Hypoxia causes a reduction in hepcidin expression, resulting in increased enteral iron absorption and increased iron release from macrophages. The resulting elevated plasma iron concentrations are the prerequisite for an increase in erythropoiesis in chronic hypoxia. Refer also to Tab. 15.4-5 – Diseases and conditions with increased HCT.

Chronic pulmonary disease

Serum EPO is elevated in 69% of patients with chronic hypoxic pulmonary disease and secondary polycythemia /26/.

Pregnancy

The red blood cell mass falls during the first trimester of pregnancy, increases during the second trimester, and reaches its maximal volume close to the due date. At that time the red blood cell mass is some 125% of that seen in non-pregnant women. The plasma volume increases by 150%. EPO levels first increase significantly during the second trimester. In comparison with non-pregnant women with EPO values of 16.4 ± 4.1 U/L these are, during the 1st, 2nd and 3rd trimesters and postpartum, respectively 19.1 ± 6.2, 28.4 ± 15.5, 37.7 ± 24.2 and 35.1 ± 34.7 /27/.

Acute fetal distress

EPO concentrations in fetal umbilical cord blood are low, on average 1.6 U/L, increase, however, continuously from the 37th week of gestation onwards. In fetal anemia, the fetus responds from around the 24th week of pregnancy with a marked rise in EPO. This is also the case in all other conditions of deficient O2 supply to the fetus /8/. With the determination of EPO in the amniotic fluid, it is possible to distinguish acute and chronic causes of intrauterine fetal death. In acute intrauterine fetal death, amniotic fluid EPO concentrations are below 20 U/L, while with chronic causes they are 49.9–391 U/L /28/.

Autologous blood donation

With a collection of 550 mL of blood, a mass of 75 g of Hb is lost; it is replaced within, on the average, 36 days. In one study of autologous blood donation for hip replacement, consisting of five blood collections on days 1, 3, 7, 14 and 21, the Hb value dropped from 141 ± 10 g/L to 110 ± 9 g/L /29/. Serum EPO rose following each phlebotomy procedure, with a peak value of 35.8 ± 15 U/L on day 16. The rise that occurred with each donation was followed by a drop to a somewhat lower plateau. Overall, the EPO levels increased in a step-wise manner until day 16.

Autologous re transfusion of blood /30/

The collection of 20% of one’s own blood and its replacement with hydroxy ethyl starch raised EPO concentration by 4-fold within one day, before it then decreased exponentially. The reticulocyte count increased 2.4-fold within one week, and remained high for an additional week. The Hb value declined by 15% for a period of 2 weeks. Soluble transferrin receptor (sTfR) rose within 14 days by 60%. The re transfusion of 800 mL of erythrocytes 4 weeks after blood collection acutely increased the Hb by 8%; 7 days later, the initial value prior to blood collection was reached. EPO concentration remained unchanged, the reticulocyte count decreased by 30%, sTfR remained elevated for 3 days until re transfusion and then decreased.

Anemia

In anemia with adequate EPO response, there is an inverse logarithmic relationship between the Hb level and the HCT, on the one hand, and EPO concentrations, on the other hand (Fig. 15.10-2 – Expected range of EPO concentration as a function of the hematocrit value/8/.

Aplastic anemia: in patients with a Hb value of 90 ± 14 g/L (x ± s) the mean EPO concentration was 408 U/L (range: 180–2700 U/L) /25/.

Iron deficiency anemia, hemolytic anemia: in uncomplicated iron deficiency anemia and in auto-immune hemolytic anemia, with a Hb value of some 90 g/L, EPO concentrations were 50–100 U/L. In the study, mean EPO concentrations in healthy volunteers with a Hb value of 130–150 g/L were 11–12 U/L /32/.

Sickle cell anemia: in chronic non-compensated hemolytic anemia in adults, including sickle cell anemia, EPO values are lower than in acute hemolytic syndromes. The reason is that in chronic hemolytic anemia, the tissue oxygen supply is better accounted for by means of a reduction in O2 affinity for heme or by compensatory mechanisms such as heart rate increase than in the acute hemolytic syndromes /31/. Refer also to Section 15.3.5.8 – Oxygen binding by EPO.

β-thalassemia syndrome: high EPO values are measured in β-thalassemia/HbE disease which, in comparison with isolated β-thalassemia or isolated HbE disease, causes severe anemia. Thus, in a study /32/ in children aged 4–10 years, the following mean EPO values were determined: β-thalassemia 24 U/L, HbE disease 16 U/L, β-thalassemia/HbE disease 372 U/L, healthy controls 19 U/L.

Myelodysplastic syndrome: EPO values are always greater than 100 U/L.

Cancer-related anemia: in children with malignant tumors, anemia is not based upon inadequately low EPO production. This is true of solid tumors as well as of hemoblastosis. In adults, however, decreased EPO synthesis is believed to be present /32/.

Table 15.10-3 Diseases and conditions with inadequately low increase in EPO concentration

Clinical and laboratory findings

Chronic kidney disease

The kidneys are the key site of EPO synthesis. Chronic kidney disease (CKD) is associated with normocytic, normochromic anemia if the glomerular filtration rate is below approximately 30 [mL × min–1 × (1.73 m2)–1]. This is usually the case with serum creatinine values of greater than 1,5–2,0 mg/dL (133–177 μmol/L). Due to damage to the renal cells that are responsible for EPO synthesis, no adequate rise in serum EPO occurs in patients with anemia caused by CKD /25/. Patients with renal insufficiency and anemia have normal EPO concentrations. However, this is not sufficient to stimulate the erythroid precursor cells to an extent allowing 2 × 1011 senescent erythrocytes to be replaced daily in the circulation, as required. In the absence iron deficiency, the hemoglobinization of the red cells proceeds normally. The feedback mechanism between the O2 sensor and EPO synthesis is, however, still functional in patients with CKD (Fig. 15.10-7 – Erythropoietin feedback control loop). Thus, acute bleeding and acute hypoxia result in an increase in EPO concentration. Similarly, viral, drug-associated or toxic hepatic disease lead to a temporary rise in serum EPO concentrations, due to the cell regeneration /30/. Patients with renal insufficiency associated with polycystic kidney have significantly higher Hb and EPO values than other hemodialysis patients. In acute kidney failure, EPO concentrations are low during the anuric phase, but they normalize once again following restoration of renal function.

With a functional graft, following renal transplantation, EPO is normalized if the HCT exceeds 0.32. The values are lower in cases of chronic rejection reaction. In one study /34/, EPO values in situations of stable grafts, acute rejection and chronic rejection were, respectively, 24.0 ± 19.7 U/L, 35.6 ± 33.9 U/L and 6.2 ± 3.4 U/L.

Anemia of chronic disease /35/

In anemia of chronic disease (ACD), which includes anemia of infection, anemia of chronic inflammation, renal anemia and cancer-related anemia (CRA), systemic elevation of pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6 are believed to inhibit messenger-RNA synthesis for EPO /36/. Impairment of EPO binding to its receptor on the erythroid precursor cells is also suspected. Altogether, the result is EPO secretion that is inadequately low in relation to the extent of the anemia. The feedback mechanism between the O2 sensor and EPO synthesis is, however, functional.

Multiple trauma

The pathogenesis of anemia in patients with multiple trauma is based on a combination of gastrointestinal blood loss, renal and hepatic insufficiency, and reduced erythrocyte life span. Relative to the anemia, serum EPO concentrations in these patients are inadequately low in the presence of elevated pro-inflammatory cytokines /37/.

Neonatal anemia

During the first week of life, all newborns experience a physiological decrease in the red blood cell mass, with the development of anemia. In healthy newborns, the nadir of the Hb value is seldom below 90 g/L at the age of 10–12 weeks. This fall occurs earlier and is more marked in premature infants, particularly in those who are ill. The Hb value usually falls to 80 g/L in those with a birth weight of 1–1.5 kg, and to 70 g/L in those with a birth weight of less than 1.0 kg. While the Hb decline is well tolerated in full-term babies, a blood transfusion is frequently required in pre term neonates. The cause of anemia is diminished EPO synthesis. At term, 70–75% of the EPO is formed in the liver. However, the liver is believed to respond in a less sensitive manner than the kidneys to anemia and tissue hypoxia, and this in particular in premature infants, resulting in inadequate stimulation of erythropoiesis /38/.

Primary familial and congenital polycythemia (PFCP)

Primary familial and congenital polycythemia (PFCP), also known as benign erythrocytosis or familial erythrocytosis, is relatively rare; its inheritance is autosomal dominant. It is characterized by increased erythroid mass, elevated Hb value, increased erythroid precursor cell sensitivity for EPO, and decreased serum EPO concentrations /39/.

Polycythemia vera (PV)

Low EPO concentrations are measured in PV, before and after treatment. At presentation, patients with Hb levels of ≥ 180 g/L usually have an EPO value of below 2.9 U/L. This can, however, also be the case in relative polycythemia. In this apparent erythrocytosis, however, normalization of the EPO value, or even a slight increase thereof, occurs following phlebotomy accompanied by a decrease in the HCT. In PV, on the other hand, the EPO value remains low /5/.

Multiple myeloma (MM)

Anemia is a frequent complication in MM, especially in advanced disease. The cause of the anemia is less a reduction in erythropoiesis due to plasma cell infiltration of the marrow than intrinsic hypo proliferation of erythropoiesis and inadequately low EPO synthesis. This occurs in 60% of MM patients with renal insufficiency, but it is also seen with normal renal function /40/.

Table 15.10-4 Diseases and conditions with a disproportionate rise in EPO concentration

Clinical and laboratory findings

Malignant tumor

EPO that is formed ectopically can, as a para neoplastic syndrome, trigger polycythemia /41/. This is the case in 63% of patients with renal cell carcinoma and 23% of patients with hepatocellular carcinoma /42/. Likewise, Wilms tumor, cerebellar hemangiomas and uterine fibromyomas can be associated with elevated EPO levels. Polycythemia does not necessarily have to emerge clinically, due to chronic hemorrhage or tumor-associated anemia. Following successful surgery, the excessive EPO formation usually normalizes; if however, there is a recurrence of the tumor, EPO formation will again be elevated. Nonetheless, the EPO value cannot be taken as an indicator of complete surgical resection, since clearly defined threshold values do not exist. EPO is, however, suitable for monitoring purposes; a continuous increase allows for the identification of a relapse /41/.

Secondary erythrocytosis

Secondary erythrocytosis must be differentiated from polycythemia vera. Elevated EPO values which go against a diagnosis of polycythemia vera (primary erythrocytosis) have to be excluded /5/:

Presence of a high-affinity Hb mutation (Hb Köln, Hb York): more than 50 mutants of the αHb gene and βHb gene have been described. Due to their enhanced O2 binding the high-affinity mutants on the α12 region cause diminished O2 release in the tissues, resulting in a compensatory elevation of EPO and the erythroid mass. See Fig. 15.3-3 – Structure and function of hemoglobin.

Hereditary methemoglobinemia: O2 binds reversibly to deoxyhemoglobin Fe2+, but not, however, to methemoglobin (Fe3+). Furthermore, in some subunits of the Hb tetramer, Fe3+-heme increases the O2 affinity of Fe 2+-heme. In consequence, the O2 dissociation curve is displaced to the left (Fig. 15.3-2 – O2 dissociation curves), and less O2 is released to the tissues. This leads to a compensatory increase in EPO synthesis and in the erythroid mass.

Hereditary 2,3-diphosphoglycerate (2,3-DPG) deficiency due to a defect in 2,3-diphosphoglycerate mutase (DPGM): the O2 affinity for Hb is increased, with the result that less O2 is released to the peripheral tissues. A compensatory elevation in EPO occurs, and the erythroid mass in increased.

Table 15.10-5 Tests for the assessment of erythropoiesis under ESA therapy

Clinical and laboratory findings

Blood count

Basic investigation: the complete blood count serves for the assessment of the extent of the anemia and its classification. Findings which may lead to consideration of ESA therapy include normocytic, normochromic anemia with an Hb concentration of 80–110 g/L, a platelet count of > 100 × 109/L, an EPO concentration < 100 U/L or an O/P ratio of less than 1.2, and absence of leukocytosis.

Monitoring: a rise in the Hb concentration is a criterion of erythroid proliferation and Hb formation and, thereby, an indicator of therapeutic success.

Chronic kidney disease /20/: the Hb value should be determined every 1–2 weeks, at the start of treatment as well as with changes in the ESA dose, until a stable value has been reached. The goal is an increase in the Hb concentration of 10–20 g/L/month. If, following the initiation of therapy or an increase of the ESA dose, no increase by 7 g/L is achieved within 2–4 weeks, the ESA dose should be raised by 50%. If, following the initiation of therapy or an increase of the ESA dose, the Hb value increases by more than 25 g/L/month, or if the Hb level exceeds the target value, the weekly ESA dose should be reduced by 25–50%. The lack of an increase of at least 10 g/L/month is a sign of an inadequate response. The most important reasons for this is functional or absolute iron deficiency or inflammation.

Cancer patients /12/: with adequate ESA therapy, an Hb increase of 10–20 g within 6–8 weeks is expected. Patients who do not meet this target are considered to be non-responders. The causes are, primarily, functional or absolute iron deficiency, or tumor progression.

Reticulocyte count, reticulocyte Hb (CHr, Ret-He)

Basic investigation: reticulocytes are released by the bone marrow 18–36 hours before they mature into erythrocytes. They provide insight into the functional status of the bone marrow (hypo-, normo- or hyper regenerative erythropoiesis). The CHr or Ret-He provide information as to whether normochromic or hypochromic red blood cells will be formed.

Monitoring: in the early phase of ESA stimulated erythropoiesis (days 1–5) reticulocytosis, reflecting the release of immature reticulocytes from the marrow rather than an increase in erythropoiesis, occurs. An increase in the reticulocyte count of > 40 × 109/L following 4 weeks of therapy is an indication of a positive erythroid response /10/. If functional iron deficiency develops under ESA, the CHr or Ret-He decrease within 10 days after the start of treatment.

Soluble transferrin receptor (sTfR)

Under ESA therapy, the serum concentration of sTfR is primarily an indicator of erythroid proliferation rather than of functional iron deficiency. Proliferation of erythropoiesis leads to a rise in sTfR concentration /42/.

Basic investigation: the most important therapeutic effect of ESA is the enhancement of erythropoiesis. In normal iron supply, there is a positive correlation between erythropoiesis and the sTfR concentration. The basal sTfR value is, therefore, an indicator for the assessment of erythropoiesis prior to the start of therapy. Elevated values are indicative of hyper proliferative erythropoiesis, that is to say, of an increase in the erythron. The absence of an elevation relative to the basal value indicates no response to ESA.

Monitoring: a rise of the sTfR value of > 20% within 4 weeks is an indicator of responsiveness; the increase often takes place as early as within 10 days following the start of therapy.

Erythropoietin (EPO)

The determination of endogenous EPO formation should be performed, for the assessment of erythroid proliferation, as the basic evaluation prior to a planned ESA therapy in all patients, with the exception of those with renal anemia. The EPO values in anemic patients cannot simply be compared with the reference interval in healthy individuals. The relation to the Hb concentration or the HCT, and the determination of the expected (predicted = P) value by means of a reference curve (Fig. 15.10-2 – Relationship between HCT and EPO concentration) is important. In individual patients, with the generation of the O/P ratio, the measured (observed = O) value is then set in relation to the expected value. The 95% confidence interval for the O/P ratio is 0.8–1.2 /10/. An O/P ratio of below 0.8 is indicative of inadequately low endogenous EPO formation and apparently good responsiveness to ESA. In myelodysplastic syndrome the O/P ratio is high, and the response to ESA is low.

Ferritin

Ferritin is a marker of iron stores. With ESA therapy, ferritin levels fall in comparison with the basal value. Under ESA therapy, the ferritin value reflects the provisioning of the functional iron compartment (which can increase 3-fold) with iron. Many patients have inappropriately high ferritin values, relative to their available iron stores because the synthesis of ferritin is increased as a result of an acute phase response, or because ferritin is released due to chronic damage of the hepatocytes. Conditions such as these are seen, for (e.g., infection, chronic inflammation, hepatocellular injury, with malignant tumors, in hyperthyreosis, alcoholism, and with the ingestion of oral contraceptives). In order that EPO resistance due to iron deficiency does not develop with EPO therapy, ferritin concentrations must always be sufficiently high.

Basic investigation: in patients with renal insufficiency, ferritin levels before the start of ESA therapy must be > 100 μg/L (optimally 200–500 μg/L) /20/.

Monitoring: in the USA, 50% of hemodialysis patients who are receiving ESA therapy have ferritin values of > 800 μg/L; in Europe, the corresponding value is 500 μg/L. In routine therapy, ferritin values should be > 500 μg/L. In order to prevent iron overload, values of > 800 μg/L should be avoided /43/. At the initiation of ESA therapy, ferritin should be monitored every 3 months in patients receiving iron intravenously, and every 4–6 weeks in those not being treated with iron.

Transferrin saturation (TfS)

TfS is an indicator of functional iron, but it is applicable only in the absence of an acute phase response (APR), because transferrin is a negative acute phase protein and TfS is low in patients with APR. This is the case with CRP levels of > 5 mg/L.

Basic investigation: in order to assure a sufficient iron supply for erythropoiesis, TfS should be > 20% prior to treatment with ESA; 30–40% is better, and under ESA therapy TfS should be some 30%.

Monitoring: TfS of < 20% is an indication of a need for iron. In order to continuously maintain values > 20%, the therapeutic goal of iron substitution in patients with CKD is a TfS value of 30–40% /20/. Monitoring intervals as described for ferritin.

%HYPO

The proportion of hypochromic erythrocytes (%HYPO) is a valuable marker for iron demand of erythropoiesis and the erythropoietic response to ESA after intravenous administration of iron /20/. %HYPO is one of the APR independent markers of the iron demand.

Basic investigation: %HYPO should be below 10 and, optimally, below 2.5 as an indicator of an adequate iron supply prior to ESA therapy.

Monitoring: %HYPO > 10 points to iron demand for erythropoiesis. In infection or chronic inflammation, ferritin may be normal and TfS may not be useful. In this situation, a %HYPO of > 10 is indicative for functional iron deficiency. However, significant increases are not seen earlier than 3 weeks after the onset of the functional iron deficiency. Monitoring intervals as described for ferritin.

Reticulocyte Hb

Reticulocyte Hb content (CHr, Ret-He) is a real-time APR independent indicator of the iron demand for erythropoiesis.

Basic investigation: a CHr or Ret-He ≥ 28 pg rule out iron-restricted erythropoiesis. At the start of ESA therapy, intravenous iron should also be substituted /44/.

Monitoring: in patients receiving ESA treatment, a decrease in CHr or Ret-He to below 28 pg, or a relative decrease of 2 pg with values ≥ 28 pg, after some 5 days, is indicative of functional iron deficiency. This may be due to over stimulation of erythropoiesis relative to the iron supply. An increase in the CHr or Ret-He , or an unchanging normal CHr or Ret-He under the therapy of ESA and intravenous iron, are suggestive of adequate iron supply. In dialysis patients, CHr or Ret-He are better markers of the response to iron therapy than ferritin, TfS and %HYPO /44/. Monitoring intervals as described for ferritin.

C-reactive protein (CRP)

CRP is an indicator of APR, which leads to a hepcidin induced disorder of iron distribution, and inhibits adequate erythroid proliferation via an inflammatory blockade /45/.

Basic investigation: apart from iron deficiency, APR is the most important cause of ESA resistance. If the CRP value is > 20 mg/L in patients with CKD, a 30–70% increase in the ESA dose must be taken into account /45/.

Monitoring: the determination of CRP is necessary if the expected response to ESA therapy does not materialize. In such a case, an ESA dose increase is imperative. In patients with chronic renal failure and ESA treatment, this means a dose increase of 1.5–3 fold /20/.

Diagnostic diagram (Iron plot) /46/

The iron plot (see Fig. 15.6-5 – Diagnostic diagram for the assessment, monitoring and therapy of the iron status) has been evaluated for the identification of patients who require ESA therapy, for the early detection of responders, for monitoring under therapy and for the identification of the balance between ESA dose and iron requirement. Out of 286 patients /47/:

  • Two hundred and four with anemia of chronic disease (ACD), adequate storage iron and a CHr ≥ 28 pg were treated, according to recommendations of the plot, with ESA monotherapy; 56% of these patients manifested a rise in Hb ≥ 10 g/L
  • Twenty two patients with ACD and iron-restricted erythropoiesis (CHr < 28 pg) were treated with ESA and intravenous iron. All of these manifested a rise in Hb of > 10 g/L.
  • Sixty patients with empty iron stores (estimated as an elevated ferritin index) and a CHr < 28 pg were treated with iron only, orally in most cases. An Hb increase was seen in 73% of these patients.
  • The study /47/ showed that the diagnostic diagram is an effective concept for the differential diagnosis and treatment of anemia in cancer patients receiving cytostatic therapy.

Table 15.10-6 Erythropoieses stimulating agents /49/

Recombinant human Erythropoietin: rHuEPO is the artificial equivalent of the endogenous hormone erythropoietin (EPO). RHuEPO has the capacity to initiate the production of red blood cells and is commercially available for the treatment of anemia. RHuEPO stimulates the erythropoietic bone marrow compartment and is used for therapy of anemia of chronic kidney disease and myelodysplastic syndrome. RHuEPO products differ in their biological composition which results in differences in their clinical efficiencies and adverse events. They not only have benefits (reducing red cell transfusion requirements, improved quality of life) but also, they have the risks of thromboembolic complications of arterio-venous fistulas, apoplexia, and increased mortality in malignant disease.

Epoetin alpha: Epoetin alpha is produced in cell culture using rDNA technology. Epoetin alpha is synthesized in Chinese hamster ovary cells, and the amino acid sequence is similar to endogenes erythropoietin. The half-life time of these erythropoietins is relatively short (8 hours intravenous and 19–24 hours subcutaneous administration).The administration is 2–3 times weekly via intravenous or subcutaneous route. Epoetin alpha is a first generation erythropoietin.

Epoetin Beta: This erythropoietin is effective to stimulate the red cell compartment and reduces transfusion requirements. Epoetin beta does not increase mortality in malignant disese. Epoetin beta is a first generation erythropoietin.

Darbopoetin alpha: This erythropoiesis stimulating agent (ESA) is synthesized in Chinese hamster ovary cells by means of rDNA technology and its amino acid sequence is rHuEPO. Darbopoetin alpha is a long-acting ESA and has a higher molecular weight than endogenous erythropoietin because it has a greater carbohydrate content. Its half-life time is 3 to 4 times longer than rHuEPO because its affinity to the erythropoietin receptor is 4,3-times higher than of rHuEPO. The half-life time ist 3–4 fold as long as of Epoetin alpha or Epoetin beta. Darbopoetin alpha is administerd once every 2 weeks.

Epoetin DELta: This erythropoietin is sythesized in a human cell lines using rDNA technology. Epoetin delta has a similar amino acid sequence to endogenous human erythropoietin. In comparison to other erythropoietins epoetin delta has lower concentrations of certain nonhuman carbohydrate residues (N-glycolylneuraminic acid). Epoetin delta is a third generation erythropoietin.

Continuous ErythropoIetin Receptor Agonist (CERA): CERA is an ESA product produced by adding a large water-soluble polyethylene glycol moiety to epoetin beta. CERA is a continuous erythropoietin receptor agonist. It has 50 to 100 times lower affinity and much slower binding rate to the erythropoietin receptor compared to epoetin beta. CERA promises both lower dosing and less frequent injections for patients (a 5-fold longer half-life time than recombinant human erythropoietin). Hemoglobin levels can be maintained with the target range (100–135 g/L) using beweekly or even monthly CERA administration. In patients undergoing maintenance hemodialysis the hemoglobin concentrations were 109 ± 8 g/L in the weekly group. Ferritin and hepcidin concentrations were maintained /50/. Some authors reported that CERAs suppress hepcidin levels, inducing the release of stored iron and effective erythropoiesis.

ESA Mechanisms Of Action: The mechanims of action of ESAs are comparable to Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors (PHIs), which effectively increase the level of hemoglobin in patients undergoing maintenance hemodialysis /51/ and in patients with chronic kidney disease for various reasons /52/.

Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor (PHI): These factors (e.g.,daprodustat) are a new class of compounds that stabilize HIF, which in turn stimulates endogenous erythropoeitin (EPO) production and thus increases hemoglobin (Hb) concentrations in patients with chronic kidney disease (CKD). HIFPHIs are as effective as ESAs in increasing Hb levels. Among patients with chronic kidney disease undergoing dialysis daprodustat was noninferior to ESAs regarding the change in the Hb level from baseline and cardiovascular outcomes /53/.

Table 15.11-1 Thrombocyte reference intervals

 

Platelet count adults (109/L), instrument dependent /8/

Abbott CD 4000

168–405

150–346

Advia 120

203–445

166–389

Coulter LH 750

166–387

137–327

Horiba ABX, Pentra 12

179–443

168–355

Sysmex XE -2100

176–391

146–328

Values are 2.5th and 97.5th percentiles

Premature infants, neonates, small children: values as in adults /9/

School children: values as in adults /10/

Mean Platelet Volume (MPV), data in fL /11/

Abbott CD 4000

6.9–10.6

Advia 120

6.4–9.7

Coulter Gen S

7.6–10.7

Horiba ABX, Pentra 12

6.8–10.0

Sysmex SE-9500

9.4–12.9

Values are 2.5th and 97.5th percentiles

Table 15.11-2 Differences between primary and secondary thrombocytosis /12/

Criterion

Thrombocytosis

Primary

Secondary

Age
(years)

Frequently > 20, often > 40

All ages

Complications

Thrombosis, hemorrhage

Very rare

Splenomegaly

Often

Rare

Thrombocyte
count

Usually ≥ 1000 × 109/L

Frequently < 1,000 × 109/L

Duration

> 2 years

Days to weeks

Thrombocytes

  • Function

Disturbed

Normal

  • Morphology

Large, dysmorphic

Large, normal

Cause

Genetic defect

Reactive

Table 15.11-3 Differentiation of the mechanism of thrombocytopenia /15/

Examination

Increased
consumption

Reduced
formation

MPV

Elevated

Normal

PDW

Increased

Normal

Thrombocyte
life span

Decreased

Normal

Thrombocyte-
bound IgG

Detectable

Undetectable or
hardly detectable

Bleeding time

Usually normal

Prolonged

Other blood cells

Usually normal

Often normal

Megakaryocytes

Normal/increased

Decreased

Other cell lines
in the bone marrow

Normal

Frequently
decreased
or abnormal

Table 15.11-4 Medication-associated autoimmune thrombocytopenia, according to Ref. /16/

Abciximab

Acetaminophen

Acetazolamide

Allopurinol

Aminoglutethimide

Amiodarone

Amrinone

Aspirin

Carbamazepine

Carbenicillin

Cefotiam

Cephalexin

Cefalotin

Cefamandole

Quinine

Quinidine

Chlordiazepoxide

Chlorpheniramine

Chlorthalidone

Chlorthiazide

Cimetidine

Cocaine

Codeine

Danazol

Desipramine

Diazepam

Diclofenac

Difunisal

Digitoxin

Digoxin

Diltiazem

Eptifitabid

Erythromycin

Fenoprofen

Furosemide

Gentamicin

Gold salts

Heparin

Hydrochlorothiazide

Hydroxychloroquine

Imipramine

Interferon

Isoniazide

Ketoprofen

Levamisole

Lidocaine

Meperidine

Meprobamate

Methicillin

Methyldopa

Minoxidil

Morphine

Naproxen

Nitrofurantoin

Nomifensine

p-Aminosalicylic acid

Penicillamine

Penicillin

Pentamidine

Phenylbutazone

Phenytoin

Piperacillin

Piroxicam

Procainamide

Prochlorperazine

Prothetazine

Ranitidine

Rifampicin

Spironolactone

Sulfasalazine

Sulfonyureas

Tetracycline

Thioguanine

Tirofiban

Ticlopidine

Trimethoprim/sulfamethoxazole

Valproic acid

Vancomycin

Table 15.11-5 Recommendation for thrombocyte transfusion /17/

Surgical
interventions

Thres-
hold*

Hematology/
Oncology

Limit
value**

Low risk of bleeding

10

No hemorrhagic
complications

5

Major surgery

50

Solid tumors

10

Cardiac surgery

20

Leukemia

10

Neurosurgery

70–100

Chemotherapy

10

Epidural anesthesia

80

Tumors and
thrombopenia

20

Spinal anesthesia

50

Necrotic tumor

50

Diagnostic interventions

Miscellaneous

Lumbar puncture

10

Hepatic
insufficiency

10

Needle biopsy
of liver

10

Liver failure

20

Arthrocentesis

20

Central vein
catheter

20

Dental treatment

20

Major dental surgery

50

Hemorrhage
requiring
transfusion

100

Gastrointestinal
endoscopy

20

Coagulopathy
due to loss of
blood (prophylaxis)

100

Bronchoscopy

20

Preterm infants

50

Transbroncial
biopsy

50

Neonates

30

Neonates ill

50

Angiography

20

*Threshold in 109/L below which a thrombocyte transfusion should be provided; ** Limit value, bleeding is likely

Table 15.11-6 Diseases and conditions with primary thrombocytosis

Clinical and laboratory findings

Myeloproliferative disease – Generally /23/

The myeloproliferative diseases polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (MF) are clonal diseases of pluripotent hematopoietic stem cells. This leads to an uncontrolled proliferation of erythropoiesis, leukopoiesis and thrombopoiesis, alone or in combination.

Clinically, the tendency for arterial or venous thrombosis, bone marrow fibrosis, splenomegaly, or transformation to acute leukemia, is observed with varying frequency. Hemorrhagic and thrombotic complications are common in the myeloproliferative diseases of ET, PV, MF and chronic myeloid leukemia (CML).

One reason for the commonality of PV, ET and MF is an activating mutation (V617F) in the JAK2 (Janus kinase 2) gene, which codes for tyrosine kinase. JAK2 is a member of the Janus kinase family. In the hematopoietic system, these enzymes phosphorylate tyrosine residues on receptors (erythropoietin, thrombopoietin and GM-CSF) following binding of the ligand, thereby ensuring signal transmission to the cell nucleus (Fig. 15.4-2 – Homodimer erythropoietin receptor showing phosphorylated tyrosines). As a rule, JAK2 is activated when the ligands (for e.g., erythropoietin) bind to their corresponding receptors. In myoproliferative syndrome a point mutation leads to the exchange of phenyl alanine with valine (V617F) at the regulatory domain, thereby inducing a long-term activation of signal transmission in the absence of ligand-receptor binding. The activated kinases stimulate cell proliferation and are resistant to apoptosis. In this way, cells expressing a mutant JAK2 have a growth and survival advantage over normal cells.

The result of the V617F mutation is that thrombocyte formation in ET and PV is increased by 2 to 15-fold and, in MF, by 2 to 8-fold. In spite of the fact that an elevated blood thrombocyte count results from the increased thrombocyte formation, there is no direct relationship between megakaryopoiesis and the peripheral thrombocyte count. This is because of the enlarged spleen and the abbreviated life span of the thrombocytes in myeloproliferative disease. It is particularly striking that in MF, the spleen is very large and the thrombocyte life span is markedly shortened. The thrombocyte counts (109/L) are the average, 660 (in PV), 910 (in ET), 610 (in CML) and 290 (in MF).

Laboratory findings: the demonstration of the JAK2 V617F mutation confirms the tentative clinical diagnosis of myeloproliferative syndrome. However, since the mutation occurs in PV, ET and MF, differentiation by means of molecular biological determination is not possible. Since, nonetheless, the mutation is present is almost all PV patients, PV should be considered in the first place upon demonstration of the mutation, and the presence of erythrocytosis should be chosen as an important diagnostic criterion. Since it has become possible to determine the JAK2 V617F mutation, the incidence of diagnosed ET has increased significantly /24/.

– Essential thrombocythemia (ET)

The incidence of ET is 2.5 per 1 million in the general population. ET is the most indolent myeloproliferative neoplasm and is characterized by thrombocytosis with an otherwise normal complete blood count. The age distribution is bimodal, with a peak at 60 years of age in both women and men, and a smaller peak at early adulthood that concerns mainly women. ET is caused by JAK2 V617F, CALR or MPL mutations and infrequently by germline single-nucleotide variants. Diagnosis is made according to the positive criteria and the exclusion criteria of the WHO /26/. ES can present as artery thrombosis and elevated concentration of lactate dehydrogenase (LD).

Laboratory findings in ET:

  • Thrombocyte count ≥ 600 × 109/L
  • Bone marrow with predominant proliferation of megakaryopoiesis and a significantly increased number of mature megakaryocytes.

ET exclusion criteria:

  • No evidence of polycythemia vera (normal red blood cell mass or Hb below 185 g/L in men and 165 g/L in women, detectable iron in the bone marrow, normal serum ferritin or normal MCV)
  • No evidence of chronic myeloid leukemia (absence of the Philadelphia chromosome or the BCR/ABL fusion gene)
  • No evidence of chronic idiopathic myelofibrosis
  • No evidence of myelodysplastic syndrome; no del(5q)-, t(3;3)(q21;q26.1)-, inv(3;3)(q21;q26.1) abnormality and no significant granulocytic dysplasia; few, if any, scattered micromegakaryocytes
  • No evidence of secondary (reactive) thrombocytosis
  • Megakaryocyte morphology 80%
  • Splenomegaly (ultrasound or CT) 26%
  • Abnormal thrombocyte function 83%.

The clinical course of ET is, like that of PV, characterized by an elevated incidence of thrombosis, and progression to myelofibrosis and to acute myeloid leukemia. The basal leukocyte count at the time of disease diagnosis is a risk factor for severe thrombosis, particularly acute coronary syndrome. Thus, ET patients with the JAK2 V617F mutation and a leukocyte count of below 8.0 × 109/L had a hazard ratio (HR) for severe thrombosis of 1.5, while the HR in those patients with a leukocyte count greater than 11.0 × 109/L was 2.0. In ET patients, thrombosis occurs at an increased rate with thrombocyte counts of ≥ 1000 × 109/L. However, the prognosis depends exclusively upon the leukocyte count, and is better if the basal leukocyte count is below 11 × 109/L. Thus, the rates of vascular events (data expressed as 109/L) were /27/:

  • Leukocytes below 11, thrombocytes above 1,000; rate 1.59
  • Leukocytes below 11, thrombocytes below 1,000; rate 2.26
  • Leukocytes above 11, thrombocytes above 1,000; rate 2.88
  • Leukocytes above 11, thrombocytes below 1,000; rate 2.95.

Response criteria following therapy of ET /28/:

  • Complete response: thrombocyte count ≤ 400 × 109/L, leukocytes ≤ 10 × 109/L, no disease-related symptoms, no enlarged spleen. Reduction of the molecular abnormalities to an undetectable level.
  • Partial response: thrombocyte count ≤ 600 × 109/L or a decrease of over 50% compared to the starting value, leukocytes ≤ 10 × 109/L, no disease-related symptoms. Reduction of the molecular abnormalities of more than 50%.

Laboratory findings /29/: 1. Thrombocyte count ≥ 600 × 109/L; 2. Hematocrit below 0.40; 3. Bone marrow storage iron present or normal MCV value; 4. No Philadelphia chromosome or bcr/abl gene rearrangement; 5. Bone marrow fibrosis (a) absent or (b) less than one third and without significant splenomegaly or leukoerythroblastosis in the blood smear; 6. No morphological or cytogenetic indication of myelodysplastic syndrome; 7, No cause for reactive thrombocytosis.

The prevalence of the findings is /30/:

  • Thrombocyte count below 600 × 109/L = 4%; (600–999) × 109/L = 59%; (1000–1499) × 109/L = 29%; over 1500 × 109/L = 8%. With thrombocyte count (> 1000–1500) × 109/L, there is an elevated risk of bleeding, since von Willebrand syndrome is acquired due to a decrease in high molecular weight multimers of VWF. The daily ingestion of over 300 mg of aspirin represents a further danger of bleeding /31/.
  • Leukocytosis of greater than 10 × 109/L = 32%.
  • Bone marrow puncture; increased cellularity 17%, elevated megakaryocyte count 79%, abnormal megakaryocyte morphology 80%
  • Splenomegaly (ultrasound or CT) 26%
  • Abnormal platelet function 83%.

– Familial essential thrombocythemia (FET) /32/

FET is a rare myeloproliferative disease, characterized by autonomously activated megakaryopoiesis and associated with excessive thrombocyte production. The gene c-MPL that codes for the thrombopoietin receptor and its ligand, thrombopoietin (TPO), regulate the proliferation and differentiation of megakaryocytes. FET is caused by an activating point mutation in the TPO receptor gene. This leads to the exchange of asparagine with serine at position 505 of the transmembrane domain (Ser505Asn).

– Hereditary thrombocythemia /33/

The hereditary thrombocythemia is an autosomal dominant disease with the clinical features of sporadic ET. It is characterized by proliferation of megakaryopoiesis and overproduction of thrombocytes. Mutations in the TPHO gene, which codes for TPO, lead to enhanced translation of TPHO mRNA, thereby resulting in enhanced TPO synthesis and increased formation of thrombocytes.

– Polycythemia vera (PV) /23/

PV presents with erythrocytosis, often microcytic, leukocytosis, and thrombocytosis. It is also present if only the combination of erythrocytosis and leukocytosis, or of erythrocytosis and thrombocytosis, occurs along with splenomegaly. However, 7–20% of PV patients do not have thrombocytosis, and up to 17% have only erythrocytosis. The determination of the JAK2 V617F mutation, which is demonstrable in some 92% of the patients, is, therefore, a diagnostically important criterion. For further information, see Tab. 15.4-5 – Diseases and conditions with increased HCT.

Table 15.11-7 Diseases and conditions with secondary thrombocytosis

Clinical and laboratory findings

Secondary thrombocytoses – Generally

In adults, the most frequent causes of thrombocytoses above 500 × 109/L /34/ are infections 21.9%, reactive processes (e.g., after acute bleeding) 19.4%, tissue damage 17.9%, chronic inflammatory diseases 13.1%, malignant tumors 5.9%, and multiple factors 6.1%.

The following etiologies are relevant in children up to the age of 16 years: infections 30.6%, hemolytic anemia 19.3%, tissue damage 15.2%, reactive thrombocytosis 14.8%, chronic inflammation 4.1%, kidney disease 4.1%, malignant diseases 2% /35/.

In inflammatory disease, the elevation in thrombocytes is the result of enhanced synthesis of TPO in the liver, stimulated by IL-6.

– Physical exertion

Upon vigorous physical exertion, rise of 50% compared with the basal value following approximately 15 min., return to basal value following some 30 min. This is, for example, also the case with delivery.

– Surgical intervention

With major surgical interventions, decrease in thrombocyte count during intervention, normalization within 2–6 days, then, in the following days, increase by 35–150% with peak values between postoperative weeks 1 and 2 /36/.

Thrombocytosis can also occur following cardiopulmonary bypass surgery and corrective surgery of fractures.

– Acute infection

This is the most frequent cause of thrombocytosis. Most prominent are purulent infections of the respiratory, gastrointestinal and urogenital tracts, hepatobiliary infections, meningitis, sepsis and pyogenic abscesses /3435/.

In children, thrombocytosis of over 700 × 109/L favors the likelihood of an infection /37/.

– Chronic infection

In adults, most prominent are rheumatoid arthritis, inflammatory intestinal diseases, tuberculosis, and autoimmune diseases; with reference to the latter, Kawasaki syndrome in particular.

In children, rheumatoid arthritis, rheumatic fever, Henoch-Schönlein purpura and, again, Kawasaki syndrome, are to be mentioned /3435/.

– Reactive thrombocytosis

Acute bleeding, iron deficiency anemia, and hypoxemia (anemic, pulmonary, cardiac) are the most frequent causes in adults and children /3435/.

– Hemolytic anemia

In children, hemolytic anemia is a frequently diagnosed cause of thrombocytosis. The majority of these children have sickle cell anemia or thalassemia syndrome /12/. In adults, hemolytic anemia is less relevant than in children for the etiogenesis of thrombocytosis.

– Renal disease

Approximately 5% of adults with thrombocytosis have acute or chronic kidney disease, or nephrotic syndrome /12/. In children, the frequency of the etiogenesis is similar /13/.

– Malignant disease

Within the context of malignant disease, thrombocytosis occurs more frequently in Hodgkin’s- and non-Hodgkin lymphoma than in solid tumors /12/. Approximately 30–60% of patients with advanced solid tumors with metastases manifest thrombocytosis, thrombopathy or thromboembolic events /38/.

– Splenectomy

Post splenectomy thrombocytosis is not a risk factor for thrombosis.

Table 15.11-8 Thrombocytopenia of various origins

Clinical and laboratory findings

Pseudo thrombocytopenia

In pseudo thrombocytopenia, erroneously low thrombocyte count is determined with hematology analyzers. The explanation is found in the blood smear assessment. Causes of pseudo thrombocytopenia are:

  • Formation of thrombocyte aggregates or agglutinates (e.g. EDTA induced thrombocytopenia)
  • Presence of giant thrombocytes, either inherited or acquired
  • Satellite phenomenon. In EDTA blood, thrombocytes are coating the surface of segmented granulocytes, and are not counted
  • Cold agglutinins, which can induce thrombocyte agglutination in an EDTA independent manner.

The frequency of occurrence of EDTA induced thrombocytopenia is about 0.1%; it is likely based upon the baring of thrombocyte membrane glycoproteins by EDTA. The glycoproteins react with heterophile antibodies and form agglutinates. If the thrombocyte determination is performed immediately following the blood sampling, the observed thrombocytopenia is usually only slight, or even non-existent. It only occurs when the blood is left standing for an extending period of time.

In order to minimize the misdiagnosis of thrombocytopenia, the measurement of EDTA, citrate or heparin blood is always recommended, as is microscopic testing for agglutinates, for the verification of EDTA induced thrombocytopenia

Pregnancy /39/

Mean thrombocyte count decreases during pregnancy in all the women, beginning in the first trimester. In women who have a thrombocyte count less than 100 × 109/L a cause other than pregnancy or its complications should be considered. Refer to Tab. 15.11-9 – Thrombocytopenia in pregnancy. According to a study /39/ at the time of delivery 9.9% of the women with uncomplicated pregnancies had a thrombocyte count below 150 × 109/L. During the course of the uncomplicated pregnancies and deliveries only 1% had a thombocyte count below 100 × 109/L. Thrombocyte counts of less than 150 × 109/L at the time of delivery were more common among women who had pregnancy related complications (11.9% versus 9.9%).

Neonatal thrombocytopenia

Neonatal thrombocytopenia is a relatively rare condition with a prevalence of 0.9% in unselected populations. Newborns with severe thrombocytopenia may suffer from hemorrhage that leads to death or to severe lifelong defects. In one study /40/, in which umbilical cord blood from over 15,000 newborn infants and their mothers was investigated, 19 neonates (0.12%) had a thrombocyte count of below 50 × 109/L. Of 756 mothers with incidental thrombocytopenia, 1414 with hypertension and 46 with idiopathic thrombocytopenic purpura (ITP), 1,5 and 4 newborns in each group manifested thrombocytopenia of (20–50) × 109/L. Six newborns had values below 20 × 109/L; all were children who were at risk of neonatal alloimmune thrombocytopenia. The study showed that severe thrombocytopenia in newborns of mothers with incidental thrombocytopenia, hypertension and ITP are rare; if thrombocytopenia occurs, it is often associated with fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) /41/

FNAIT occurs when a women becomes alloimmunized against fetal platelet antigens inherited from the fetus’s father (which are absent on maternal platelets), leading to fetal thrombocytopenia (below 150 × 109/L). Mothers who do not possess this antigens develop specific IgG antibodies. As of the 14th week of pregnancy the antibodies cross the placenta and bind to fetal thrombocytes, which are then removed from the blood prematurely. The incidence is 1 in 800–1000 births. About 24 human thrombocyte-specific allo antigens are recognized. In white populations (approximately 80%) the most common antibody is anti-HPA-1a. The antibody binds to the polymorphic leu/pro residue of glycoprotein IIIa on the thrombocyte membrane. With a proportion of 15%, anti-HPA-5b is the second most common antibody. Some 10% of HPA-1a-negative women who have been pregnant with an HPA-1a-positive child have antibodies.

The medical history provides an indication of possible FNAIT, if sisters or close relatives have given birth to children with thrombocytopenia and an anti-HPA-1a was determined. The mothers have a normal thrombocyte count. Alloimmunization is associated with the HLA-DRB3*0101 allele, which is expressed by 90% of immunized women.

Clinical findings: Die FNAIT is the most common cause of severe thrombocytopenia in the newborn, accounting for 3% of all fetal and neonatal thrombocytopenia and 27% of severe cases defined as < 50 × 109/L or intracranial hemorrhage (ICH). The clinical symptoms defined as from isolated petechiae to intracranial hemorrhage (ICH), which occurs in 7–26% of the cases. Approximately 80% of ICH occurs intrauterine, and up to 42% of the cases occur before the 32nd week of pregnancy. In newborns with signs of hemorrhage and thrombocyte count below 30 × 109/L, the infusion of banked thrombocytes is indicated. Most of the other causes of neonatal thrombocytopenia are infection, and autoimmune thrombocytopenia

Laboratory findings: the maternal HPA antibody titer is determined, for (e.g., in the 22nd and/or the 34th week of pregnancy) using the MAIPA method (see Section 17.4 – Alloimmune thrombocytopenia); in the neonate, the thrombocyte count is determined. There is an association between the severity of the thrombocytopenia and the antibody concentration. In one study /42/, with an anti-HPA-1a cutoff value of > 3.0 IU/mL for severe FNAIT (thrombocyte count below 50 × 109/L) the diagnostic sensitivity was 93% with a specificity of 63%. The positive and negative predictive values were 54% and 95%, respectively. Of the 338 newborns with NAIT, 105 had thrombocyte counts of below 50 × 109/L, and 233 had higher values. The normalization of the thrombocyte count occurred within 4 weeks and, even after only 7–10 days. The determination of the maternal thrombocyte characteristics elicited the HPA-1A1/1A2 genotype; the baby and the father were positive for the HPA-1A1.

Surgical intervention

Thrombocyte counts of higher than 50 × 109/l do not necessitate pre-operative thrombocyte substitution, because the bleeding complications that only seldom occur can be treated intraoperatively. With counts of (50–80) × 109/L, surgical interventions, in which hemorrhage can easily be identified or stopped, can be performed. With operations within body cavities (bladder, thorax, abdomen), in which bleeding is only recognized later, it is necessary to increase the pre-, peri- and post-operative thrombocyte level to above 80 × 109/L /43/.

Postoperative thrombocytopenia

Postoperative thrombocytopenia is seen particularly following cardiovascular surgery, surgical interventions for aneurysms or peripheral vascular grafting, as well as orthopedic interventions such as hip replacement surgery /44/. Thus, in intensive surgical care units, thrombocytopenia of below 100 × 109/L is seen at least once during a stay in 20–40% of patients, and thrombocytopenia of less than 50 × 109/L is observed in 10–20% of patients /45/.

Mild to moderate thrombocytopenia occurs following the transfusion of large quantities of blood. Thus complete replacement of erythrocytes with 11 red blood cell units in a patients weighing 75 kg leads to a decrease in thrombocytes from 250 × 109/L to 80 × 109/L. Generally, however, postoperative hemorrhage only occurs if the thrombocyte count falls to below 60 × 109/L and, additionally, there is a clotting factor deficiency due to vitamin K deficiency, diminished liver function or plasma dilution /44/.

Thrombocytopenia can also be of relevance in postoperative thrombosis prophylaxis with heparin. If the patient has a deficiency in platelet factor 4, heparin is neutralized too slowly and accumulates in the blood, with consequential bleeding.

A postoperative thrombocyte count below 60 × 109/L raises the suspicion of sepsis, even in the absence of fever, of DIC, and of heparin-associated thrombocytopenia /44/.

With the administration of thrombocyte units and in the presence of a mixed population of native and foreign thrombocytes in the circulating blood, the therapeutic effect lasts for a maximum of 4 days. The duration of the substitution effect of transfused thrombocytes is only 1 day.

Sepsis /45/

In sepsis patients, the thrombocyte count decreases during the first 4 days following admission to the intensive care unit. The incidence of sepsis is 35–44% for a thrombocyte count of below 150 × 109/L, 20–25% for below 100 × 109/L, and 12–15% for below 50 × 109/L.

Post-transfusional purpura (PTP)

PTP involves a delayed transfusion reaction. Approximately 5–10 days following the transfusion of thrombocytes or of thrombocyte-containing red cell units severe hemorrhage, due to massive thrombocytopenia, occurs. The cause is immunization, often dating to many years earlier, against a thrombocytic allo antigen, usually HPA-1A. The antibodies were formed during pregnancy or following a thrombocyte transfusion. Re-exposure leads to an immune reaction and the elimination of thrombocytes. In this manner, not only are the incompatible thrombocytes damaged, but the autologous platelets are also involved in the reaction as innocent bystanders /46/.

Clinical findings: with a delay of 1 week following transfusion, massive hemorrhagic diathesis occurs. Women in particular are affected.

Laboratory findings: the thrombocytopenia can be below 10 × 109/L and return to above 100 × 109/L after 3–75 days.

Immune thrombocytopenia (ITP) – Generally

ITP is triggered by IgG antibodies that cause destruction of the thrombocytes and inhibition of their formation. The major clinical symptom is bleeding, which correlates with the extent of the thrombocytopenia. Primary or secondary forms of ITP can occur. An international working group /47/ has standardized the terminology and definitions.

  • Primary ITP is an autoimmune disorder characterized by isolated thrombocytopenia (below 100 × 109/L) in the absence of other causes or disorders that may be associated with thrombocytopenia. The diagnosis of primary ITP remains one of exclusion; no robust clinical laboratory parameters are currently available to establish its diagnosis with accuracy. The main clinical problem of primary ITP is an increased risk of bleeding, although bleeding symptoms may not always be present.
  • Secondary ITP: all forms of immune-mediated thrombocytopenia except primary ITP
  • Newly diagnosed ITP: within 3 months of diagnosis
  • Persistent ITP: within 3–12 months of diagnosis. It includes patients without spontaneous or complete therapeutic remission.
  • Chronic ITP: present for longer than 1 year
  • Severe ITP: patients present with newly occurring hemorrhage, or bleeding again following therapy

Primary ITP comprises some 80% of all ITP cases, and secondary ITP some 20%, if medication-dependent ITP is not taken into consideration /48/.

– Primary ITP

Primary ITP is caused by an immune-mediated mechanism. In some patients the antibodies are directed against a single glycoprotein; in others, against a pattern of glycoproteins. The antigens in question are glycoproteins that are located on the thrombocyte membrane (e.g., GPIIb/IIIa, GP Ia/IIa, GPI/IX). With the demonstration of antibodies against these proteins, it must be ensured that one is dealing with autoantibodies, and not with allo antibodies such as the common HLA class I antibodies. For further information, see Section 17.2 – Autoimmune thrombocytopenia.

– Secondary ITP /48/

The various forms of secondary ITP are caused by different immunological preconditions and infections.

– Acute immune thrombocytopenia of childhood

Some two thirds of children with ITP have a prior febrile disease. It is believed that thrombocytes express viral antigens or bind immune complexes against which viral antibodies are formed. As a result, cross reaction with thrombocyte antigens occurs. The thrombocytopenia is also believed to be due to a macrophage infection with resulting hemo phagocytosis and enhanced megakaryocyte clearance. About 80% of the cases of thrombocytopenia normalize within 6–12 months.

– Measles, mumps, and rubella (MMR) vaccine

Acute ITP may occur following vaccination against various viruses such as MMR, as well as Streptococcus pneumoniae, Hemophilus influenzae, Hepatitis B virus and Varicella-zoster virus. Following vaccination with the MMR vaccine, the prevalence of ITP is increased by a factor of 6. The thrombocytopenia is often severe; it develops within 72 days, and normalizes after 2 months.

– Helicobacter pylori (HP)

There is a relationship between HP infection and ITP. The pathogenesis is based upon molecular mimicry, whereby HP-induced antibodies cross-react with thrombocyte antigens. It is believed that in patients with HP antibodies, altered mucosal permeability and/or bacterial eradication following treatment with proton pump inhibitors and antibiotics initiate the immune reaction against thrombocytes. The thrombocytes are activated by the binding of HP antibodies to FcγIIa, or by an interaction of HP antibody-bound von Willebrand factor and the thrombocyte glycoprotein GPIb. Under bacterial eradication, thrombocytopenia has been reported at a rate of 0–7% in the USA, and 100% in Japan. Patients with ITP and a thrombocyte count of over 30 × 109/L should be observed, while those with a count lower than that should be treated with corticosteroids /49/.

– Cytomegaly (CMV)

CMV infection leads to severe congenital IPT, and to delayed initiation of thrombopoiesis in bone marrow transplantation. Immune-suppressed and immune-incompetent individuals with CMV infection can also suffer with ITP-like symptoms.

– Varicella-zoster virus (VZV)

VZV-ITP develops in 5% of children with acute Varicella-zoster virus infection within 5 days of the outbreak of exanthema. In non fulminant cases, spontaneous remission occurs within 2 weeks. In rare cases, the ITP does not develop until a number of weeks after the outbreak of exanthema.

– Hepatitis C

Some 20% of patients with ITP have HCV antibodies. By binding to CD81, the HCV structural protein E2 is believed to activate polyclonal B cells and to lead to activation of B cells. The result is the development of antibodies against thrombocyte glycoproteins. The thrombocyte count is below 50 × 109/L.

– HIV infection

Thrombocyte count below 150 × 109/L is reported in 5–30% of HIV patients. In a study of 36,515 patients, the 1-year incidence of HIV-associated ITP with a thrombocyte count below 50 × 109/L was 3.7%. The causes of HIV-associated ITP are multifactorial.

Chronic lymphocytic leukemia (CLL)

CLL-associated ITP develops in 1–5% of CLL patients; its incidence is approximately one tenth that of autoimmune hemolytic anemia. The ITP develops, on the average, 13 months following diagnosis of CLL; it can, however, also precede the CLL, and can develop at any time.

Hodgkin’s lymphoma

The incidence of ITP in Hodgkin’s lymphoma is approximately 0.2–1%.

Large granular lymphocyte leukemia (LGL)

This involves the clonal proliferation of CD8+T cells. Anemia, neutropenia, and mild thrombocytopenia occur frequently in these patients. Severe ITP is seen in 1% of the patients.

Common variable immunodeficiency (CVID)

More than 10% of CVID patients develop ITP, with a thrombocyte count of about 20 × 109/L. The pathogenesis of the antibody development is unknown.

Autoimmune lymphoproliferative syndrome (ALPS)

ALPS is a hereditary disease, characterized by defective apoptosis of T- and B cells. Benign hepatosplenomegaly and lymphadenopathy are found. Approximately 20% of the patients develop ITP.

Evans syndrome

In Evans syndrome, ITP and autoimmune hemolytic anemia (AHA), caused by warm antibodies, and usually directed against the Rh system, occur simultaneously. Some 50% of the patients have neutropenia, and 10% have pancytopenia. The ITP can precede the AHA, but it often also occurs subsequent to the AHA.

Antiphospholipid syndrome (APS)

Many APS patients have thrombocytopenia. The question arises as to whether the thrombocytopenia is caused by antibodies or by activation of coagulation. In ITP, antibodies to thrombocyte glycoproteins are detected, and their titer correlates better with the extent of the thrombocytopenia than the phospholipid antibodies. The thrombocytopenia is mild to moderate. Severe thrombocytopenia correlates with the risk of thrombosis. The thrombocyte values should be maintained at (40–50) × 109/L.

Systemic lupus erythematosus (SLE)

In SLE, thrombocytopenia is generally not severe and is diagnosed in one third of patients. Conversely, 2–5% of ITP patients have SLE. The thrombocytopenia develops during the course of the disease. The pathogenesis of SLE-associated ITP is multifactorial; autoantibodies against thrombocyte glycoproteins and against DNA, phospholipids and phospholipid-binding proteins, CD40 ligand, as well as thrombopoietin and its receptor, can be present.

Post-transplantation ITP

ITP can develop following bone marrow and organ transplantation. This may occur as early as after 1 day, or only 1 year later. The course can be fatal, but remission is also possible.

Medication-induced thrombocytopenia (MIT)

Medications can induce an immune response that leads to the degradation of thrombocytes. Substances associated with this undesirable side effect are listed in Tab. 15.11-4 – Medication-associated autoimmune thrombocytopenia. It is difficult to diagnose MIT in patients who are taking a number of medications. Approximately 1 out of 100,000 patients taking medicine develop immune thrombocytopenia /50/. A difficulty lies in the differential diagnosis, since immune thrombocytopenia also develops within the framework of disease, and thrombocytopenia often occurs in severely ill patients, even in the absence of exposure to medication.

In medication-associated immune thrombocytopenia, antibodies against one or both of the glycoproteins GPIb and GPIIb/IIIa are often present. The thrombocyte count may be below 10 × 109/L, and petechiae or purpura are common. The majority of patients have cutaneous bleeding, gastrointestinal or urogenital hemorrhage occurs in 10%, and 2% manifest intracranial bleeding. The thrombocyte decline usually occurs during the first week following the medication, and a rise is usually seen within 2 weeks of dis continuation of treatment /16/.

Glycoprotein IIb/IIIa-receptor antagonists

Thrombocytopenia of below 100 × 109/L or below 50 × 109/L occur under medication /51/:

  • With abcicimab at the rate of 2.5–6% or 0.4–1.6%; following dis continuation and re-exposure thrombocytopenia of below 20 × 109/L may occur
  • With eptifibatid, at the rate of 1.2–6.8% or 0.2%; following dis continuation as early as 2 h following re-exposure, thrombocytopenia of below 20 × 109/L may occur
  • With tirofiban, 1.1–1.9% or 0.2–0.5%.

Approximately 1/3 of the cases of severe thrombocytopenia following abcicimab are pseudo thrombocytopenia.

Heparin-induced thrombocytopenia (HIT)

For further information, see Section 17.5 – Heparin-induced thrombocytopenia and Lit. /52/.

Cyclic thrombocytopenia

Cyclic thrombocytopenia (CTP) is a rare disease. The hallmark of CTP is a periodic fluctuation of the platelet count. The severity of the thrombocytopenia and the duration of the cycles may vary. Mean cycle duration were 14 to 34 days. Bleeding complications can occur and patients suffer from mucocutaneous bleeding. Comorbidities may be hypothyroidism. CTP is often misdiagnosed as immune thrombocytopenia. In some patients thrombocytopenia is followed by rebound thrombocytosis with peak thrombocyte counts well about the upper reference range value /53/

Malignant disease

Thrombocytopenia occurs frequently in patients with solid tumors and in malignant hematological disease /54/. It can also be caused by:

  • Displacement of megakaryocytes in the bone marrow, which is the case in metastatic solid tumors, leukemia, multiple myeloma, and advanced lymphoma
  • Tumor-associated hypersplenism with thrombocyte sequestration or splenic metastases (rare)
  • Tumor-associated disseminated intravascular coagulation in promyelocytic leukemia, mucinous adenocarcinoma of the prostate, or pancreatic carcinoma with the release of enzymes that lead to activation of coagulation
  • Chemotherapy or radiotherapy. This is the most common cause of tumor-associated thrombocytopenia, since, daily, some 300,000 patients worldwide receive a course of chemotherapy that leads to thrombocytopenia. Approximately 1–3 weeks following suppression of the thrombopoiesis, a recovery in the count is observed. The administration of mitomycin C or nitroso ureas may lead to long term thrombocytopenia /55/.

Table 15.11-9 Thrombocytopenia in pregnancy

Clinical and laboratory findings

Incidental thrombocytopenia

The incidental thrombocytopenia is first diagnosed at the time of delivery; it concerns some 5% of pregnant women, causes 75% of pregnancy-associated thrombocytopenia, and is associated with a platelet count of over 100 × 109/L. Following delivery, the platelet count is normalized once again. It can reoccur during subsequent pregnancies. Bone marrow findings are normal. Hemostasis is not affected. Incidental thrombocytopenia is a diagnosis of exclusion /56/.

Preeclampsia

In otherwise normal pregnancy, thrombocytopenia develops in association with pregnancy-associated hypertension (preeclampsia). Preeclampsia and thrombocytopenia are seen in 1–2% of pregnancies; thrombocytopenia occurs in 50% of the cases of preeclampsia. Approximately 13–15% of the cases of thrombocytopenia occurring in pregnancy are due to preeclampsia. The platelet count is above 75 × 109/L. Thrombocytes increase within 72 hours of delivery /56/. Further pathological laboratory findings are an elevation of LD and the demonstration of schistocytes in the blood smear. Schistocytes are also detected in micro angiopathy-associated thrombocytopenia.

Immune thrombocytopenia

The incidence of immune thrombocytopenia is 1–2 per 10,000 pregnancies; it comprises 1–2% of the cases of pregnancy-associated thrombocytopenia. The onset of ITP at the beginning of pregnancy is not uncommon. The thrombocyte count is usually below 50 × 109/L, while a count of over 30 × 109/L is seldom associated with bleeding. Caesarean sections can be performed without bleeding complications with thrombocyte counts of greater than 70 × 109/L /57/.

HELLP syndrome

This syndrome comprises micro angiopathic hemolytic anemia with edema, hypertension and proteinuria.

Laboratory findings: schistocytes in the blood smear, thrombocytopenia of less than 100 × 109/L, in severe cases less than 50 × 109/L, elevated bilirubin, LD and AST. Since many patients with HELLP syndrome have hypertension and proteinuria, there is clinical overlapping with preeclampsia. Approximately 4–12% of preeclampsia patients also manifest the diagnostic criteria of HELLP syndrome /58/.

HIV infection

Thrombocytopenia occurs frequently with HIV infection, even early on. In one study /59/, out of 26 HIV positive pregnant women, 18 had thrombocytopenia and the thrombocyte count in 12 of these patients was below 50 × 109/L. Out of 13 caesarean sections, postoperative bleeding complications occurred in 4 cases.

Table 15.12-1 Leukocyte reference intervals (data expressed in 109/L or %)

Age

Total

Neutro-
phils

Eosino-
phils

Baso-
phils

Lympho-
cytes

Mono-
cytes

Fetus /3/

3.4 ± 0.9

0.2 ± 0.1

0.08 ± 0.1

0.02 ± 0.02

2.6 ± 0.7

0.2 ± 0.1

Umbilical
cord blood /4/

6.2–17.7

Cesarean section (Cs)

7.3–48.0

Spontaneous delivery

4.2–40.3

Emergency Cs

Children /56/

1–14 d

6.5–15.0

2.0–10.0

0–0.8

3.0–7.5

0–3.0

20–60%

0–8%

0–2.5

18–55%

2–20

15–180 d

6.5–15.0

1.5–6.5

0–0.5

 

2.0–8.0

0–3.0

15–60%

0–5%

0–1.2%

18–65%

5–20%

0.5–< 2 yrs

6.5–15.0

2.0–9.0

0–0.3

 

1.6–7.0

0.4–2.0

20–70%

0–4%

0–1.1%

18–60%

5–15%

2–< 6 yrs

5.0–12.0

2.0–8.0

0–0.3

 

1.5–4.5

0.3–1.2

30–75%

0–4%

0–1.0%

13–55%

4–10%

6–< 12 yrs

4.5–11.0

2.0–7.5

0–0.4

 

1.2–3.6

0.3–0.9

40–75%

0–5%

0–1.0%

13–50%

4–10%

12–< 18 yrs

4.5–10.5

2.0–7.5

0–0.3

 

1.0–3.2

0.4–1.3

40–75%

0–5%

0–1.0%

13–45%

4–8%

Adults /7/

 

 

+

+

Abbott
CD 4000

4.0–11.2

2.0–7.7

0.03–0.45

0.007–0.09

1.2–3.6

0.23–0.82

3.9–10.1

1.7–7.0

1.1–3.2

0.25–0.94

Advia
120

4.0–11.2

2.1–7.7

0.03–0.47

0.02–0.11

1.2–3.5

0.20–0.65

3.8–10.3

1.8–7.0

1.1–3.1

0.24–0.73

Coulter
LH 750

4.0–11.2

2.2–7.5

0.04–0.40

0.00–0.10

1.1–3.5

0.26–0.81

3.9–10.9

2.0–6.7

1.2–3.0

0.29–0.86

Horiba
Pentra12

4.0–11.5

2.2–7.9

0.06–0.78

0.02–0.27

1.1–3.5

0.22–0.93

3.9–10.3

1.8–6.7

1.1–3.1

0.25–0.90

Sysmex
XE-2100

3.9–10.4

1.9–7.3

0.03–0.44

0.01–0.08

1.2–3.6

0.25–0.85

3.7–9.9

1.8–6.2

1.1–3.2

0.25–0.85

Relative % /8/

40–75

0.5–7

0.2–1.5

17–47

4–12

The values for fetuses aged 20–27 weeks (of pregnancy) are expressed as x ± s. The values for children were averaged from three studies since the variation was considerable. Values are 2.5th and 97.5th percentiles. The childrens’ values were obtained with the following hematology analyzers: Bayer H3, Siemens Advia 120, Coulter STKS. Values for adults are 2.5th and 97.5th percentiles.

Table 15.12-2 Kinetics of the neutrophil granulocytes /10/

Progenitor cells

  • Mean maturation time in the mitotic pool (myeloblast to myelocyte)

7–9 days

  • Mean time in the post-mitotic and storage pool (metamyelocyte to PMN)

3–7 days

Polymorphonuclear neutrophil (PMN)

  • Mean half life time in the circulation

6 h

  • Neutrophil count in the total body pool*

4.6 × 1011

  • Circulating pool in the vascular system*

2.2 × 1010

  • Marginal pool of the vascular system*

2.3 × 1010

  • Daily turnover*

1.0 × 1011

* Based upon body weight of 70 kg.

Table 15.12-3 Criteria of neonatal sepsis

Manroe criteria /9/

Two or more criteria must be fulfilled.

  • I/T neutrophil ratio greater than 0.16
  • Neutrophil count below 7.5 × 109/L or above 14.5 × 109/L
  • Immature neutrophil count above 1.4 × 109/L

Rodwell criteria /14/

Three or more criteria must be fulfilled.

Manroe criteria, plus:

  • I/M ratio ≥ 0.30
  • Leukocyte count ≤ 5.0 × 109/L or above 25 × 109/L
  • Degenerative neutrophil changes
  • Thrombocyte count ≤ 150 × 109/L

I, immature granulocytes; T, total granulocytes; M, mature granulocytes

Table 15.12-4 Diseases and conditions with neutrophilia /10/

Clinical and laboratory findings

Physical work, situations of mental and physical stress, normal delivery

Neutrophilia is caused by the release of catecholamines, because the proportion of marginated PMNs is reduced. The pulmonary capillary bed is the main site of the marginated PMNs. The lymphocyte and monocyte counts also increase. Leukocyte counts of 20 × 109/L are achieved. In leukocytosis associated with acute infection and glucocorticoid administration, no increase in lymphocytes or monocytes is seen. Following a marathon run, the leukocyte, neutrophil and immature granulocyte counts increase respectively by 230%, 330%, and 150% /34/.

Smoker

On the average, the leukocyte count is 10% higher in smokers than in non-smokers. The increase is seen with as few as 4–5 cigarettes per day. Heavy smokers may have values of up to 15 × 109/L. Almost all leukocyte sub populations are increased to the same extent /35/.

Pregnant women /36/

The leukocyte count is elevated in pregnancy, mainly due to the rise in PMNs. Accordingly, the following neutrophil values in 109/L were observed in women: non-pregnant 3.70 ± 1.43; pregnant 1st trimester 6.46 ± 1.64; pregnant 2nd trimester 7.70 ± 1.67; pregnant 3rd trimester 7.37 ± 1.76.

Neonate

The leukocyte counts are (5–30) × 109/L. Higher or lower values are indicative of bacterial infection. The I/T ratio is also indicative. The ratio of immature (I) to total (T) leukocytes of ≥ 0.5 on the first day of life, and of > 0.2 from the second day of life onwards can be indicative for sepsis.

Bacterial infection

Acute bacterial infections result in neutrophilia primarily the PMNs, but also bands are increased. Metamyelocytes are found only following marked stimulation. Bacterial infections lead to neutrophilia of (15–25) × 109/L, and less often of up to > 50 × 109/L. A left shift and toxic granulation are common. In sepsis, especially sepsis caused by gram negative bacteria, neutropenia may develop. Acute infections that are associated with splenomegaly, such as enteric fever, only lead initially, if at all, to mild neutrophilia.

Leukemic reaction: this reaction occurs in severe systemic infection (e.g., sepsis, miliary tuberculosis) as well as in severe hemolysis. The leukocytosis is > 25 × 109/L. Cells such as myelocytes, promyelocytes and myeloblasts pass from the mitotic compartment into the blood. In contrast to chronic myeloid leukemia, all precursor cells of granulopoiesis are present, and there is no leukemic hiatus.

Fungal, parasitic and viral infection

These infections lead to leukocytosis of only up to 20 × 109/L; neutrophilia is frequently present only in the early phase. Viral infections are often associated with neutropenia.

Chronic inflammatory disease

An increase in PMNs of up to 3-fold the upper reference interval value may occur, for example, in rheumatoid arthritis, rheumatoid fever, bronchitis, colitis, dermatitis, or pyelonephritis /10/.

Metabolic disease

Coma diabeticum, coma uremicum, coma hepaticum, acute gout attack, eclampsia, and acute thyrotoxicosis lead to endogenous toxic neutrophilia.

Cushing’s disease /37/

Glucocorticoid receptors are expressed in leukocytes and are known to play a role in cell adhesion and leukocyte recruitment from bone marrow. Approximately 40% of the patients present with leukocytosis of (10.5 ± 2.6) × 109/L. and dropped to (8.4 ± 1.9) × 109/L. Mean neutrophil count was (7.6 ± 2.6) × 109/L and dropped to (5.3 ± 1.7) × 109/L.

Coronary heart disease (CHD) /38/

Leukocytosis is an independent risk factor for CHD. Re infarction studies also show that the basal leukocyte count correlates with the frequency of re infarction. In patients with angina pectoris or myocardial infarction, mortality is increased if the leukocyte count is greater than 10 × 109/L.

Intoxication

Corticosteroids, lead, mercury, benzene, and carbon monoxide lead to exogenously or endogenously induced neutrophilia. The neutrophils increase shortly or a few hours after exposure.

Acute loss of blood

Post-hemorrhagic neutrophilia. Variable increases in neutrophils up to 25 × 109/L on day 3–5. Frequently concurrent thrombocytosis. Major surgery leads to similar findings.

Malignant tumor

Neutrophilia is encountered with many gastrointestinal and pulmonary tumors, particularly those with liver and lung metastases. Neutrophilia is either caused by the inflammatory reaction to destruction of the tumor tissue, or is associated with the production, by some of the malignant tumors, of granulopoiesis-activating colony-stimulating factors (G-CSF).

Chronic myeloid leukemia

Proliferative leukocytosis. Leukocyte count increases from 20 × 109/L to higher than 50 × 109/L with left shift (blasts, fraction < 20%), proliferation of eosinophils and basophils. Optional thrombocytosis, demonstration of the Philadelphia chromosome in 90% of the patients, splenomegaly.

Myelofibrosis

Proliferative leukocytosis with leukocytes up to 50 × 10 9/L with pathological left shift (appearance of blasts) and extramedullary hematopoiesis. Reduction, to varying degrees, of the erythrocyte and thrombocyte life spans. ANP index low-normal to increased /10/.

Polycythemia vera

Mean proliferative neutrophilia of 20 × 109/L due to enhanced granulopoiesis. The ANP index may be elevated. Erythrocytosis and thrombocytosis are characteristic /10/.

Splenectomized patient

According to one study /39/, 42.5% of patients have leukocyte counts above 10 × 109/L, and 9% have counts of over 13.5 × 109/L. An disproportionate neutrophil increase occurs. Approximately 46% of the patients have a thrombocyte count of greater than 350 × 109/L, with a normal thrombocyte distribution.

Table 15.12-5 Drugs that can cause neutropenia

Analgetics and anti-
inflammatory drugs

  • Indomethacin
  • Gold salts
  • Pentazocine
  • Acetaminophen
  • Phenacetin
  • Aminopyrine
  • Butasone
  • Valproic acid

Antimalarial drugs

  • Chloroquine
  • Pyrimethamine
  • Dapsone

Antibiotics

  • Cephalosporins
  • Clindamycin
  • Gentamicin
  • Isoniazide
  • Metronidazole
  • Nitrofurantoin
  • PAS
  • Penicillin
  • Rifampicin
  • Streptomycin
  • Sulfonamides
  • Tetracycline
  • Vancomycin

Anticonvulsants

  • Carbamazepine
  • Ethosuximide
  • Phenytoin
  • Valproic acid

Antidepressants

  • Amitriptyline
  • Desipramine
  • Doxepine
  • Imipramine

H2-blockers

  • Cimetidine
  • Ranitidine

Cardiovascular
medications

  • Ajmalin
  • Alprenolol
  • Captopril
  • Quinidine
  • Disopyramide
  • Flecainide
  • Hydralazine
  • α-Methyldopa
  • Oxprenolol
  • Procainamide
  • Propafenone
  • Propranolol
  • Tocainide

Antidiabetics

  • Chlorpropamide
  • Tolbutamide

Diuretics

  • Acetazolamide
  • Chlorthalidone
  • Chlorthiazide
  • Ethacrynic acid
  • Hydrochlorothiazide
  • Spironolactone

Others

  • Allopurinol
  • Levamisol
  • Penicillamine

Antithyroid drugs

  • Carbimazole
  • Methimazole

Hypnotics, sedatives

  • Benzodiazepines
  • Chlorpromazine
  • Clozapine
  • Chlordiazepoxide
  • Meprobamate

Others

  • Pindolol
  • Ticlopidine
  • Interferon-α
  • Ribaverin

Table 15.12-6 Congenital neutropenia

Clinical and laboratory findings

Severe congenital neutropenia (SCN) /40/

SCN was first discovered by Kostmann in the 1950s and is therefore also known as Kostmann syndrome. SCN is a heterogeneous group of diseases, with severe, life-threatening bacterial infection beginning at an early age. In addition to a reduced granulocyte count, some patients manifest a spectrum of qualitative changes such as defective production of bactericidal proteins or defective granulocyte activation. Furthermore, a key feature of many forms of SCN is the termination of maturation at the level of myoblast to promyelocyte. The cells manifest atypical nucleoli and cytoplasmic vacuolization. More than 95% of patients react to rHuGCSF with a rise in granulocytes to over 1 × 109/L.

The following forms of SCN are distinguished:

  • Kostmann form (autosomal recessive SCN). Functionally inactive mutations in the HAX1 gene are present. The gene codes for the mitochondrial protein of the same name; its role is the prevention of cellular apoptosis.
  • Autosomal dominant gain-of-function mutations in ELANE, which encodes neutrophil elastase, lead to the mislocalization and intracellular accumulation of unfolded proteins, creating endoplasmic reticulum stress The stress in turn affects the survival and differentiation of granulocytic progenitors with few or no mature neutrophils in the peripheral blood. The patients have life-threatening bacterial infections shortly after birth. Approximately 50% of patients with congenital neutropenia harbour autosomal dominant gain-of-function mutations in ELANE. There are more than a hundred mutations in ELANE that span its five exons. Daily subcutaneous administration of recombinant human granulocyte-stimulating factor, bone marrow transplantation or correction of a single mutation in ELANE with the use of CRISPR-Cas9 gene editing of primary hematopoietic stem cells (HSCs) are methods of therapy /71/.
  • Autosomal dominant inherited mutation in the GFI1 gene, a transcription factor. GFI1 is a zinc finger molecule with transcriptional repressor and splicing functions. It regulates the differentiation of hematopoietic and non-hematopoietic cells. Monocytosis, lymphopenia and disturbed lymphocyte function occur in addition to granulocytopenia.
  • X chromosomal recessive inherited mutations in the WAS genes that encode the Wiskott-Aldrich protein. In addition to neutropenia, monocytopenia, T-lymphocyte activation and severe hypoplasia of bone marrow myelopoiesis (which is resistant to GCSF treatment) occur.
  • A mutation in the G6PC3 gene, which encodes the catalytic unit 3 of glucose-6-phosphatase. The result is premature apoptosis of the neutrophils. Apart from that, the patients manifest constitutional problems such as cardiac defects and abnormal urogenital development /41/.
  • Autosomal dominant inherited mutation in the CSF3R gene, which codes for the G-CSF receptor.
  • Shwachman-Diamond syndrome: defect in the SBDS gene. Autosomal recessive disease with enzymatic deficiencies of the gastrointestinal tract and skeletal abnormalities.
  • Barth syndrome: defect in the Taz1 gene. X-linked disease with disturbances of lipid metabolism, cardiomyopathy and muscle weakness.
  • WHIM syndrome: defect in the CXCR4 gene. Autosomal dominant disease with wart formation, hypogammaglobulinemia and recurrent bacterial infection.
  • Chediak-Higashi syndrome: defect in the LYST (CHS1) gene. Autosomal recessive systemic disease with hypopigmentation, prolonged bleeding time and peripheral neuropathy.

Clinical findings: overall, SCN is a are rare disease, often associated with severe bacterial infection and fever starting in the first months of life. Some 20% of the children have splenomegaly in the first year of life, around 40% by the age of 10. With rHuGCSF treatment, the neutrophil count can be maintained at above 1 × 10 9/L, and the clinical symptomatology can be reduced significantly. The most important differential diagnosis in children aged 1-3 years are autoantibodies, which can cause destruction of the neutrophils. Nonetheless, these children do not suffer from severe infections.

Laboratory findings: often severe neutropenia, usually below 0.2 × 109/L. Monocytes are elevated 3–5-fold, mild anemia, thrombocytes and IgG slightly elevated. In the bone marrow presence of myeloblasts and promyelocytes, but marked reduction in myelocytes and metamyelocytes, bands and neutrophils. Marrow eosinophils and monocytes are increased, other cell lines are normal.

Cyclic neutropenia

Genetic molecular and cellular studies have demonstrated that autosomal dominant cyclic neutropenia and the sporadically occurring form involve mutations in the gene ELA2 that codes for neutrophil elastase. This enzyme is formed in the granulation of the precursor cells. In cyclic neutropenia an atypical enzyme that causes apoptosis of the granulocytic precursor cells is formed, leading to effective oscillatory cell production /42/.

Clinical findings: suspicion sets in during the first year of life in children with recurrent fever, pharyngitis, mouth ulcers, lymphadenopathy or recurrent cellulitis. Rare adult cases manifest similar symptomatology, often with painful cervical lymphadenopathy. Not infrequently, these patients present with acute peritonitis, ileus or septic shock.

Laboratory findings: typically, oscillations of the PMN and monocytes occur every 21 days (i.e., every 21 days the neutrophil count falls to below 0.2 × 109/L for 3–5 days) and 10–15 days later reaches a peak with subnormal numbers of some 2.0 × 109/L. Some patients manifest oscillations of the thrombocytes and the reticulocytes, other, in addition, of the lymphocytes and eosinophils.

Glycogen storage disease

Glycogen storage disease (GSD) is a rare autosomal recessive disease that leads to elevated glycogen concentrations or to an abnormal glycogen structure. Based upon the causative defect, GSD is subdivided into 10 types. GSD1b is caused by a glucose-6-phosphatase translocase deficiency. This enzyme transports glucose-6-phosphate to the endoplasmic reticulum, where the conversion of glucose-6-phosphate to glucose and phosphate takes place. A deficiency in this enzyme causes deficient glucose formation from gluconeogenesis and glycogenolysis. The exact mechanism underlying the neutropenia in these patients is unknown /43/.

Clinical and laboratory findings: hepatomegaly, seizures, coma, hypoglycemia, hyper lactatemia, acidosis, hyperuricemia, hyperlipidemia. See also Tab. 5.6-6 – Hereditary metabolic disorders causing lactic acidosis and Tab. 3.2-2 – Hypoglycemia syndromes in childhood and infancy).

Primary immunodeficiency syndromes

The primary immunodeficiency syndromes are a heterogeneous group of more than 75 diseases, some of which are associated with neutropenia /44/ (see also Tab. 21.2-9 – Frequency and inheritance of severe combined immunodeficiency (SCID) forms).

X-linked agammaglobulinemia (XLA): some of these patients have significant neutropenia, and are at risk of suffering from a fungal or a Pneumocystis carinii infection.

Hyper-IgM syndrome: according to a European Society for Immunodeficiencies report, 68% of patients with hyper-IgM syndrome have neutropenia. Approximately 45% of these cases are chronic, and a few of these are cyclic.

Common Variable Immunodeficiency (CVID): the neutropenia may be autoimmune in nature.

IgA deficiency: the basis for the neutropenia is likely autoimmune.

Cartilage-hair hypoplasia: moderate to severe neutropenia with cell neutrophil counts of (0.1–2.0) × 109/L.

Reticular dysgenesis: this is the most severe form of SCID, with early interruption of differentiation of the granulocyte cell lineage and severe lymphopenia. Moderate to severe neutropenia.

Table 15.12-7 Secondary neutropenia

Clinical and laboratory findings

Pre-term delivery

Approximately 50% of newborns weighing less than 2 kg have neutropenia below 1.5 × 109/L. In some newborns of mothers with preeclampsia, the neutropenia may be severe, and may be associated with gram negative infection. The presence of a proliferative disorder of regulation of the myeloid lineage, with a reduction of the post mitotic pool, is assumed /45/.

Chronic idiopathic neutropenia

This is an acquired selective neutropenia of unknown etiology that occurs in all age groups and lasts for more than 3 months. Some of the patients are symptomatic, while in others the disease course is associated with relatively few infections. A neutrophil count of (1.5–0.5) × 109/L is typical, while in severe cases it may fall to below 0.5 × 109/L. With regard to other cells, the blood count is normal. The bone marrow is also often normal; otherwise, it may show a decrease in post mitotic granulocytes. Long-term studies demonstrate that chronic idiopathic neutropenia does not convert to leukemia, myelodysplastic syndrome or aplastic anemia /46/.

Alloimmune neonatal neutropenia (AINN)

AINN is caused by IgG allo antibodies that are directed against neutrophil alloantigens. They are formed by the mother against fetal neutrophil granulocytes, they cross the placenta and are detected in maternal blood and the umbilical cord /47/. The neutropenic newborn is prone to infections during the neonatal period. AINN occurs only infrequently; anti neutrophil antibodies were detected in 4 out of 1016 sera collected from newborn infants, but none of these infants had AINN /48/.

Laboratory findings: selective neutropenia in umbilical cord blood, often with compensatory monocytosis and eosinophilia. Evidence for presence of allo antibodies (e.g., anti-HNA-1a, anti-HNA-1b, anti-HNA-2a, anti-HNA-3a, anti-pan-FcRγIIIb; ND1).

Autoimmune neutropenia (AIN)

AIN is a chronic neutropenia in which autoantibodies to neutrophils are detected. AIN is a relatively benign, non-familial disease. It is found primarily in children < 2 years of age. AIN begins 1–15 months following birth with recurrent oropharyngeal and cutaneous infections, and ends spontaneously 7–73 months later /49/. The leukocyte count can be normal in spite of neutropenia < 0.25 × 109/L. AIN in adults is rare.

Laboratory findings: the diagnostic investigation involves the demonstration of autoantibodies in the neutrophil agglutination test, the neutrophil immunofluorescence test, or the Monoclonal Antibody Immobilization of Neutrophil Antigen (MAINA) assay /47/.

Autoimmune disease

Autoimmune diseases such as rheumatoid arthritis (RA), Felty syndrome, large granular lymphocyte (LGL) leukemia and systemic lupus erythematosus (SLE) may be associated with chronic neutropenia /50/.

Felty syndrome: this syndrome is defined by arthritis, splenomegaly and neutropenia. Patients who develop Felty syndrome often have long standing RA with nodes, ulcers, splenomegaly and hyper pigmentation of the lower extremities. With disease progression, vasculitis, neuropathy, pulmonary fibrosis and hepatomegaly can develop.

Laboratory findings: neutropenia < 1,5 × 109/L, mild anemia, thrombocytopenia and lymphopenia, high level of rheumatoid factor, hypergammaglobulinemia, positive antinuclear antibodies /28/.

LGL leukemia: LGL are microscopically large lymphocytes with rough eosinophilic inclusions. LGL can be CD8+T cells or natural killer (NK) cells. Some of the RA patients have clonal proliferation of CD8+LGL and neutropenia. Virtually all patients with Felty syndrome, and the RA patients with LGL leukemia, are HLA-DR4-positive which renders a uniform disease process likely /50/.

SLE: neutropenia is common in SLE, although infections due to severe neutropenia are infrequent. In one study /51/, 47% of the patients had neutropenia, 13% had hemolytic anemia, 20% lymphopenia and 27% thrombocytopenia. Causes of neutropenia were neutrophil granulocyte reactive IgG, enhanced apoptosis, and intrinsic hyper proliferation of myelopoiesis /50/.

Medication-dependent neutropenia – Generally

The granulopoiesis disorder can affect the mitotic compartment or the pluripotent stem cells; immune-mediated neutropenia may also occur /52/. Some of the medications that can cause neutropenia are shown in Tab. 15.12-5 – Drugs that can cause neutropenia.

– Isolated neutropenia

Suppression of the mitotic compartment: the granulocyte storage pool contains a 5-day supply of neutrophil granulocytes. In mitotic compartment dysfunction and with a granulocyte half-life time of 6 hours, a continuous decline in the granulocyte count occurs within 5 days. If the toxic substance is discontinued, recovery takes place within 36–72 hours.

– Aplastic anemia

Suppression of the pluripotent stem cells: the result is a decline of the maturation of all hematopoietic cell lines a condition that is called aplasia. In acute aplastic anemia, neutropenia and thrombocytopenia (petechiae and/or ecchymosis) occur during the first 2–3 weeks following the start of medication. Since the life span of the erythrocytes is longer, anemia is not seen at this point in time.

Hematopoiesis is diminished in chronic aplastic anemia, but thrombocytes and neutrophils are at levels which prevent hemorrhage and sepsis.

– Immune neutropenia

Immune-mediated neutropenia: the medication or a metabolite thereof alters the antigen pattern of the neutrophil membrane by attaching as a hapten. The immunization can occur either following a single administration or only after long term ingestion. The pathogenesis is believed to be based upon an antibody or a cell-mediated immune response. Immune regulation may, possibly, also be influenced through the formation of autoantibodies /52/. Neutropenia may develop rapidly, within hours, but it may also develop slowly. The neutrophil count can stabilize at a low level, but it may also normalize again.

Chemotherapy of malignant tumors

Chemotherapy-induced suppression of granulopoiesis is dependent upon the cytotoxic substance. A decrease in neutrophil granulocytes first occurs when the granulocyte storage pool is empty and the maturation of CFU-GM to myeloblasts stops. Following chemotherapy, neither a shortened neutrophil half-life time nor ineffective granulopoiesis are usually observed /53/.

Cytotoxic substances which act during the M phase such as vinblastin, cytarabin or methotrexate lead to more rapid neutropenia and thrombocytopenia, followed by quick normalization. Only longer term infusion of these phase-specific cytostatic agents leads to permanent neutropenia. Non-phase-specific cytostatics (anthracyclines and dactomycin) with effects on the mitotic pool are associated with an onset of neutropenia similar to that induced by the phase-specific agents, but the duration of the neutropenia lasts longer. Cytostatics with no effect on the mitotic pool (busulfan) cause a more delayed nadir and recovery of granulocytes.

After chemotherapy, granulocyte-colony stimulating factor (G-CSF) such as filgastrim, lenogastrim, and pegylated filgastrim is used to accelerate neutrophil recovery and thus prevent complications due to neutropenia.The European Organisation for Research and Treatment of Cancer, the American Society of Clinical Oncology, and others have established evidence based guidelines for the prophylactic use of G-CSF. However, there is either a lack of use or incorrect application of the guidelines in up to 90% of patients with lung cancer /58/.

Methotrexate (MTX) therapy of chronic polyarthritis

In chronic rheumatoid arthritis, MTX is administered as basic therapy at weekly doses of 7.5–25 mg. Aplastic anemia occurs in some cases, with a leukocyte count of (0.2–2.6) × 109/L, a thrombocyte count of (3–63) × 109/L, and an Hb concentration of 49–121 g/L /54/.

Metamizole

Metamizole is employed in the initial treatment of hyperthyreosis. Agranulocytosis with fatal consequences may occur. In a treatment study with metamizole / neutropenia or agranulocytosis occurred in 1.6% of the cases treated with a daily dose of 30 mg; at a daily dose of 15 mg, the frequency of these side effects was only 0.47%, with no attenuation of the therapeutic efficacy /55/.

Acute febrile infection

Acute febrile infection in adults may be associated with leukopenia and thrombocytopenia (Tab. 15.11-8 – Thrombocytopenia of various origins). Thus, in diarrhea caused by Campylobacter jejuni, 30% of the patients had a leukocyte count below 4 × 109/L, in 25% the thrombocyte count was below 130 × 109/L, and 1 patient (5%) manifested both of these /56/.

Myelodysplastic syndrome (MDS)

Obligatory are leukocytes below 4 × 109/L, thrombocytes below 100 × 109/L, and Hb levels below 120 g/L. Reticulocytes are often decreased, LD and ferritin increased. Possibly bone marrow mutation analyses for diagnosis confirmation: tet2, runx1, asxl1, sf3b1, srsf2, tp53, u2af1, dnmt3a, zrsr2, ezh2, nras, kras. See also Section 15.16 – Myelodysplastic syndrome.

Acute leukemia (AL)

ALs are malignant diseases, the origins of which are to be found in a myelomonocytic precursor cell (AML), a lymphoid precursor cell (ALL), or in the stem cells. Characteristic laboratory findings in these patients are neutropenia, anemia, and thrombocytopenia.

Cirrhosis of the liver

Anemia, leukopenia, and thrombocytopenia occur frequently in chronic liver disease, especially cirrhosis of the liver. Presumed mechanisms are hypersplenism, hemolysis, and humoral inhibition of hematopoietic precursor cells.

Megaloblastic anemia

Due to a DNA synthesis disorder, anemias of severe folate and vitamin B12 deficiency are associated with neutropenia and pancytopenia. The marrow is hyperc ellular, and a maturation dissociation of the hematopoietic cell lineages occurs. Hematopoiesis is ineffective, since a considerable fraction of the cells does not mature and become apoptotic.

Table 15.12-8 Acute febrile infections with leukopenia and/or thrombocytopenia /5657/

Bacterial infections

Viral infections

Severe sepsis, particularly gram-negative pathogens

Herpes viruses: EBV, CMV, HHV6, VZV, HSV

Toxic shock syndrome (staphylococci, streptococci)

Measles, rubella

Typhoid fever (S. typhi, S. paratyphi)

Hepatitis A and B

HIV and AIDS

Shigella enteritis, campylobacter infections

Parvovirus B19

Dengue virus infection

Rickettsia infections (spotted fever, typhus)

Severe acute respiratory tract infection due to Coronavirus

Ehrlichiosis, anaplasmosis

Influenzavirus

Coxiella burneti infection

Nonspecific viral infection

Bartonella infection (cat scratch disease)

Following protective vaccination against viral infections

Brucellosis, leptospirosis, tularemia

Borrelia burgdorferi infection

Parasitosis

Recurrent fever (Borrelia spp.)

Toxoplasmosis

Tuberculosis (M. tuberculosis)

Malaria

Mycoplasma pneumoniae infection

Visceral leishmaniasis

Table 15.12-9 Diseases and conditions with lymphopenia

Clinical and laboratory findings

Congenital immunodeficiency

Different forms of severe combined immunodeficiency (SCID), ataxia teleangiectasia, malnutrition, and zinc deficiency may be associated with varying degrees of lymphopenia. The formation of lymphocytes is either not in place due to a lack of stem cells, or is disturbed for other reasons /20/.

HIV infection

The selective destruction of CD4+T cells and the reversal of the CD4+/CD8+ ratio occurs /58/.

Chemotherapy

Chemotherapy can lead to severe lymphopenia. Alkylating substances have very different actions /20/. Thus, cyclophosphamide causes lymphopenia even at low to moderate doses. The lymphopenia may persist for years following dis continuation of therapy. CD4+T cells are more severely affected than other T cells. A decrease in CD4+T cells to below 0.2 × 109/L should not be permitted to occur, due to the severe deficiency in the defense against infections that will occur.

Radiotherapy

Daily therapy with low radiation doses is more destructive to the lymphocytes than higher twice weekly doses /20/.

Systemic lupus erythematosus (SLE)

Not only in SLE, but also in mixed connective tissue disease (MCTD) and dermatomyositis, does lymphopenia occur. Anti-lymphocytic antibodies, which lead to lymphopenia via complement-mediated lysis, are detected in SLE. The antibody titer correlates with the extent of the lymphopenia /20/.

Tuberculosis

Lymphocytopenia with a particularly marked reduction of CD4+T-cells occurs. Normalization returns under successful therapy.

Influenza virus infection

Typically, lymphopenia develops following recovery from infection /57/.

Bacteremia

In intensive care patients, lymphopenia and the neutrophil/lymphocyte cell ratio (NLCR) and CRP can be predictors of bacteremia. The lymphocyte count is often below 1.0 × 109/L and the NLCR is above 10. Thus, in a study /59/ in patients with positive blood cultures:

  • CRP with a concentration higher than 50 mg/L demonstrated positive and negative predictive values of 54.3% and 59.6%, respectively
  • Lymphocyte counts below 1.0 × 109/L showed positive and negative predictive values of 63.6% and 59.6%, respectively
  • NLCR of greater than 10 had positive and negative predictive values of 67.6% and 73.4%, respectively.

Miscellaneous

Lymphopenia can occur in sarcoidosis, uremia, Cushing’s disease, with corticosteroid treatment, in inflammatory intestinal disease, following snake bites, burns, anesthesia, surgery including cardiopulmonary bypass surgery. The causes are, in some cases, lymphocyte recirculation disorders and, to a lesser extent, a reduction in the organism’s total lymphocyte count /20/.

Table 15.12-10 Diseases and conditions with monocytosis /61/

Clinical and laboratory findings

Infection /60/

Monocytosis is found in:

  • Chronic infections such as tonsillitis, dental infection, liver abscess, endocarditis, tuberculosis, candidiasis
  • Acute bacterial infections in the recovery phase (e.g., in the 2nd week of pneumococcal pneumonia).

Autoimmune disease

Mild monocytosis occurs with low prevalence in autoimmune vasculitis, rheumatoid arthritis, myositis, and arteritis temporalis.

Hematological systemic disease

  • Myelodysplastic syndrome, approximately one third of the patients have monocytosis
  • Chronic myeloid leukemia; a high monocyte fraction may be present; if it is considerable, then myelomonocytic leukemia or monocyte leukemia are present
  • Some of the diseases with chronic neutropenia are associated with monocytosis
  • Medication-induced neutropenia; transient monocyte elevations occur in the acute phase, but the elevations during the recovery phase (e.g., following a chemotherapy cycle) are characteristic
  • Hodgkin’s disease; one fourth of the cases is associated with monocytosis, but there is no correlation for purposes of prognosis
  • Histiocytosis, multiple myeloma
  • Non-hematological solid tumors are believed to be associated with monocytosis, independent of metastases, in up to 70% of the cases.

Various causes

Glucocorticoid therapy, splenectomy, poisoning, Hand-Schüller-Christian disease, Niemann-Pick disease, M. Gaucher.

Table 15.12-11 Differential diagnosis of Eosinophilia

Clinical and laboratory findings

Parasitosis /62/

Following a stay in the tropics, helminthiasis should always be taken into consideration if eosinophilia is present. In infection with intestinal worms, mild eosinophilia is usually found (below 1.5 × 109/L), in infection with tissue-invasive worms, severe eosinophilia occurs (above 5 × 109/L). The positive predictive value of eosinophilia with regard to the diagnostic investigation of helminthiasis is believed to lie in the range of 10%.

Tissue-invasive parasitosis includes infection with Strongyloides, Trichinella, Toxocara, Echinococcus, Cysticercus and Schistosoma. Suspicion of a parasitic infection exists if the patient originates from a region in which certain parasites are endemic, or if he returns from a trip to such a region. Parasitosis normally leads to moderate eosinophilia, with a cell count of (1–5) × 109/L. In infection caused by Toxocara species, which humans usually acquire from dogs and cats, eosinophilia of up to 50 × 109/L can occur if visceral Larva migrans is present /63/.

Tropical eosinophilia: this disease is a variant of human filarial infection, which results from a hypersensitivity reaction to substances that are released from the micro filariae Wucheria bancrofti and Brugia malayi. Endemic regions are the coastal regions of India, Pakistan, Sri Lanka, Burma, Thailand, Malaysia, the Philippines, Southern China, Korea, and Central Africa. The eosinophil count is greater than 3 × 109/L and in hyperacute cases it can reach (30–50) × 109/L. Total serum IgE is elevated, and filaria-specific IgG and IgE antibodies are detectable /64/.

Allergy

Allergic diseases which must be taken into consideration are bronchial asthma, allergic rhinitis, atopic dermatitis, urticaria, and angioneurotic edema. Allergic reactions to aspirin, sulfonamide, allopurinol, and L-tryptophan can also lead to eosinophilia. The eosinophil count is about (0.5–1) × 109/L.

Asthma /65/

Up to 40% of severe asthma start later in life, the eosinophilic form of asthma probably represents less than 5% of the total number of cases of adult-onset asthma. Patients with asthma who have eosinophilia have greater airway remodeling and more exacerbations, whereas those without eosinophilia have more airway obstruction. Eosinophils play a role in exacerbations and contribute to the pathophysiology of asthma. The eosinophils and the eosinophil cationic protein are elevated in the bronchial lavage of eosinophilic asthma /66/.

Eosinophilic- pneumonia (EP) /67/

EP includes a broad spectrum of lung diseases characterized by peripheral blood eosinophilia of over 1 × 109/L and/or alveolar eosinophilia over 25%. Blood eosinophilia may be lacking, as in the early phase of idiopathic acute EP or in patients taking oral corticosteroids. EP may present with varying severity, ranging from almost asymptomatic infiltrates to the acute respiratory symptom. Possible causes of EP are drugs and a variety of parasitic infections, however chronic EP remains idiopathic in many cases.

Hyper eosinophilic syndrome (HES) /68/

Hyper eosinophilic syndromes are a group of disorders characterized by persistent and marked hyper eosinophilia of over 1.5 × 109/L not due to an underlying disease to cause eosinophil expansion (allergic drug reaction, parasitic infection), and which is directly implicated in damage or dysfunction of at least one target organ. Pathogenic mechanisms leading to hyper eosinophilia in patients fulfilling classical HES diagnostic criteria involve:

  • Stem cell mutations leading to expression of PDGFRA-containing fusion genes with constitutive tyrosine kinase activity (mainly the FIP1L1-PDGFRA fusion gene)
  • Sustained overproduction of IL-5 by activated T cell subsets with unusual phenotypes, and/or clonal T cell receptor rearrangement patterns
  • Unknown molecular mechanisms leading to acquisition of abnormal phenotype by T cells such as CD3CD4+, CD3+CD4CD8 or CD3+CD4+CD7.

Eosinophilia-associated muscular disorders /69/

Various skeletal muscular pathologies are associated with blood and/or tissue eosinophilia. In addition to L-tryptophan related eosinophilia-myalgia syndrome and toxic oil syndrome, muscle involvement may occur in parasitic infections, Churg-Strauss vasculitis, hematological and non-hematological malignancies, and Shulman’s eosinophilic fasciitis. Idiopathic eosinophilic myopathies have also been described, encompassing three main subtypes: focal eosinophilic myositis, eosinophilic perimyositis and eosinophilic polymyositis. Skin involvement, in the form of an erythematosus rash, subcutaneous indurations and angioedema, is common. In a study /69/, 7 eosinophilic myopathies (without parasitosis) were published; the blood eosinophil count was (132–1464) × 109/L.

Adrenal insufficiency

Primary and secondary adrenal insufficiency are associated with eosinophilia. The cell count is usually (0.5–1) × 109/L. This condition is suspected in patients with hyper pigmentation, fatigue, hypotension, anorexia, nausea, and stomach disorder. Laboratory diagnostic evaluation includes cortisol and/or ACTH.

Malignant disease

Some 0.5% of malignant tumors are associated with eosinophilia which has been described in the following carcinomas: lung, breast, cervical, ovarian, liver, pancreatic, thyroid, Hodgkin’s, T cell lymphoma, multiple myeloma /70/.

Idiopathic hyper eosinophilia (IHES)

IHES is a largely heterogenous group of disorders defined as persistent marked hyper eosinophilia of unknown origin generally complicated by end-organ damage. With the concept of IHES, empirical diagnostic criteria have been defined, including /26/:

  • Eosinophilia of at least 1.5 × 109/L for longer than 6 months
  • No identifiable etiology of the hyper eosinophilia in spite of intensive diagnostic investigation
  • Presence of functional disorders or injury to organs due to the idiopathic hyper eosinophilia.

The organs that are most commonly affected are the skin, the heart, and the central nervous system. Observations indicate that distinct primitive hematological disorders involving either myeloid or lymphoid cells account for hyper eosinophilia in patients fulfilling the initial diagnostic criteria of IHES. Many patients can be classified in one of two diverse variants:

  • The myeloproliferative variant with clinical and biological features reminiscent of chronic myelogenous leukemia. Prognosis has been considered poor.
  • The lymphocytic variant. T-cell clones display eosinophilopoietic activity. This variant can be defined as a primitive lymphoid disorder characterized by nonmalignant expansion of a T-cell population able to produce eosinophilopoietic cytokines, especially IL-5.

DRUG RASH WITH EOSINOPHILIA AND SYSTEMIC SYMPTOMS (DRESS)

The Drug Rash with Eosinophilia and Systemic Symptoms (DRESS) syndrome is a rare but severe, potentially fatal idiosyncratic, adverse drug reaction with cutaneous and systemic manifestations /74/. Treatment with trimethoprim-sulfamethoxazole of a patient caused eosinophil count of 3028/μL, lymphocytic infiltration, alanine aminotransferase of 989 U/L, and aspartate aminotransferase of 162 U/L /74/.

Table 15.13-1 Artifacts and morphological changes of leukocytes in the blood smear

Clinical and laboratory diagnostics

Granulocytes – Generally

Morphological changes in the granulocytes are often of diagnostic value.

– Drumstick

A drumstick is characteristic of the female gender (Fig. 15.13-3 – Morphological granulocyte alterations).

– Hyper segmentation of segmented granulocytes

Polymorphonuclear neutrophils (PMNs) with more than five segments indicate hyper segmentation. Hyper segmented granulocytes in combination with giant bands is typical of a megaloblastic anemia. Hyper segmented granulocytes are often found in body cavity fluids (e.g., ascites) in which the granulocytes are separated from the circulation.

– Toxic granulation, Döhle bodies, cytoplasmic vacuolization

Toxic granulation, Döhle bodies and cytoplasmic vacuolization of granulocytes are nonspecific toxic manifestations of infection /11/.

Toxic granulation: appearance of increased dark granules in the cytoplasm of neutrophils. They occur following infection. Toxic granulation is found in two thirds of sepsis, but it’s diagnostic specificity is low.

Döhle bodies: these pale blue inclusion bodies, located in the peripheral cytoplasm, are aggregates of the endoplasmic reticulum. They are seen in infection and in association with toxic granulation. Approximately one third of sepsis patients have Döhle bodies. They also occur in pregnancy and postpartum.

Toxic vacuolization: toxic vacuolization of the cytoplasm of neutrophils occurs in 90% of patients with bacterial sepsis. The vacuoles are large, opaque, and numerous, the entire cell is often deformed. Artificial vacuolization, which occurs when old EDTA blood samples are smeared, must be kept in mind. These vacuoles are usually smaller, more uniform in shape, do not deform the cell structure.

– Pelger-Huet anomaly /12/

Pelger-Huet anomaly is a hereditary peculiarity of the neutrophil granulocytes whose nuclear segmentation does not exceed two segments. Approximately 30% of the cells have a band-shaped or peanut-shaped nucleus (bilobed or dumbbell-shaped nucleus). The prevalence is approximately 1: 6000. The function of the cells is normal, and they have a normal life span. Pelger anomaly is not gender-related. The distinction from an infection-dependent left shift is recognizable by means of the homologous cell morphology in Pelger, in contrast to the polymorphous appearance of bands.

The acquired form of the Pelger-Huet anomaly (Pseudo-Pelger) is found in polycythemia vera, myeloid leukemia, osteomyelofibrosis, Hodgkin’s disease, and multiple myeloma, and can occur for the first time following bone marrow transplantation. It is also found in the loss of the short arm of chromosome 17 (17p-syndrome). Under therapy with taxane /8/ and ibuprofen /13/ a transient occurrence of Pseudo-Pelger occurs. The Pseudo-Pelger is distinguished from congenital Pelger-Huet anomaly based on the fact that in Pseudo-Pelger:

  • Only a certain percentage of granulocytes manifests the characteristic changes
  • Neutropenia, and a decrease in neutrophil granules, may be present
  • There are no Pelger-type monocytes as there are in the hereditary form of the disease.

– Chediak-Higashi syndrome

In this abnormality, giant lysosomes in the form of eosinophilic inclusions are present in neutrophil granulocytes and other blood cells. Pseudo-Chediak-Higashi syndrome occurs in acute myeloid leukemia and myelodysplastic syndrome /8/.

– Howell-Jolly-body-like inclusions

Howell-Jolly-body-like inclusions in the cytoplasm of neutrophils are nuclear fragments that are identical to those found in erythrocytes. They occur in HIV infection, and must be distinguished from other unusual inclusions like those seen in infection or those which occur in Chediak-Higashi syndrome /14/.

– Leukocyte agglutination

Easily recognizable granulocyte agglutinates, which cause pseudo leukopenia, can be caused /8/:

  • EDTA induced and affects not only neutrophils, but also eosinophils and basophils, as well as lymphocytes
  • By cold agglutinins. The agglutination can be eliminated by incubation of the samples at 37 °C.

Lymphocytes – Generally

For the purpose of lymphocyte differentiation, they are primarily classified as “typical” or “atypical”. Typical means that morphologically, the lymphocytes are normal (cell diameter approximately 10 μm, nuclear diameter approximately 7 μm, heterogeneous lumpy nuclear chromatin, light-basophilic cytoplasm with smooth margins). Large granular lymphocytes (LGL) contain azurophilic granulation, and are considered to be typical if their proportion does not exceed 10%. All lymphocytes, apart from typical lymphocytes, as well as LGL, are designated as atypical if their proportion exceed 10%. The classification of atypical lymphocytes is made according to the categories of: “atypical, presumably reactive” or “atypical, presumably neoplastic”. Lymphocytes that cannot be classified are designated as “diverse”. For these, a commenting description such as, for (e.g., hair cells is recommended).

– Diagnostic investigation of leukemia and lymphoma

The role of the blood smear in the diagnosis of leukemia and lymphoma lies in the making of a tentative diagnosis as a basis for immunophenotyping. Examples:

  • In acute leukemia, Auer rods (fused granules) are indicative
  • Bilobular leukemia cells with fine granules and with faggots or bundles of fine Auer rods are indicative of acute promyelocytic leukemia. The same holds true for abnormal promyelocytes that are studded with rough azurophilic granules (hypergranular form)
  • Hair-like cytoplasmic lymphocyte filaments. In the presence of low normal values of leukocytes, thrombocytes, granulocytes and hemoglobin, they are indicative of hairy cell leukemia.
  • Large basophilic vacuolized lymphoma cells are indicative of Burkitt lymphoma.
  • Philadelphia (Ph) chromosome: Ph-chromosome is associated with chronic myeloid leukemia (CML) in about 90% of cases and in acute lymphoblastic leukemia in 20–25% of cases /24/. Ph-chromosome is not seen microscopically in the stained and colored specimen of the bone marrow. Ph-chromosome is a rearrangement between ABL1 of chromosome 9 and BCR of chromosome 22. ABL1 (v-abl; Abelson murine leukemia viral oncogene homolog 1), originally located on chromosome 9 is translocated within the BCR (breakpoint cluster region) gene on chromosome 22 under formation of the fusion gene BCR-ABL1. In the normal location on chromosome 9 the ABL1 proto-oncogene encodes a cytoplasmic and nuclear tyrosine kinase that is implicated in cell differentiation, cell division and cell adhesion. The t (9, 22) translocation results in the fusion of the ABL1 and BCR gene on chromosome 22. The fusion gene encodes an unregulated cytoplasmic tyrosine kinase that allows cells to proliferate without being regulated by cytokines. Patients with CML, treated with tyrosine kinase inhibitors, have a life expectancy similar to that of the general population /25/.

Table 15.13-2 Artifacts and morphological red blood cell changes in the blood smear

Clinical and laboratory diagnostics

Anisochromia

There are differences in red blood cell staining intensity. These are dependent upon the red blood cell hemoglobin concentration. Hypochromic and normochromic erythrocytes are distinguished from one another. Hypochromic anisocytosis is typical of iron deficiency. Hyperchromic red blood cells do not exist, since erythrocyte hemoglobin concentrations cannot be greater than 360 g/L. Cells more intensively stained as normal appear to be hyperchromic. However, these cells have an atypical form like spherocytes or oval macrocytes in disorders of DNA synthesis /1/.

Polychromasia

Polychromatic red blood cells are stained pale blue. These are cells in the transformation of reticulocytes to erythrocytes. An increase of polychromatic erythrocytes indicates an enhanced consumption of erythrocytes (acute hemorrhage) /1/.

Anisocytosis

Variability in the size of red blood cells. With the determination of red cell distribution width (RDW), hematology analyzers provide more precise information than the blood smear. Anisocytosis is found in iron deficiency anemia, DNA synthesis disorder (vitamin B12 or folic acid deficiency), and in anemia that is associated with reticulocytosis (e.g., acute hemorrhage, acute hemolysis).

Microcytosis

Microcytes are cells with a reduced volume. Microcytosis is associated with a decrease in erythrocyte hemoglobin content (MCH). Microcytes are pale in color and may have reduced MCHC. All microcytic red cells are hypochromic and all hypochromic cells are microcytic /1/. Hypochromic microcytic anemias are caused by iron deficiency and thalassemia syndromes. The blood smear is generally less important in the differential diagnosis of the microcytic anemias. However, it is important to note that the presence of Pappenheim bodies and red cell dimorphism in the sideroblastic anemias and of basophilic stippling in cases of lead poisoning and in some types of thalassemia is diagnostically significant /2/.

Macrocytosis

Macrocytes are erythrocytes with a diameter of greater than 8 μm and normal thickness, while megalocytes have a diameter of over 9 μm. If, along with an increased diameter, the MCV is also elevated, it is necessary to rule out reticulocytosis as well as a sample from a newborn. Pseudo macrocytosis on the blood film is caused by the presence of thin red cells (leptocytes). If macrocytes are seen on the blood film and confirmed by an elevation in MCV, the presence of an increased reticulocyte count should be excluded. If in an adult patient with macrocytosis and without reticulocytosis the macrocytes are round, then the macrocytosis is likely due to chronic liver disease; if they are oval, they have megaloblastic maturation (vitamin B12 deficiency, folate deficiency) /1/. In such cases, anisocytosis, poikilocytosis and hypogranular or hypo lobulated neutrophils are often present. In the elderly, myelodysplastic syndrome is a frequent cause of macrocytic anemia.

Nucleated Red Cells (NRC)

The presence of NRC on the peripheral blood smear suggests an abnormality in the bone marrow or in the red cell production. NRC are especially common in hemolytic anemia, massive blood loss, severe oxygen deficiency, acute and chronic leukemias, malignant tumor, osteomyelofibrosis.

Poikilocytosis

Appearance of deformed red blood cells with many possible variations of shape.

Anulocytes

Hypochromic microcytic red blood cells, in which the hemoglobin is concentrated in a thin rim around a pale center /1/. Typical for chronic severe iron deficiency.

Elliptocytes

In healthy individuals, some 1% of the erythrocytes have an elliptical shape. The shape varies from a slightly discernible ellipse to cells with a rod-like shape. Elliptocytes are present in vitamin B12 deficiency or folate deficiency, they can result from chemotherapy agents that disturb DNA and RNA synthesis or occur in patients with dyserythropoietic anemia /1/.

Hereditary elliptocytosis: this is a rare autosomal dominant disease, in which up to 90% of the erythrocytes have an elliptical form. Hemolytic anemia is present. In Southeast Asian ovalocytosis, the cells are about twice as large as normal, and in addition, stomatocytes with two Y-shaped or V-shaped stomata are present /2/.

Spherocytes

The spherocyte has a smaller diameter than the erythrocyte, but the volume is identical. The cause is the loss of cell membrane. The spherocyte appears dense or darker due to the greater thickness of the cells in comparison to normocytes /1/. Young cells are not spherocytic in appearance. Spherocytosis is indicative of hemolytic anemia. The presence of spherocytes is not diagnostically specific, since this may result from hereditary spherocytosis, autoimmune hemolytic anemia, or alloimmune hemolytic anemia. In hereditary spherocytosis, a cell membrane synthesis disorder is present. Microspherocytes (cells that are both hyperchromic and significantly reduced in size and therefore in diameter) may be present in low numbers in patients with hemolytic anemia, but are also characteristic of burns and of microangiopathic hemolytic anemia /2/.

Burr cells

Burr cells frequently have bizarre shapes reminiscent of animal shapes. The finding of Burr cells lacks clinical specificity /1/.

Heinz bodies

Heinz bodies are precipitated hemoglobin and may be visible with Giemsa or Wright’s stain but not with ordinary Romanowsky stained films. Heinz bodies may be seen in /1/:

  • Unstable hemoglobin disorders
  • After the use oxidative drugs such as dapsone
  • In red cell metabolic deficiencies (methemoglobin reductase deficiency).

Keratocytes

In the diagnosis of acute hemolysis the detection of keratocytes (have a horn-like shape) or bite cells is important /1/. The erythrocytes have changed their shape due to oxidative damage. Due to a drug, hemoglobin of the erythrocytes precipitates as a dense mass, causes Heinz bodies and leads to changes in the shape of the cells. The erythrocytes with irregular outline either have horn-like protuberances or they present as bit cells or the shape is still more or less normal, but they manifest large hemoglobin-free areas /2/. Oxidant-induced hemolysis is often seen in glucose-6-phosphate dehydrogenase (G6PD) deficiency, or disorders of the pentose phosphate shunt. Worldwide, millions of people are affected by G6PD deficiency. See also Section 15.8 – Erythrocyte enzymes).

Echinocytes

Thorn apple-shaped erythrocytes. Echinocytes are artifacts of blood collection and blood film preparation. Exposure to an alkaline pH, usually the result of glass effect, results in the formation of echinocytes. The slow drying of the blood smear on the glass slide is the most common cause of echinocyte formation /1/.

Stomatocytes

Stomatocytes are usually an artifact on blood films, since hereditary stomatocytosis is very rare. Exposure of erythrocytes to an acid pH results in the formation of stomatocytes. This is also the case in the presence of drugs such as phenothiazines or of cationic and amphiphilic substances /1/. Stomatocytes are also present in liver disease, particularly those in which the pathogenesis is related to alcohol.

Acanthocytes

Acanthocytes are erythrocytes with a lot of spines. They develop from normal red cells in the circulation and the nucleated precursor cells are normal. Acanthocytes occur in hereditary acanthocytosis, which is characterized by abetalipoproteinemia. Acanthocytes also develop in severe liver failure with hypo cholesterolemia /1/. Acanthocytosis can also develop with parenteral nutrition with fish oil; it is reversed following dis continuation /16/. Drugs such as benzodiazepines and 5-fluorouracil have also been described as causes acanthocytosis.

Dacrocytes

The erythrocytes have the shape of a teardrop. These cells are observed in chronic idiopathic myelofibrosis, and in myelophthisic blood pictures associated with bone marrow replacement by malignant tumor cells /1/.

Sickle cells

These cells represent the ultimate spiculated red cells /1/. They are observed in the blood of homozygous hemoglobin S disease. Sickle cells are rarely seen in heterozygous trait carriers /17/.

Schizocytes

Schizocytes are erythrocyte fragments or remnants after fragments have been removed. The features are small size, dense appearance, and sharp pointed edges /1/. Many schizocytes are triangular shaped fragments. Schizocytes are formed by mechanical destruction (e.g., following artificial heart valve replacement, in hemolytic uremic syndrome, or in disseminated intravascular coagulation).

Target cells

Target cells have the form of a shooting target, because hemoglobin is concentrated in a rim and in the center of the cell. They occur in hypochromic anemia, hemolytic anemia, thalassemia, HbC disease, and following splenectomy.

Erythrocyte agglutinates

Irreversible erythrocyte agglutination due to heterophile IgM antibodies. They are formed in infectious mononucleosis (EBV), infection with Mycoplasma pneumoniae, and in the presence cold agglutinins of unknown etiology.

Rouleaux formation

Rouleaux formation is also known as the rouleaux phenomenon. In thick parts of the smear, the term rouleaux phenomenon refers to two and more cells that form a chain; in thin areas none are seen. The rouleaux phenomenon is reversible, it resolves when the blood is diluted with saline. For this reason, rouleaux formation does not affect the MCV measurement with hematology analyzers. Hyper fibrinogenemia and hypergammaglobulinemia are causes of rouleaux formation /1/.

Basophilic stippling

Basophilic stippling is seen in stained erythrocytes as fine dark inclusions and appear with Wright and Pappenheim staining. The inclusions are precipitated ribosomal RNA that developed during drying and staining of the smear /1/. Basophilic stippling is a sign of impaired regeneration of erythropoiesis; it may be seen in megaloblastic anemia, alcoholism, lead poisoning (impaired heme synthesis), arsenic poisoning, thalassemia (impaired globin synthesis), porphyria, and pyrimidine 5’-nucleotidase deficiency.

Howell-Jolly bodies

Small, round, violet-colored chromatin bodies in the erythrocytes. These are nuclear DNA residues which are normally removed by the spleen. They occur in hyper regenerative erythropoiesis with compromised reticulocyte nuclear expulsion. Howell-Jolly bodies are found in splenomegaly and chronic graft versus host disease.

Cabot’s rings

Reddish rings in the erythrocytes, in some cases as single rings or complex loops, such as a figure eight. Cabot rings are observed in DNA synthesis disorders (megaloblastic blood films) /1/.

If, during erythropoiesis, iron that is taken up by the erythroid precursor cells is not immediately utilized for the synthesis of hemoglobin but rather, is stored as ferritin and hemosiderin, this can be shown using iron staining. Erythocytes containing siderosomes are called siderocytes, and erythroblasts that contain siderosomes are known as sideroblasts. Erythroblast mitochondria are organized as rings around the nucleus. Defects in erythroblast protoporphyrin synthesis lead to the accumulation of iron in the mitochrondria. Ring sideroblasts are seen in the hereditary form of sideroblastic anemia, in its acquired variant, namely, myelodysplastic syndrome (see Section 15.16 – Myelodysplastic syndrome), and in idiopathic forms of sideroblastic anemia /1/.

Table 15.13-3 Artifacts and morphological platelet changes in the blood smear in thrombocytopenia and thrombocytosis

Clinical and laboratory diagnostics

Platelet aggregation

Platelet aggregation is often EDTA induced, and causes pseudo thrombocytopenia. Depending upon the literature, a frequency of 0.1–1.9% is estimated. Most patients have no characteristic underlying disease. However, EDTA induced platelet aggregation is noted to occur more frequently in autoimmune rheumatoid arthritis, Sjögren’s syndrome, Guillain-Barre syndrome, neoplasms, atherosclerosis and in critically ill patients /8/. Treatment with the glycoprotein IIb/IIIa antibody abciximab can lead to platelet aggregation and pseudo thrombocytopenia. Some 5% of treated patients develop this symptomatology, while one third has EDTA induced thrombocytopenia /18/.

Platelet satellitosis

Platelets have coated neutrophil granulocytes, basophils, eosinophils, lymphocytes, and lymphoma cells. Satellitosis is mediated by IgG antibodies that are directed against the glycoprotein IIb/IIIa thrombocyte receptor and the FcRIII receptor on the leukocyte membrane /19/. Mild thrombocytopenia and leukopenia can be measured with the hematology analyzer.

Giant platelets

Giant platelets are seen in myeloproliferative disease, hypersplenism, and hereditary conditions like Bernard-Soulier syndrome and May-Hegglin anomaly /8/. When present in largely numbers giant platelets can falsely elevate the automated white blood cell and red blood cell counts while falsely lowering the automated platelet count /4/.

Gray platelet syndrome

The platelets look gray or pale blue, due to a deficiency in α-granules. The phenomenom is seen in hereditary syndromes such as Bernard-Soulier syndrome or May-Hegglin anomaly, but also may be present in myeloproliferative disorders, hypersplenism, following cardiopulmonary bypass surgery, and in hairy cell leukemia /8/.

Other particles

Erroneously high platelet counts are measured with hematology analyzers in the presence of red blood cell fragments of leukemia cells, and in mycosis-related sepsis /2/.

Table 15.13-4 Morphological blood smear findings in infectious disease

Clinical and laboratory diagnostics

Clostridium perfringens

The presence of bacteria in routinely prepared blood smears is an uncommon phenomenon in the hematology laboratory. Sepsis caused by bacteria and fungi can lead to intravascular hemolysis, but is a rare phenomenon. Despite its rarity, the most well-known cause of bacterial hemolysis is associated with C. perfringens /11/. Sepsis caused by this pathogen occurs preferentially in patients with gastrointestinal tumors. The hemolysis is dependent upon the formation of bacterial lecithinase with direct destruction of erythrocyte membranes. The C. perfringens sepsis in tumor patients is almost always fatal /20/. In the smear there is prominent spherocytosis and red cell fragmentation /11/.

Malaria

See Section 44.5.2.3 – Malaria. In malaria infection, the size of the affected erythrocytes is altered as follows: not enlarged in infection with P. falciparum and P. malariae, significantly enlarged (1.5 to 2-fold) in infection with P. vivax, and enlarged by 1.25 to 1.5-fold in P. ovale infection.

N. meningitidis, S. aureus

Generally, bacteria are seldom found in blood smears. If they are found, this speaks for severe sepsis with poor prognosis. The bacteria are located in neutrophil granulocytes, tend to clusters and usually lie within a distinct vacuole. In the routine diagnostic investigation, Staphylococcus aureus and Neisseria meningitidis are most commonly found /11/. If the bacteria are found only outside of the leukocytes, contamination of the dye solution must be taken into consideration.

Ehrlichia /11/

Ehrlichia are obligatory intracellular growing coccoid, pleomorphic, gram negative bacteria belonging to the Rickettsiaceae family. The vectors are ticks. Relevant are human monocytic Ehrlichosis (HME) caused by E. chafeenis, and granulocytic Ehrlichosis (HGE) caused by an unknown species. Ehrlichia are phagocytized by granulocytes and monocytes and multiply to form 1–3-μm, membrane bound clusters of pale-blue staining coccobacillary organisms. These inclusions are known as morulae and their detection establishes the diagnosis of Ehrlichiosis /21/. In 25–80% of patients with HGE but only 1–2% of those with HME detectable morulae in leukocytes are present. The morulae may be easily overlooked. Ehrlichosis is a mild and self-limiting disease. Older persons and patients with severe underlying disease are affected by a symptomatic course, which can be mild, severe, or fatal. This also concerns immune-suppressed patients (HIV, post-organ transplantation, high-dose corticosteroid therapy) /22/.

Babesia /11/

Babesia are protozoa, the vectors are ticks. There are two forms of babesia a mild American form, the pathogen is B. microti, and a severe European form that is caused by B. divergens. Following a tick bite, Babesia merozoites enter the erythrocytes, mature to trophozoites, multiply asexually, and can cause lysis of the erythrocytes. Trophozoites that are found in the erythrocytes are ring-shaped; they can have very small rings, of diameter as small as 2 μm. One cell may contain 1 to 12 rings. In most patients, 1–20% of the erythrocytes are affected; in splenectomized patients, this figure can be as high as 85%. The duration of parasitemia ranges from 3–12 weeks. The main differential diagnosis of babesiosis is malaria. Clinical symptoms present 1–4 weeks following a tick bite, and can lead to a broad spectrum of flu-like symptoms as well as to moderately severe malaria-like symptoms. Fever, weakness, sweating attacks, myalgia, arthralgia and nausea in early summer (tick season) should also bring to mind the possibility of Babeosis /22/.

Spirochetes /11/

During acute febrile episodes of recurrent fever (not in afebrile periods) spirochetes are detected in blood smears in 70% of the cases. Buffy coat investigations with acridine-orange staining has been advocated for detection. The organisms are extracellular; they have a length of 8–40 μm, a width of 0.2–0.6 μm, and 3–10 loose, irregular coils. The recurrent fever can be triggered either by Borrelia recurrentis (vector is the louse) or by any one of 16 Borrelia species (vector is the tick).

Trypanosoma /11/

There are two geographic variants: African trypanosomiasis (sleeping sickness), caused by T. brucei gambiense or T. brucei rhodesiense, and South American trypanosomiasis (Chagas disease), caused by T. cruzi. The vector for the African variant is the tsetse fly, while a bed-bug is the vector for the South American disease. Both disorders begin with a localized inflammatory reaction at the site of the infection bite (chancre). In acute phase of Chagas disease trypomastigotes are detectable in the blood smear. This is not the case in chronic Chagas disease. The circulating parasite in tryposomiasis is the trypomastigote, an extracellular, non invading form. The organisms measure 14–33 μm in length and 1.5–3.5 μm in width.

Filariae

Five species of filariae produce a circulating microfilarial form in humans: Wucheria bancrofti, Brugia malayi, Loa Loa, Mansonella perstans, Mansonella ozzardi. Microfilaria have a length of 230–300 μm, a thickness of some 7 μm, and a ribbon-like structure. They contain a chain of nuclei extending most of the length of the body. Because their demonstration in a normal blood smear is insensitive, concentration methods are recommended /23/.

Table 15.14-1 Methods for investigation of bone marrow in primary diagnosis or suspected diagnosis of leukemia and lymphoma /8/

Method

AL

MPN/
CML

MDS

Lym-
phoma

Re-
lapse

Cyto-
morphology

+

+

+

+

+

Cyto-
chemistry

+

+

+

Histology

(+)

+

(+)

+

(+)

Immuno-
phenotyping

+

(+),*

(+)

+

#

Cytogenetics

+

+

+

(+)

+

FISH

§

§

+

§

§

24 color FISH

§

§

§

§

§

PCR

+

+

(+)

#

#

+, obligatory; (+), optional, for special issues; #, only within framework of studies with clearly-defined issues; §, if necessary to supplement other methods (e.g., cytogenetics). AL, acute leukemia; CML, chronic myeloid leukemia; MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasia; * blast crisis

Table 15.14-2 Percentage of bone marrow cells in healthy individuals according to Ref. /5/

Bone marrow cell

95% range

Blast cells

0–3.0

Promyelocytes

3.2–12.4

Myelocytes

3.7–10.0

Metamyelocytes

2.3–5.9

Bands and segments

  • Men

21.9–42.3

  • Women

28.8–45.9

  • Eosinophils

0.7–6.3

  • Basophils

0–0.4

Erythroblasts

  • Men

16.2–40.1

  • Women

13.0–32.0

  • Lymphocytes

6.0–20.0

  • Plasma cells

0–1.2

  • Monocytes

0–2.6

  • Macrophages

0–1.3

Myelopoiesis/erythropoiesis ratio

  • Men

1.1–4.1

  • Women

1.6–5.2

Table 15.15-1 Methods for the diagnostic investigation of acute leukemia

Methods

AML

ALL

Cytomorphology
(blood, bone marrow)

+

+

Cytochemistry

+

+

Histology

0/+

0/+

Immunophenotyping

+

+

Cytogenetics

+

+

FISH

bB

bB

PCR

bB (+): (+)

bB

+, should be part of the routine; 0, must not be part of the routine; bB, as necessary in accordance with other results; FISH, fluorescence in situ hybridization; PCR, polymerase chain reaction

Table 15.15-2 Previous FAB classification of AML (can be performed as initial diagnostic step before WHO classification)

AML-M0

Mostly undifferentiated acute myeloid leukemia, immunophenotype necessary for confirmation

AML-M1

Granulocytic leukemia without tendency for maturation

AML-M2

Granulocytic leukemia with maturation of at least 10% of the leukemia cells to promyelocytes, myelocytes or further mature cells

AML-M3

Hypergranular promyelocytic leukemia

AML-M3v

Similar to M3, with bilobular nuclei and hypogranulated cytoplasm, corresponding to microgranular promyelocyte leukemia

AML-M4

Leukemia with evidence for a granulocytic and a monocytic component

AML-M4Eo

Similar to M4, but with pathological eosinophil granulocytes

AML-M5a

Acute monoblastic leukemia

AML-M5b

Acute monocytic leukemia

AML-M6

At least 50% erythroblasts, among the other cells ≥ 30% blasts

AML-M7

Megakaryoblastic leukemia, immunophenotype necessary for confirmation

Table 15.15-3 WHO classification of AML (2008)

AML with specific genetic aberrations

  • AML with t(8;21)(q22;q22); RUNX1-RUNKX1T1
  • AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22)

CBFB-MYH!!

  • Acute promyelocytic leukemia with t(15;17)(q22;q12)

PML-PARA

  • AML with t(9;11)(p22;q23); MLLT3-MLL
  • AML with t(6;9)(p23;q34); DEK-NUP214
  • AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2);RPN1-EVII
  • Megakaryoblastc AML with t(1;22)(p13;q13)

RMB15-MKLI

  • AML with mutated NPMI
  • AML with mutated CEBPA

AML with myelodysplasia-associated changes

  • AML with prior MDS or MDS/MPN
  • AML with MDS-specific cytogenetic aberrations
  • AML with multilineage dysplasia

Therapy-induced myeloid neoplasia

  • AML following alkylating agents
  • AML following epipodophyllotoxin
  • Post-radiation AML

AML with no other classification criteria

  • Minimally differentiated AML
  • AML without maturation
  • AML with maturation
  • Acute myelomonocytic leukemia
  • Acute monoblastic and monocytic leukemia

Acute erythrocytic leukemia

  • Acute megakaryoblastic leukemia
  • Acute basophilic leukemia
  • Acute panmyelosis with myelofibrosis

Myelosarcoma/chloroma

Down syndrome-associated myeloid proliferation

Blastic plasmacytoid dendritic cell neoplasm

Table 15.15-4 Differentiation of blasts (type I and II) from abnormal promyelocytes (in AML M3) and from normal promyelocytes

Criterion

Blasts
in AML
type I/II

Abnormal
promyelocytes
in AML M3

Normal
promyelocytes

Nuclear
position

Central

Central

Excentric

Nuclear
chromatin

Fine

Mean

Roughly

Nucleoli

0–2

0–2

0–1

Golgi zone

None or
suggested

Rare

Emphasized

Cytoplasm
basophilia

+++

+
up to reddish

+

Azurophilic
granules

0/15–20

Very much,
emphasized

Usually > 20

Auer rods

Possible

In bundles
(faggot cells)

No

Table 15.15-5 Laboratory results in acute myeloid leukemia /14/

Leukocytes

In 30–40% of patients, the white blood cell (WBC) count > 100 × 109/L is present diagnosed with acute leukemia and presence of blasts

Hämoglobin

Normocytic to hypochromic anemia

Thrombocytes

(50–150) × 109 platelets

Fever

> 38 °C

Potassium

2–3 mmol/L; leukemic leukocytes have augmented sodium-potassium pump activity resulting in movement of potassium from plasma into leukocytes, decreasing the plasma concentration of potassium

Sodium

135–145 mmol/L

Creatinine/eGFR

eGFR < 70 [mL × min–1 × (1.73 m2)–1]. In leukemia of monocytic differentiation lysozymuria, is a rare paraneoplastic complication. Lysozyme is released in the blood circulation, filtered by the glomerula and reabsorbed by renal tubules leading to toxic tubular injury and loss of potassium. The use of Cisplatin, aminoglycosides or loop diuretics is another cause of renal potassium loss.

Urea

May be elevated

Uric acid

Hyperuricemia; leukemic infiltrates or uric acid nephropathy can reflect kidney injury

Gesamteiweiß

Hypoproteinemia

Albumin

Hypoalbuminemia

Globulin

Normal

LDH

Greatly increased

ALT

Increased

AST

Increased

ALP

Normal

Bilirubin

Normal

pH

Slightly increased

pO2

PO2 is a major criterion for evaluation of respiratory distress. Because oxygen consumption is proportional to the increase in white blood cells, arterial oxygen tension is decreased.

pCO2

Normal

Table 15.16-1 WHO classification of myelodysplastic syndrome /1/

Entity

Dysplasia
(usually only)

% Blood
blasts

% BM
blasts

% BM
Ring-Sb

Cyto-
genetics

5q-syndrome

dysE

< 5%

< 5%

< 15%

Only 5q

RCUD
(RN, RA, RT)

dysG, dysE

or dysM

< 1%

< 5%

< 15%

V

RARS

dysE

None

< 5%

≥ 15%

V

RCMD

2–3 lineages

Rare

< 5%

< 15%

V

RAEB-1

1–3 lineages

< 5%

5–9%

< 15%

V

RAEB-2

1–3 lineages

5–19%

10–19%

< 15%

V

MDS-U

1 lineage

None

< 5%

< 15%

V

BM, bone marrow; V, various; RCUD, refractory cytopenia with single-lineage dysplasia; RN, refractory neutropenia; RA, refractory anemia; RT, refractory thrombocytopenia; RCMD, refractory cytopenia with multi-lineage dysplasia; RAEB, refractory anemia with increased blasts; Ring-Sb, ring sideroblasts

Table 15.16-2 International Prognostic Scoring System: grouping according to points /4/

 

Score points

0

0.5

1

1.5

2

% Blasts in
bone marrow

< 5

5–10

11–20

21–30

Karyotype

F

I

UF

Cytopenia

0/1

2/3

Favorable karyotype: normal, -Y, del(5q), del(20q); unfavorable karyotype: complex, aberrant (≥ 3 aberrations), aberrations on chromosome 7; all other chromosome findings are intermediat; F, favorable; UF, unfavorable; I, intermediate

Table 15.16-3 International Prognostic Scoring System: prognostic classification /4/

Points

0

0,5 to
1,0

1,5 to
2,0

≥ 2,5

Groups at risk

Low

Int-1

Int-2

High

Table 15.16-4 Characteristic cytogenetic alterations in MDS, in comparison with AML

Chromosomal
abnormality

Frequency
in MDS (%)

Frequency
in AML (%)

only del(5q)

5–20%

2%

-7, only del(7q-)
allein

3–10%

2%

+8

5–10%

5%

-17, del(17p),
iso (17q)

5%

3%

del(20q))

3–5%

0,5%

-X, -Y

2%

0,5%

t(3;3)(q21;q26),
inv(3)(q21q26)

< 1%

1–2%

Complex
karyotype

10–15%

10–15%

Table 15.17-1 Clinical characteristics and orienting markers in the differential diagnosis of MPN

Findings

CML

PV

PMF

ET

Spleno-
megaly

(+)

No

+

No

Blood count
changes in
granulopoi-
esis and/or
erythropoi-
esis

Leukocytosis
with left shift,
eosinophilia,
basophilia

Leukocytosis

Hematocrit

Red cell
teardrops,

left shift of
granulopoi-
esis, normo-
blasts

No

Thrombo-
cytes

()

() Giant forms

up to ()

> 450 × 109/L, giant forms

Bone marrow
cytology

Very
hypercellular,
basophilia,
megakaryo-
cytes and
eosinophils

Markedly
hypercellular
erythropoiesis

Usually
punctio
sicca

Megakaryo-cytes markedly increased

Bone marrow
histology

Granulopoi-
esis

Megakaryo-
cytes

Cell content

Marked
fibrosis

Megakaryo-cytes increased, lying in nests

Philadelphia-
chromosom
t(9;22);

BCR-ABL1

Yes

No

No

No

Other chromos.
changes

In
acceleration
and
blast crisis

del (20q), +8. +9,
+1q

–7, +8,
+9, +1q

Very rare

Vit. B12 > 900
(ng/L)

Normal

Normal

CML, chronic myeloid leukemia; PV, polycythemia vera; PMF, primary myelofibrosis; ET, essential thrombocythemia

Table 15.17-2 Methods at the time of diagnosis in chronic myeloproliferative neoplasia

Methods

PV

ET

PMF

CML

Cytomorphology
(blood and BM)

+

+

+

+

Histology

+

+

+

+

Cytogenetics

(+)

+

+

FISH

+ (BCR-ABL1)

PCR

+

+

+

+ (BCR-ABL1)

+, should be part of the routine; –, must not be part of the routine; FISH, fluorescent in situ hybridization; PCR, polymerase chain reaction; BM, bone marrow

Table 15.17-3 WHO classification of CML based upon the morphological findings /1/

Chronic phase CML

  • Classic cytomorphological and/or histological picture BCR-ABL1 positive or demonstration of t(9;22). Blasts in blood < 2%, in bone marrow < 5–10%.

CML in accelerated phase (one or more criteria met)

  • Blasts 10–19% in blood and/or bone marrow
  • Basophils in blood > 20%
  • Platelets not therapy-related < 100 × 109/L or > 1,000 × 109/L
  • Leukocytes > 10 × 109/L or spleen increasing and not susceptible to treatment
  • Clonal evolution in cytogenetics.

CML in blast crisis (one or more criteria met)

  • ≥ 20% blasts in blood and/or bone marrow
  • Extramedullary blast cell proliferation
  • Large foci of blast nests seen on bone marrow biopsy.

Table 15.17-4 Diagnostic criteria of polycythemia vera (PV)

Main criteria

1.

Hemoglobin values > 185 g/L in men, > 165 g/L in women, or any further evidence of erythrocytosis*

2.

Demonstration of JAK2 V617F mutation or other equivalent functional mutation (e.g., JAK2 exon12)

Secondary criteria

1.

Age nonspecific, hypercellular bone marrow with three lineage growth (panmyelosis) with marked erythrocytic, granulocytic, and megakaryocytic proliferation

2.

Serum erythropoietin values below the reference interval

3.

Endogenous erythroid colony formation in vitro

* Hemoglobin (Hb) or hematocrit values above the 99th percentile of the method-specific upper reference interval value for age, gender or hemoglobin values > 170 g/L in men or > 150 g/L in women (as long as this is associated with a documented and continuous rise of at least 20 g/L from the individual basal value that is not due to therapy of iron deficiency); or erythrocytosis of greater than 25% of the hypothetical normal value

Table 15.17-5 Diagnosis criteria for primary myelofibrosis (PMF)

Main criteria

1.

Demonstration of megakaryocytic proliferation and atypiaa, usually associated with reticulin fibrosis and/or collagen fibrosis

or

if relevant reticulin fibrosis cannot be demonstrated the megakaryocyte change must be accompanied by increased bone marrow hypercellularity, characterized by granulocytic proliferation and, often, decreased erythropoiesis (e.g., prefibrotic disease phase).

2.

The WHO criteria for polycythemia vera b, BCR-ABL1+ chronic myeloid leukemia c, myelodysplastic syndromed, or other myeloid neoplasia, are not fulfilled.

3.

Presence of JAK2 V617F or other clonal markers (e.g., MPL W515K/L)

or

if a clonal marker is not present, the bone marrow fibrosis or other changes may not assumed to be indicative of an infection, autoimmune disease, other chronic disease such as hairy cell leukemia, or other lymphoid neoplasia, metastatic malignancy or toxic (chronic) myelopathye.

Secondary criteria

1.

Leukoerythroblastosisf

2.

Increase in serum lactate dehydrogenasef

3.

Anemiaf

4.

Splenomegalyf

a Small to large megakaryocytes with an abnormal nucleus to cytoplasm ratio and hyperchromatic, round or irregularly-shaped nuclei and dense clustering.

b Presumes the failure of treatment with iron to increase hemoglobin levels, in order to bring the value with low serum ferritin up to that seen in polycythemia vera. Polycythemia vera can be ruled out based upon the hemoglobin and hematocrit values, and measurement of the red cell mass is not necessary.

c BCR-ABL1 must be negative.

d Neither dyserythropoiesis nor dysgranulopoiesis may be demonstrated.

e Patients with symptoms resembling those of reactive myelofibrosis are not immune to PMF; the diagnosis should be taken into consideration if other conditions are met.

f The degree of variance can be borderline or marked.

Table 15.17-6 Diagnostic criteria of essential thrombocythemia (ET)

1.

Persistent a thrombocyte count ≥ 450 × 109/L

2.

The bone marrow biopsy manifests mainly proliferation of the megakaryocyte line with an increasing number of enlarged, mature megakaryocytes. No rise worth mentioning or left shift of neutrophilic granulopoiesis or of erythropoiesis.

3.

The WHO criteria for polycythemia verab, primary polycythemiac, BCR-ABL1 positive chronic myeloid leukemiad, or myelodysplastic syndromee, or other myeloid neoplasia are not met in each case.

4.

Demonstration of JAK2 V617F or other clonal markers, or if JAK2 V617F is not mutated no evidence for reactive thrombocytosisf.

a During the clarification process.

b If the failure of an iron treatment for raising the hemoglobin value is postulated, in order to bring the value in the presence of low serum ferritin to the level in polycythemia vera. Polycythemia vera can be ruled out on the basis of the hemoglobin and hematocrit values; measurement of the red blood cell mass is not necessary.

c If an absence of relevant reticulin fibrosis or collagen fibrosis is postulated leukoerythroblastosis in the peripheral blood, or marked hypercellular bone marrow accompanied by megakaryocytic morphology which is typical of primary myelofibrosis, including small to large hyperchromic megakaryocytes with an aberrant nucleus-to-cytoplasm ratio, round or irregularly-shaped nuclei and dense clustering.

d BCR-ABL1 must be negative.

e Dyserythropoiesis or dysgranulopoiesis must be undetectable.

f The following must be considered as putative causes of reactive thrombocytosis: iron deficiency, splenectomy, surgery, infection, inflammation, disease of connective tissue, metastatic carcinoma, and lymphoproliferative disease. If, however, the first three criteria are met, conditions that resemble reactive thrombocytosis cannot rule out the possibility of ET.

Table 15.18-1 Basic panel for the initial diagnosis in acute leukemia, recommended by the German Kompetenznetz Akute und chronische Leukämien

 

F1

F2

F3

F4

cy 1

cy-NK

cy-NK

cy-NK

cy-NK

cy 2

TdT

cyCD22

CD45

cyCD3

cy 3

MPO

LF

CD45

CD34

1

NK

NK

NK

NK

2

CD10

CD13

CD45

CD19

3

CD7

HLA-DR

CD45

CD33

4

Kappa

Lambda

CD45

CD19

5

CD2

CD1a

CD45

CD3

6

CD34

”7.1”

CD45

CD117

7

CD65

CD56

CD45

CD20

8

CD61

CD14

CD45

F1–F4, fluorescence 1–4; cy, cytoplasmic staining; NK, negative control

Table 15.18-2 Terminology, frequency and phenotype of immunological ALL subtypes

Subtype

B precursor cell ALL

B cell ALL, Burkitt lymphoma

T precursor cell ALL

 

pro-Ba

Common

pre-B

 

pro-T

pre-T

cortical

mature

Incidence (%) children/adultsb

5/11

65/51

15/10

3/4

 

12/24

 

 

Progenitor cell-associated antigens

  • TdT

+c

+

+

+

+

±

  • HLA-DR

+

+

+

+

+

  • CD10

+

±

±

±

±

±

B cell antigens

  • CD19

+

+

+

+

  • CD22d

+

+

+

+

  • CD79a

+

+

+

+

  • cyIgM

+

  • mIgM

+

T cell antigens

  • cyCD3

+

+

±

  • CD7

+

+

+

+

  • CD2

+

+

+

  • CD5

+

+

+

  • CD1a

+

  • mCD3

±

+

a Synonym pre-pre-B ALL; b Corresponding to the results of the German Multicenter ALL Therapy Study /421/; c +, positive; ±, positive or negative; –, negative; demonstration of the respective antigen on ≥ 20% of the leukemia cells (membrane-bound antigens), or in ≥ 10% of the leukemia cells (MPO, cyCD3, cyCD79a, TdT); d Intracytoplasmic expression in immature subtypes. Abbreviations: CD, Cluster of Differentiation; Cy, intracytoplasmic; m, membrane-bound; Ig, Immunoglobulin

Table 15.18-3 Correlation of the FAB classification, cytogenetics and immunophenotype

Antigen

FAB classification

M0

M2

M3

M4Eo

M5

M5

M7

t(8;21)

t(15;18)

Inv16

t(9;11)

MPO

+/–

+

+

+

–/+

CD2

+/–

CD13

+/–

+

+

+

+/–

+/–

CD14

+/–

+/–

CD15

+/–

–/+

+/–

+

CD19

+/–

CD33

+/–

+/–

+

+

+

+

+

CD34

+/–

+/–

CD56

+/–

–/+

–/+

CD61

+

CD64

+/–

+

+

+

CD65s

–/+

+/–

–/+

+

+/–

+

+/–

CD117

+/–

+/–

–/+

+/–

–/+

HLA-DR

+/–

+

+

+

+

+/–

Immunological and cytogenetic characteristics of acute myeloid leukemia in correlation with the FAB subtype. There is no definite correlation, immunophenotype is only indicative, never proof of definite subtyping of AML. –, antigen not expressed; –/+, antigen expressed in < 50% of the cases; +/– , antigen expressed in the majority of the cases; +, antigen expressed. Blank fields show only partial expression, which is nonspecific for the diagnosis, or for which no reliable data is available.

Table 15.18-4 Score for the definition of mixed phenotype acute leukemia (MPAL)

Myeloid cell series

  • MPO or
  • two markers of monocytic differentiation, like CD11c, CD14, CD64, lysozyme, NSE

T cell series

  • cyCD3 or
  • mCD3 (only a few cases known)

B cell series

  • CD19 (strong expression) accompanied by an additional marker such as CD79a, cyCD22, CD10 or
  • CD 19 (weak expression) accompanied by two additional markers such as CD79a, cyCD22, CD10

In order to diagnose MPAL, the expression criteria for at least two cell series (myeloid, B or T) must be fulfilled.

Abbreviations: CD, cluster of differentiation; cy/m, intracytoplasmic or membrane-bound expression; MPO, myeloperoxidase; NSE, neuron-specific enolase

Table 15.18-5 Antigen combinations for the diagnosis of minimal residual disease in patients with acute lymphocytic leukemia

Subtype

Phenotype

Incidence (%)

B precursor
cell ALL

CD19/CD34/CD10/TdT

30–50

CD19/CD34/CD10/CD22

20–30

CD19/CD34/CD10/CD58

40–60

T precursor
cell ALL

TdT/CD5/CD3

90–95

CD34/CD5/CD3

30–50

AML

CD117/CD33/HLA-DR

26

CD34/CD117/CD33/HLA-DR

20

Table 15.18-6 Immunological marker profile of normal B cells and chronic lymphoproliferative diseases of the B cell type

 

Norm B

B-CLL

B-PLL

LPZ

MZL

FL

HCL

HCL-V

SLVL

DLBCL

sIg

+

(+)

+

+

+

+

+

+

+

+

CD5

+

–/+

+

CD10

+/–

–/+

CD11c

–/(+)

+

+

–/+

CD19

+

+

+

+

+

+

+

+

+

+

CD22

+/–

+

+

+

+

+

+

+

+

CD23

–/+

+

–/+

–/+

–/+

N.A.

CD24

+

+

+

+/–

+

+

–/+

–/+

+

+

CD25

–/(+)

+

–/+

–/+

CD43

+

+/–

–/+

+

CD79a

+

+/–

+

+

+

+

+

+

+

+

CD103

+

+

–/+

CD200

+

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

FMC7

+

+

+

+

+

+

–/+

N.A.

Cyclin D1

+

–/(+)

–/(+)

Abbreviations: N.A. no data available; Norm B, normal circulating B cells; B-CLL, B cell chronic lymphocytic leukemia; B-PLL, B cell prolymphocyte leukemia; LPL, lymphoplasmacytic lymphoma; MZL, mantel cell lymphoma; FL, follicular lymphoma; HCL, hairy cell leukemia; HCL-V, variant form of hairy cell leukemia; SLVL, splenic lymphoma with villous lymphocytes; sIg, surface immunoglobulin; DLBCL, diffuse large cell B cell lymphoma.

Table 15.18-7 Immunological marker profile of normal T cells and T cell type lymphoproliferative diseases

CD

Norm T

Act T

LGLL/
T

LGLL
NK

T-PLL

SEZARY

ATLL

ALCL

2

+

+

+

+

+

+

+

+/–

3

+

+

+

+

+

+

+/–

4

+/–

–/+

+/–

+

+

+/–

5

+

+

+

+/–

+/–

7

+

+

+

+/–

8

–/+

+/–

+

+/–

–/+

+/–

10

–/+

+/–

16

+

+

25

+/–

+

+

30

+/–

+/–

+/–

56

+/–

+/–

57

+/–

+/–

HLADR

+

–/+

+/–

+/–

Abbreviations: Norm T, normal circulating T cells; Act T, activated T cells, LGLL/T, T cell large granular lymphocyte leukemia; LGLL/NK, NK cell large granular lymphocyte leukemia; T-PLL, T prolymphocyte leukemia; ATLL, adult T cell leukemia/lymphoma; ALCL, anaplastic large cell lymphoma

Figure 15.1-1 Hierarchy of the hematopoietic system with myelopoiesis and lymphopoiesis; courtesy of Ref. /9/. CFU, colony forming unit; BFU, burst forming unit; E, erythrocytic; G, granulocytic; M, macrophagoytic; Meg, megaloblastic.

Multipotentialstem cells Committedprogenitor cells Morphologicallyrecognizableprecursor cells TISSUE, BLOOD BONE MARROW CFU blast(pluripotentialstem cell) CFU-GEMM(hematopoieticstem cell) Lymphoidstem cell Influence by the primary lymph organs Bone marrow Thymus Pre-β-cell Pre-T-cell Proliferation insecondary lymph organs Monoblast Pronormoblast Megakaryoblast Myeloblast (Myeloblast) (Myeloblast) Basophilnormoblast Promegakaryocyte Promonocyte Promyelocyte Neutrophilmyelocyte Neutrophilmetamyelocyte Eosinophilmyelocyte Basophilmyelocyte Polychromatophilicnormoblast Orthachromatic normoblast Reticulocyte Reticulocyte Erythrocyte Granularmegakaryocyte Neutrophil band Neutrophil Neutrophil Neutrophil Neutrophil Eosinophil Eosinophil Eosinophil Basophil Basophil Basophil Monocyte Monocyte Megakaryocyte Platelets Macrophage Eosinophilmetamyelocyte Eosinophilband Immature-basophil CFU-M CFU-G CFU-Eos CFU-Baso CFU-Meg CFU-E BFU-E BFU-Meg CFU-GM B Lymphocyte T Lymphocyte T Lymphocyte B LymphocytePlasma cell

Figure 15.1-2 Erythropoiesis develops a proliferation pool (upper row) and a maturation pool (lower row). In the proliferation pool, the committed progenitor cells proliferate and differentiate stimulated by growth factors, particularly erythropoietin (EPO). EPO deficiency leads to apoptosis of CFU-E cells. The cells form hemoglobin (Hb) in the maturation pool; Hb synthesis is diminished in iron deficiency. Courtesy of Ref. /29/.

Cell divisionPresence of RNAEPO receptorTransferrin receptorHemoglobin synthesis EPO receptorTransferrin receptorHemoglobin synthesis Pro-normoblast Basophilicnormoblast Polychromatic Orthochroma-tic Reticulocyte Erythro-cyte EPO EPO EPO CFU-E (late) BFU-E CFU-GEMM IL-3IL-4IL-11GM-CSF IL-3IL-9GM-CSF IL-3IL-9GM-CSFEPO

Figure 15.2-1 Erythrogram. The hemoglobin content (HC) of the erythrocytes is plotted on the abscissa against their volume (V) on the ordinate. A normal erythrogram is depicted. In iron deficiency anemia, the point cloud is shifted to the bottom left, in pernicious anemia it is displaced toward the upper field, and in hereditary spherocytosis it is moved towards the center right.

ERY HC V

Figure 15.2-2 Histogram of red cell distribution width (RDW). The erythrocyte volume is plotted on the abscissa in fL against the relative frequency (REL%) on the ordinate.

I. Normocytic anemia with normal RDW. In thalassemia the RDW is normal or only slightly elevated.

II. In iron deficiency the distribution peak is shifted further to the right.

III. Macrocytic anemia. The RDW is broadened in the upper MCV range.

IV. Distribution in the presence of cold agglutinins at a high titer, the arrow shows the characteristic change.

ERY I REL% 50 100 200 300 fl ERY II REL% 50 100 200 300 fl ERY III REL% 50 100 200 300 fl ERY IV REL% 50 100 200 300 fl

Figure 15.3-1 Frequent causes of anemia. Modified from Ref. /18/. The hypoproliferative anemias are highlighted in dark gray.

Iron deficiency Acute bleeding Marrowdamage Maturation disorder Hemolyticanemia Neoplasia,Inflammation

Figure 15.3-2 The O2 dissociation curves in dependence of PO2 and the O2 saturation of hemoglobin. With increasing PO2, the O2 saturation (SO2) reaches a plateau at high PO2. In this portion the O2 dissociation curve is flat indicating that PO2 values in arterial blood lead to full O2 saturation of hemoglobin. The steep part of the O2 dissociation curve at low PO2 is important for O2 release from hemoglobin to the tissues, since with an only small decline in tissue PO2 a great amount of O2 is unloaded from hemoglobin /16/.

  • The curve A shows the normal O2 dissociation curve
  • The curve B depicts the situation in the presence of decreased erythrocyte 2,3-DPG content
  • The curve C presents the O2 dissociation curve if 2,3-DPG in the erythrocytes is completely absent
  • The curve D shows the situation if erythrocyte 2,3-DPG content is increased.
Oxygen saturation (%) Oxygen partial pressure (mm Hg) 10050 C B A D 0 100 50

Figure 15.3-3 Structure and function of hemoglobin (Hb), modified according to Ref. /19/. The α and β components first combine to αβ-dimers and the αβ-dimers aggregate spontaneously to form the hemoglobin tetramer α2β2. The region where the two dimers come into contact is known as the α1β2-region. Both dimers can modify their gap in the α1β2-region, depending upon the 2,3-diphosphoglycerate (DPG) content. Each monomer contains a heme molecule (+) for the binding of O2. The binding site for DPG is located between the two β-units. The erythrocyte DPG content is maintained by diphosphoglycerate mutase.

The Hb molecule oscillates between two conformations. In the deoxygenated conformation (T, taut form), the affinity for O2 is low; in the oxygenated conformation (R, relaxed form) it is high. DPG is the regulator of O2 binding. The affinity of Hb for O2 decreases, and O2 is released, if DPG binds to Hb in the peripheral tissues. The dimers move closer together, and the tetramer takes on the T configuration. If the T conformation reaches the lungs, the reverse process takes place and O2 is bound.

A mutation in the Diphosphoglycerate mutase gene leads to a decrease in intra erythrocyte DPG, whereby O2 binding of the Hb molecule remains high and insufficient O2 is released to the tissues; the result is compensatory erythropoietin mediated erythrocytosis.

OxyhemoglobinR-configuration DeoxyhemoglobinT-configuration O 2 O 2 O 2 O 2 O 2 O 2 O 2 O 2 DPG DPG β 2 β 1 α 2 α 1 β 2 β 1 α 2 α 1

Figure 15.4-1 Classification of erythrocytosis. EPO, erythropoietin; 2,3-DPG-Mutase, 2,3-diphosphoglycerate mutase; HVL gene, von Hippel-Lindau gene; PHD, prolylhydroxylase; HIF2α, transcription factor

Erythrocytosis(polycythemia) Not clonally (polycythaemia vera) Congenital Hypoxia inducedEPO-increaseparaneoplastic EPO-receptor-mutation High affinity O 2 -hemoglobin 2,3-DPG- mutase deficiency Disorder of the O 2 -sensitivity – HVL-gene mutation – PHD 2 -gene mutation – HIF2α-gene mutation Clonally Acquired

Figure 15.4-2 Homodimer erythropoietin (EPO) receptor showing phosphorylated tyrosines. The tyrosines form binding sites for the components of the EPO signaling pathway. The tyrosines are phosphorylated by Janus kinase 2 (JAK2) and bind positive and negative regulators of the receptor to nucleus activated by EPO /33/. Positive and negative regulators respectively stimulate and suppress signal transmission.

LYN Y368 Y426 Y454 Y456 Y468 Y485 Y489 Y504 EPO SOCS3 CIS STAT5 JAK2 SAP-1 BMP-2 Grb2 F13K JAK2 Positive regulation Negative regulation AP5

Figure 15.4-3 Increased synthesis of erythropoietin (EPO) in von Hippel-Lindau gene mutation. The relationship between the proteins prolylhydroxylase (PHD), von Hippel Lindau protein (VHL), transcription factor HIF2α and normoxia and hypoxia are shown. Modified according to Ref. /19/. Normally O2 activates PHD, which then hydroxylates HIF2α. This leads to the binding of VHL protein and to ubiquitination and degradation of HIF2α. In hypoxia, HIF2α associates with its β subunit (not shown in figure), the complex binds to hypoxia-responsible elements (HREs) and leads to gene activation for enhanced synthesis of EPO.

EPO HRE HIF-2α PHD O H N HIF-2α O 2 Lack of O 2 HIF-2α O H N HIF-2α O H N HIF-2α O H N O Ubiquitination Normal O 2 supply Hypoxia PHD VHL PHD

Figure 15.4-4 Effect of 2,3-diphosphoglycerate (2,3-DPG) on O2 saturation curve. Courtesy of Ref. /37/ modified. A, normal erythrocyte 2,3-DPG content; B, low erythrocyte 2,3-DPG content; C, no 2,3-DPG present in the erythrocytes; D, elevated erythrocyte 2,3-DPG content.

Oxygen saturation (%) Oxygen partial pressure (mm Hg) 50 0 50 100 100 A B D C

Figure 15.6-1 Differentiation of normocytic anemia by reticulocyte count /17/.

Normocytic anemia(MCV 80–95 fL) Reticulocyte count Increased Normal or reduced Acuteloss of blood Hemolyticsyndrome Bone marrowinsufficiency Chronicinflammatorydisease

Figure 15.6-2 Differentiation of macrocytic anemia by reticulocyte count /17/.

Retikulocyte count Increased Normal, reduced Vit. B 12 -, folate deficiency Treated Vit. B 12 -, folate deficiency Acute bleeding in case of Vit. B 12 -, folat deficiency Macrocytic anemia(MCV > 95 fl)

Figure 15.6-3 Differentiation of microcytic anemia by reticulocyte count /17/.

Ferritin, Transferrin saturation Normal Reduced Microcytic anemia(MCV < 80 fL) Retic. normal Retic. increased Chronic loss of bloodNutritive iron deficiency Chronic inflam-matory disease Hemoglobino-pathy

Figure 15.6-4 Differentiation of reticulocytes in maturation stages of low fluorescence reticulocytes (LFR), medium fluorescence reticulocytes (MFR), and high fluorescence reticulocytes (HFR) of a healthy individual.

Reticulocytes MFR HFR LFR Erythro-cytes Thrombocytes Fluorescence Scattered light

Figure 15.6-5 Diagnostic diagram for the assessment, monitoring and therapy of the iron status /37/. The ferritin index (sTfR/log10 ferritin) is a marker of iron supply, CHr is an indicator of the iron demand of erythropoiesis. Ferritin indices of greater than 1.5 and 0.8 in patients without and with inflammation, respectively, reflect deficient iron supply, a CHr below 28 pg indicates the iron demand of erythropoiesis.

Over 95% of ACD patients are found in quadrants 1 and 4, those with iron-restricted erythropoiesis or functional iron deficiency in quadrant 4 and those without in quadrant 1.

The data points of patients with latent iron deficiency (iron deficiency without anemia) are found in quadrant 2, those with classic iron deficiency in quadrant 3. If sTfR is determined with the Siemens assay, the threshold values of the ferritin indices are 1.5 and 0.8., for the Roche assay 3.2 and 2.0. Treatment recommendations are shown in the 4 quadrants.

Q1 Q2 Q4 Q3 0 3.2 (2.0) sTfR/log Ferritin 20 28 CHr (pg) ESA + Iron Iron Iron ESA 36 Anemia of a chronic disease and ironrestrictive erythropoiesis Microcytic erythropo-iesis in iron deficiencyanemia Normocytic erythropo-iesis in anemia of a chronic diseaseNo iron deficiency Normocytic erythropo- iesis in latentiron deficiency

Figure 15.7-1 Clinical laboratory diagnosis of thalassemia syndromes and hemoglobinopathies.

Detection of an anomalyComparison of the morbidity with those of known anomalies. On suspected HbS: Solubility test Separation/quantification Hemolysate If necessary DNA sequencing Evaluation of all data and findings Assessment of all data and results Blood sample Diagnosis of β-thalassemia DNA analysisif indicated Electrophoresis HPLC: HbA 2 , HbF Diagnosis ofβ-thalassemias Diagnosis of α-thalassemia ElectrophoresisHPLC, DNA analysis Diagnosis ofα-thalassemias Anamnesis, ethnic lineageclinical symptomatology Blood count including erythrocyte morphology Diagnosis of the hemoglobino-pathy Evaluation of all data and findings including blood count, ethnic lineage Alkaline electrophoresis,acid electrophoresis, HPLC Anomalous hemoglobins

Figure 15.7-2 Use of RDW values in diagnostic investigation of thalassemia minor.

Normal RDW Increased > 15 fL Thalassemia diagnostics Ferritin, Iron deficiency diagnostics AnemiaMicrocytosisHypochromia

Figure 15.7-3 Separation schema for normal and abnormal hemoglobin on micro zone electrophoresis.

HbA HbA 2 Hbl; HbH HbN; Hb Bart‘s HbJ HbA HbF HbS; HbD; HbG; HbA 2 ; HbC; HbE; HbO

Figure 15.10-1 Compensation of O2 deficiency in patients with acute hemorrhage induced anemia. O2 deficiency in the kidney activates hypoxia inducible factor HIF2α to stimulate the production of erythropoietin (EPO). Erythropoietic activity is stimulated by EPO. The erythroid regulator erythroferrone is produced by erythroid precursors and facilitates compensatory acquisition of iron. Erythroferrone acts directly on the liver to decrease hepcidin production, and thereby increases iron availability for new erythrocyte synthesis.

HIF2a EPO Normochromic erythropoiesis Proliferation of erythropoiesis Intestinal iron resorption Availability of RES iron Erythroferrone Hepcidin synthesis

Figure 15.10-2 Expected range of serum EPO level as a function of the hematocrit (HCT) value. With a decrease in the HCT, the EPO level increases exponentially. The 95% confidence interval is shown. Modified from Ref. /5/.

Hematocrit Erythropoietin (U/L) 1000 100 10 1 0.2 0.25 0.3 0.35 0.40 0.45

Figure 15.10-3 Relationship between HCT and serum EPO level /7/. 1, β-thalassemia; 2, hemolytic anemia; 3, renal anemia; 4, anemia of chronic disease; 5, pregnancy; 6, polycythemia vera; 7, normal individuals; 8, renal cell carcinoma. The line represents the standard EPO increase in patients with acute bleeding.

1,000100101 Erythropoietin(U/L) 0.2 0.3 0.4 HCt (fraction) 0.5 0.6 8 6 7 2 5 1 4 3

Figure 15.10-4 Activation of the immune system in acute phase response (APR) as the result of the formation of inflammatory cytokines (IFN-γ, IL-6) by activated immune cells (TH-lymphocytes, monocytes/macrophages). Erythroid precursor cells of the bone marrow proliferation pool need the binding of some 10 EPO molecules per cell in order to enter the maturation pool and not to become apoptotic. EPO exerts anti-apoptotic effects, while inflammatory cytokines such as IFN-γ are pro-apoptotic. The survival of the erythroid precursors is dependent upon the balance between EPO and inflammatory cytokines. In severe inflammation (high CRP values), apoptosis predominates and erythropoiesis becomes hypoproliferative, mediated by hepcidin, and the result is normochromic anemia /17/.

APR and otherinflammatory conditions Immune activation Iron-metabolism: Increased Fe-storage Decreased Fe-resorption Mono-cyte Pro-apoptotic IFN-γ Anti-apoptotic Erythroid progenitor cell TH 1 - cell Epoetin

Figure 15.10-5 Site of action of erythropoietin (EPO) and other hematopoietic growth factors in erythropoiesis. Modified from Ref. /21/. BFU-E , Burst forming unit erythroid; CFU-E, Colony forming unit erythroid; IL-3, interleukin-3; GM-CSF, granulocyte-macrophage colony-stimulating factor; SCF, stem cell factor; RBC, red blood cells.

Bone marrow Blood Proliferative pool Maturation pool Maturecells Stem cells Multipotent Committed BFU-E CFU-E CFU-E EPO IL-3GM-CSFSCF

Figure 15.10-6 Erythropoietin feedback control loop modified according to Ref. /24/.

Stem cells → BFU-E → CFU-E → Erythroblast Bone marrow Erythropoietin Red blood cell mass ← Atmospheric O 2 ← Cardiopulmonary function ← Blood volume ← Hemoglobin concentration ← O 2 affinity Renal blood flow → Renal O 2 consumption → Erythropoietin ← Oxygen sensorformation Kidney

Figure 15.10-7 Homodimer erythropoietin (EPO) receptor showing phosphorylated tyrosines. The tyrosines form binding sites for the components of the EPO signaling pathway. The tyrosines are phosphorylated by Janus kinase 2 (JAK2) and bind positive and negative regulators of the receptor to nucleus activated by EPO /34/. Positive and negative regulators respectively stimulate and suppress signal transmission.

LYN Y368 Y426 Y454 Y456 Y468 Y485 Y489 Y504 EPO SOCS3 CIS STAT5 JAK2 Grb2 Positive regulation Negative regulation SAP-1 BMP-2 F13K JAK2 AP5

Figure 15.12-1 Hematogram of leukocyte differentiation using volume, conductivity and scatter technology. N, neutrophils; E, eosinophils; B, basophils; L, lymphocytes; M, monocytes.

Conductivity scattered light signal WBC (volume) L M N B E

Figure 15.12-2 Hematogram of leukocyte differentiation with combined flow cytometry and cytochemistry. In the leukogram (left diagram), the following leukocytes are counted: 1, neutrophils; 2, eosinophils; 3, monocytes; 4, lymphocytes and blasts; 5, large unstained cells (LUC). In the nucleogram (right diagram), the following leukocytes are differentiated after stripping of cell cytoplasm: 6, mononuclear cells; 7, polynuclear cells; 8, basophils.

Figure 15.12-3 Neutrophile count in the newborn, according to Ref. /9/. The upper and lower reference interval numbers are shown.

Time (h) Neutrophil count/μL 18,000 16,200 14,400 12,600 10,800 9000 7200 5400 3600 1800 0 0 6 12 18 24 30 36 42 48 54 60

Figure 15.12-4 Age-dependency of the lymphocyte count in children according to Ref. /21/. Values are 2.5th and 97.5th percentiles.

Age in months/years 0 4 8 12 2 4 6 8 10 12 14 16 18 20 Lymphocyte count (10 9 /L) 161412108642

Figure 15.13-1 Blood smear method.

Figure 15.13-2 Relevant areas for the evaluation of the blood smear.

Direction of the smear Area of the optimal density Blooddrop Front Feathered edge

Figure 15.13-3 Morphological granulocyte alterations.

Nuclear abnormalities Neutrophil granulocyte with a drumstick Pelger Huet anomaly Peanut shape Granulation anomalities Toxic granulation Döhle bodies Auer rods Oversegmentation ”Faggot“ shape

Figure 15.13-4 Morphological lymphocyte alterations.

Normal lymphocyte ReactiveLymphocyte Diffuse largeB Cell Gumprechtnuclearshadow

Figure 15.13-5 Morphological thrombocyte alterations.

Normal Agranular platelet Giant platelet Platelet satellitosis

Figure 15.13-6 Morphological erythrocyte alterations.

Normal Cell size Microcyte Macrocyte Cell staining Normal Hypochromia Hyperchromia Cell shape Elliptocyte Target cell Spherocyte Stomatocyte Leptozyte Sickle cell Acanthocyte Fragmentocytes Teardrop cell Cell inclusions Howell-Jolly body Basophilic stippling Cabot rings Malaria Cell distribution Rouleau formation Aggregation

Figure 15.14-1 Transverse section at the level of the biopsy site.

Arteries Veins Nerves M. psoas major M. gluteus minimus M. gluteus medius M. gluteus maximus M. erector trunci Horizontal section through the upper posterior iliac spine

Figure 15.14-2 Jamshidi needle (above) in comparison with a usual puncture needle.

Figure 15.14-3 Preparation of a smear of bone marrow fragments.

Figure 15.15-1 Previous FAB classification: bone marrow blast threshold between AML and MDS is now 20%.

Blasts % of allnucleated cells Blasts % of all non-ery-thropoietic cells Erythroblast % of all nucleated cells < 50% Erythroblasts ≥ 50% Erythroblasts Blasts ≥ 30% Blasts ≥ 30% Blasts < 30% Blasts < 30% AML MDS AML M6

Figure 15.15-2 Standard diagnostic investigation of acute lymphocytic leukemia.

Differentanomalies,normalkaryotype No describable result or less metaphases of inadequate quality Screening with PCRand/or FISHon BCR-ABL- andMLL-rearrangement MRD examinationswith PCR for receptorrearrangements Suspicion of relapsemorphology + cytogenetics MRD examinationswith PCR for fusion transcripts PCR BCR-ABLPCR MLL-AF4 FISH IGH-C-MYC Fully developedB-ALL † (9;22),† (4;11) Morphology + cytogenetics + immunophenotypingasservation of DNA for MRD examinations

Figure 15.17-1 Algorithm for molecular genetic testing in myeloproliferative neoplasia.

TET2 10% + + TET2 5–10% 95% JAK2V617F JAK2V617F JAK2V617F JAK2V617F + 5% 50% + + + 50% 50% 50% PMF BCR-ABL CML 90% + + 10% family:– VHL– EPO– HIF2A– PHD2 family:– MPL– THPO FIP1L1-PDGRFA:– PDGFRA– PDGFRB FGFR1 TET2 CBL20% NRASTET2EZH2RUNX1 TET2 MPN? CMML PV ET JAK2exon12 MPLW515 Eosino-philia? + NHL? HES/CEL

PV, polycythemia vera; ET, essential thrombocythemia; PMF, primary myelofibrosis; MPN, myeloproliferative neoplasia; CMML, chronic myelomonocytic leukemia; TET2, tet oncogene family member 2; MPL, myeloproliferative leukemia virus oncogene/thrombopoietin receptor; CBL, Casitas B-lineage lymphoma proto-oncogene; VHL, von Hippel-Lindau tumor suppressor gene; EPO, erythropoietin; HIF2A, HIF-1-alpha-like factor; PHD2, HIF prolyl hydroxylase 2; THPO, thrombopoietin; PDGFRB, Platelet-derived growth factor receptor beta; PDGFRA, platelet-derived growth factor receptor alpha; EZH2, Enhancer of zeste homolog 2 (Drosophila); RUNX1, runt-related transcription factor 1.

Figure 15.18-1 Flow diagram for immunophenotyping in acute leukemia.

Acute leukemia – Diagnosis according to FAB criteria

Assignment to B or T lymph cell or myeloid cell series /20/

B cell lineage: CD19, (cy) CD22, (cy) CD79a

T cell lineage: cyCD3, CD7

Myeloid lineage: CD13, CD33, CD65, CD117, MPO

NK celllineage CD56, CD57

Diagnosis: B precursor cell ALL, B-ALL/Burkitt’s lymphoma, T-lineage ALL, AML

Definition of stage of maturity or immunological subtype
(see Tab. 15.17-1 – Clinical characteristics and orienting markers in the differential diagnosis of MPN)

B-lineage ALL: CD10, CD20, CD24, cyIgM, mIg; CD34, HLA-DR, TdT

T-lineage ALL: CD1a, CD2, mCD3, CD4, CD5, CD8, CD10, TCRα/β or γ/δ; CD34, HLA-DR, TdT

AML: CD14, CD15, CD41, CD61, CD64, Glycophorin A; CD34, HLA-DR, TdT

Abbreviations: CD, cluster of differentiation; cy/m, intracytoplasmic or cell surface expression; MPO, myeloperoxidase; Ig, Immunoglobulin; TCR, T-cell receptor; TdT, terminal deoxynucleotidyl transferase

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