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⁺DR^–lin^–. 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 hyperchromic
• 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 /1, 21/:

• 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 Therapeutic measures that compromise hematopoiesis

Refer to Tab. 15.1-4 – Therapeutic measures that compromise hematopoiesis.

15.1.12 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.

15.1.13 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).

15.1.14 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, Ret_He), 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).

15.1.15 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

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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.

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38. Gonzalez-Lopez T, Jarque I, Siniscalchi C, DI Micco P. Editorial: Infectious diseases and hematology. Frontiers Med 2024. doi: 10.3389/fmed.2024.1385874.

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 /2, 3, 4/.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 /27, 28/:

• 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:

• 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:

• MCHC (mean cellular hemoglobin concentration). MCHC is expressed as red cell hemoglobin (g/dL or g/L) and is calculated as follows:

• 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 /2, 3, 5, 13, 14/ 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 /3, 4, 5, 6/ 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 /7, 8/:

• 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:

• Tab. 15.3-5 – Correction for anemia during an extended stay at high altitude
• Tab. 15.3-6 – Correction for anemia in smokers.

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/:

• Sideroblastic anemias (refer to Tab. 15.3-9– Classification and differentiation of microcytic anemia)
• Erythropoietic protoporphyria (refer to Tab. 14.6-6)
• Congenital erythropoietic porphyria (refer to Tab. 14.6-6).

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:

• Tab. 15.3-8 – Blood cell status in certain groups of individuals and patients
• Tab. 15.3-9 – Classification and differentiation of microcytic anemia
• Tab. 15.3-10 – Hematological data in patients with β-thalassemia
• Tab. 15.3-11 – Classification and differentiation of normocytic anemias
• Tab. 15.3-12 – Causes of hemolytic anemias
• Tab. 15.3-13 – Laboratory findings in sickle cell anemia

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:

• Tab. 15.3-14 – Classification and differentiation of macrocytic anemias
• Section 13.3 – Vitamin B₁₂ deficiency
• Section 13.4 – Folate deficiency.

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-H_e) 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 /16, 83/. 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/.

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

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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.

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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 /1, 2, 3, 4, 5, 6, 7, 8/ 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 /19, 20/. PFCP is characterized by an isolated primary polycythemia in which an increased red cell mass is associated with subnormal erythropoietin levels.

Refer to:

• Tab. 15.4-5 – Diseases and conditions with elevated HCT
• Tab. 15.4-6 – WHO criteria for polycythemia vera.

A genetic abnormalites in concern to the EPO level are:

• Defects of the EPO signalling pathway (EPO receptor mutations). Refer to Fig. 15.4-2 – Homodimer erythropoietin receptor
• High oxygen affinity hemoglobins
• Bisphosphoglycerate mutase deficiency. Refer to Fig. 15.4-4 – Effect of 2,3 diphosphoglycerate on O₂ saturation
• Mutations in genes in the oxygen sensing pathway. Refer to Fig. 15.4-3 – Increased synthesis of erythropoietin.

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) /19, 20/. 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

1. 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.

2. Fairbanks VF, Tefferi AY. Normal ranges for packed cell volume and hemoglobin concentration in adults: relevance to apparent polycythemia. Eur J Hematol 2000; 65: 285–96.

3. Kujala UM. Hemoglobin and packed-cell volume in endurance athlets prior to rhEPO. Int J Sports Med 2000; 21: 228.

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

5. Mordechai S, Merlob P, Reisner SH. Neonatal polycythemia: I. Early diagnosis and incidence relating to time of sampling. Pediatrics 1984; 73: 7–10.

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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 /3, 5/:

• 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:

• Tab. 15.5-1 – Agents capable of inducing methemoglobinemia
• Tab. 15.5-2 – Correlation of clinical symptoms with increasing proportions of metHb.

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:

• CO and, therefore, COHb, accumulate increasingly due to the shortened lifespan of erythrocytes
• COHb increases the affinity of other binding sites for O₂, an effect originally described by Haldane. This shifts the oxygen dissociation curve to the left makes it less S-shaped and more hyperbolic (See Fig. 15.3-2 – The O₂ dissociation curves in dependence of PO₂ and the red cell content of 2,3-DPG). In sickle cell disease this shift in the dissociation curve would have the effect of reducing the level of deoxyhemoglobin and thus the tendency for HbS polymerization and sickling (see also Tab. 15.3-9 – Classification and differentiation of microcytic anemia) /14/.

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 /6, 15/.

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.

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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.

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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, Ret_He)
• 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:

• Fig. 15.6-1 – Differentiation of normocytic anemia by reticulocyte count
• Fig. 15.6-2 – Differentiation of macrocytic anemia by reticulocyte count
• Fig. 15.6-3 – Differentiation of microcytic anemia by reticulocyte count
• Tab. 15.6-4 – Diseases and conditions with reticulocytosis.

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 Ret_He)

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-H_e 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-H_e 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-H_e 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-H_e 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-H_e 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-H_e 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, Ret_He) is decreased /44/.

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

CHr and Ret_He 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 Ret_He 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 Ret_He 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-H_e 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-H_e are stable for at least 24 hours /31/.

References

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2. Heilmeyer L, Westhäuser R. Reifungsstudien an überlebenden Retikulozyten in vitro und ihre Bedeutung für die Schätzung der täglichen Hämoglobinbildung in vivo. Zschr Klin Med 1932; 121: 361–655.

3. Löwenstein LM. The mammalian reticulocyte. Int Rev Cytol 1959; 8: 135–74.

4. Perrotta AL. The polychromatophilic erythrocyte. Am J Clin Pathol 1972; 57; 471–7.

5. Pappas AA, Owens RB, Flick JT. Reticulocyte counting by flow cytometry. Ann Clin Lab Sci 1992; 22: 125–32.

6. Houwen B. Reticulocyte maturation. Blood Cells 1992; 18: 167–86.

7. Hillman RS. Characteristics of marrow production and reticulocyte maturation in normal man in response to anemia. J Clin Invest 1969; 48: 443–53.

8. Come SE, Shohet SB, Robinson SH. Surface remodeling vs whole cell hemolysis of reticulocytes produced with erythroid stimulation or iron deficiency anemia. Blood 1974; 44: 817–30.

9. NCCLS. Method for reticulocyte counting. Proposed standard. Order Code HP 16-P, Vol 5, No 10. Villanova NCCLS, 1985.

10. Davis BH, Bigelow NC. Automated reticulocyte analysis. Hematol Oncol Clin North Am 1994; 8: 617–30.

11. Herkner KR, Bock A, Mulh A, et al. Reticulocyte counting and maturity grading in pediatrics using the Sysmex R-1000 automated reticulocyte analyzer. Sysmex J Int 1992; 2: 4–9.

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

13. Butarello M, Bulian P, Farina G, Temporin V, Toffolo L, Trabuio E, et al. Flow cytometric reticulocyte counting. Parallel evaluation of five automated analyzers: an NCCLS-ICSH approach. Am J Clin Pathol 2001; 115: 100–11.

14. 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.

15. Nebe CT, Diem H, Heimpel H. Aktuelle Aspekte zur Bestimmung der Retikulozytenzahl. J Lab Med 2010; 34: 295–304.

16. Heimpel H, Diem H, Nebe CT. Die Bestimmung der Retikulozytenzahl: Eine alte Methode gewinnt neue Bedeutung. Med Klin 2010; 105: 538–43.

17. Thomas L, Messinger M. Bedeutung der Retikulozytenbestimmung zur Differenzierung und Behandlungskontrolle der Anämie. Lab Med 1994; 18: 305–10.

18. Means Jr RT, Krantz SB. Progress in understanding the pathogenesis of the anemia of chronic disease. Blood 1992; 80: 1639–47.

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

20. Bowen D, Williams K, Phillips I, Cavill I. Cytometric analysis and maturation characteristics of reticulocytes from myelodysplasic patients. Clin Lab Haem 1996; 18: 155–60.

21. Delaunay J, Iolascon A. The congenital dyserythropoietic anemias. Baillaire’s Clinical Haematology 1999; 12: 691–705.

22. Pekrun M, Lakomek M, Eber S, Konrad H, Schröter W. Diagnostik des Pyruvatkinasemangels bei erhöhter Retikulozytenzahl. Dtsch Med Wschr 1995; 120: 1620–4.

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

24. Koepke JF, Koepke JA. Reticulocytes. Clin Lab Haematol 1986; 8: 169–79.

25. Aulesa C, Ortola J, Olive T, Ortega JJ. Temporary increase in the reticulocytes after transfusion of RBC concentrates in the BMT patients. Sysmex J Intern 1994; 4: 96–8.

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.

29. Van den Bossche J, Devreese K, Malfait R, van de Vyvere M, Wauters A, Neels H, et al. Reference intervals for complete blood count determined on different hematology analyzers: ABX Pentra 120 Retic, Coulter Gen S, Sysmex SE 9500, Abbott Cell Dyn 4000 and Bayer Advia 120. Clin Chem Lab Med 2002; 40: 69–73.

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.

31. Brugnara C. Reticulocyte cellular indices: a new approach in the diagnosis of anemias and monitoring of erythropoietic function. Crit Rev Clin Lab Sci 2000; 37: 93–130.

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33. Brugnara C, Hipp MJ, Irving PJ, Lathrop H, Lee PA, Minchello EA, Winkelman J. Automated reticulocyte counting and measurement of reticulocyte cellular indices. Evaluation of the Miles H*3 blood analyzer. Am J Clin Pathol 1994; 102: 623–32.

34. Buttarello M, Bulian P, Venudo A, et al. Laboratory evaluation of the Miles H*3 automated reticulocyte counter. A comparative study with manual reference method and Sysmex R-1000. Arch Pathol Lab Med 1995; 119: 1141–8.

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

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39. Ervasti M, Kotisaari S, Sankilampi U, Heinonen S, Punnonen K. The relationship between red blood cell and reticulocyte indices and serum markers of iron status in the cord blood of newborns. Clin Chem Lab Med 2007; 45: 1000–3.

40. Ullrich C, Wu A, Rieber S, Wingerter S, Brugnara C, Shapiro D, Bernstein H. Screening healthy infants for iron deficiency using reticulocyte hemoglobin content. JAMA 2005; 294: 924–30.

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42. Brugnara C, Laufer MR, Friedman AJ, Bridges K, Platt O. Reticulocyte hemoglobin content (CHr): early indicator of iron deficiency and response to therapy (letter). Blood 1994; 83: 3100.

43. Brugnara C, Zurakowski D, DiCanzio J, Boyd T, Platt O. Reticulocyte hemoglobin content to diagnose iron deficiency in children. JAMA 1999; 281: 2225–30.

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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 HbA_1a, HbA_1b and HbA_1c 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) /1, 2, 3/.

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/β⁺-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 /2, 4/.

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) /4, 5/. 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 /4, 5, 6/. 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 /2, 7, 8, 9, 10, 11, 12/.

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 /2, 7, 8, 10, 11/.

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 /2, 7, 8, 10, 11/. 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 /10, 11, 14, 15, 16/.

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⁺/α⁰-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 a prevalent anemic condition worldwide, particularly in Southeast Asia, arises due to genetic irregularities affecting globin chain synthesis. Mutations in the β-globin genes lead to deficient or non-functional ß-globin chain production causing anemia and relate complications. A notable β-hemoglobin variant linked to a relative ß-thalassemia phenotype is hemoglobin E, resulting from GAG to AAG transition at codon 26 in the β-globin gene. The prevalence of β-thalassemia and HbE carriers in Thailand ranges from 3 to 9% and 10 to 53%, respectively.

β-thalassemia carriers experience mild to asymptomatic anemia, whereas patients with β-thalassemia major encompassing homozygous β-thalassemia and thalassemia/HbE endure moderate to severe anemia necessitating treatments such as blood transfusions and iron chelation.Given the high prevalence of these carriers, β-thalassemia major cases are likely widespread, making it a substantial public health and socioeconomic concern. Accurate quantification of hemoglobin A₂ is vital for diagnosing β-thalassemia carriers /19/.

Refer to Tab. 15.7-9 – Compilation of the most important criteria of β-thalassemias.

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 /2, 5, 10, 11, 17/:

• 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 /1, 2, 17/.

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-β^o 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 /1, 2, 10, 11/.

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:

• Tab. 15.7-12 – Characteristic features of the most important hemoglobin variants
• Tab. 15.7-7 – Basic forms of hemoglobinopathies.

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 /1, 2, 10, 11/.

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 /2, 14, 15/

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 /14, 15/

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⁺/α⁰-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 /2, 14/

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) /1, 2, 4/

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. Satthakarn S, Panyasai K, Phasit A, Panyasai S. Reliability of hemoglobin A2 value as measured by the premier resolution system for screening of β-thalassemia. J Clin Chem Clin Biochem 2023. doi: 10.1515/cclm-2023-1006.

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 /1, 2, 3/.

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 /1, 2, 3/.

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 /1, 2, 3/. 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 /1, 2/. 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 /4, 5/. 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 /4, 7, 8/. 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 /1, 2, 6/

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 /4, 5, 7, 8/

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. /1, 2/).

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 /1, 2/. 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:

• Fig. 15.10-1 – Compensation of O₂ deficiency in patients with acute hemorrhage.

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 /1, 4/ 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:

• Tab. 15.10-2 – Diseases and conditions associated with an adequate rise in EPO concentration
• Tab. 15.10-3 – Diseases and conditions with inadequately low increase in EPO concentration
• Tab. 15.10-4 – Diseases and conditions with a disproportionate rise in EPO concentration.

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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54. Hirai K, Kaneko S, Yanai K, Taisuke K, Kiyonori I Ookawara S. Effects of Roxadustat on anemia, iron metabolism, and lipid metabolism in patients with non-dialysis chronic disease. Front Med 2023; 22 February. doi: 10.3389/fmed.2023.1071342.

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

• Section 16.1-3 – Thrombocytes
• Chapter 17 – Thrombocyte diagnostics.

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

• Diseases and conditions with primary thrombocytosis are listed in Tab. 15.11-6 – Diseases and conditions with primary thrombocytosis
• Diseases and conditions with secondary thrombocytosis are shown in Tab. 15.11-7 – Diseases and conditions with secondary thrombocytosis.
• Thrombocytopenias of various etiologies are listed in Tab. 15.11-8 – Thrombocytopenia of various origins.
• Pregnancy related thrombozytopenias are listed in Tab. 15.11-9 – Pregnancy associated 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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47. Rodeghiero F, Stasi R, Gernsheimer T, Michel M, Provan D, Arnold DM, Bussel JB, et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood 2009; 113: 2386–93.

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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 /1, 2/.

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 /3, 4, 5, 6, 7, 8/ 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

• The most frequent cause of neutropenia is a toxic disorder of granulopoiesis due to medication (Tab. 15.12-5 – Drugs that can cause neutropenia).
• Congenital neutropenia (Tab. 15.12-6 – Congenital neutropenia) is usually severe in nature.
• The causes of secondary neutropenia are diverse (Tab. 15.12-7 – Secondary neutropenia) are diverse.
• Infections with neutropenia are shown in Tab. 15.12-8 – Acute febrile infections with leukopenia and/or thrombocytopenia.

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 CD3^– CD16⁺.

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 /28, 29/. 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, and invasive candidiasis /76/.

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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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 /1, 2, 3, 4/.

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. /3, 6, 7/.

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:

• Tab. 15.13-1 – Artifacts and morphological changes of leukocytes in the blood smear
• Fig. 15.13-3 – Morphological granulocyte alterations
• Fig. 15.13-4 – Morphological lymphocyte alterations.

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:

• Tab. 15.11-3 – Differentiation of the mechanism of thrombocytopenia
• Fig. 15.13-5 – Morphological thrombocyte alterations
• Tab. 15.13-3 – Artifacts and morphological platelet changes in the blood smear.

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:

• Fig. 15.13-6 – Morphological erythrocyte alterations
• Tab. 15.13-2 – Artifacts and morphological red blood cell changes in the blood smear.

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.

References

1. Pierre RV. Red cell morphology and the peripheral blood film. Clin Lab Med 2002; 22: 25–61.

2. Bain BJ. Diagnosis from the blood smear. N Engl J Med 2005; 353: 498–507.

3. Houwen B. Blood film preparation and staining procedures. Clin Lab Med 2002; 22: 1–14.

4. Gulati GL, Bong HH. Blood smear examination. Hematol Oncol Clin North Am 1994; 8: 631–50.

5. Barnes PW, McFadden SL, Machin SJ, Simson E. The international consensus group for hematology review: suggested criteria for action following automated CBC and WBC differential analysis. Lab Hematol 2005; 11: 83–90.

6. Woronzoff-Dashkoff KK. The Wright-Giemsa stain. Clin Lab Med 2002; 22: 15–23.

7. Bucher U. Labormethoden in der Hämatologie. Bern; Huber 1988; 59–65, 87.

8. Dalal BI, Brigden ML. Artifacts that may be present on a blood film. Clin Lab Med 2002; 22: 81–100.

9. NCCLS. Reference leukocyte differential count (proportional) and evaluation of instrumental methods. Villanova 1992; NCCLS Document H20–A Vol 12 No 1.

10. Nosanchuk J, Chang J, Bennett J. The analytical basis for the use of platelet estimates from peripheral blood smears. Am J Clin Pathol 1978; 69: 383–7.

11. Kroft SH. Infectious diseases manifested in the peripheral blood. Clin Lab Med 2002; 22: 253–77.

12. Constantino BT. Pelger-Huet anomaly – morphology, mechanism, and significance in the peripheral blood film. Labmedicine 2005; 30: 103–7.

13. Moreira AMB, Vieira LM, Carvalho M. Acquired Pelger-Huet anomaly associated with ibuprofen therapy. Clin Chim Acta 2009; 409: 140–1.

14. Blood images in hematology. Howell-Jolly body-like inclusions in neutrophils. Blood 2009; 114: 2860.

15. Baumann H, Bettelheim P, Diem H, Gassmann W, Nebe T. Lymphozytenmorphologie im Blutausstrich – Vorstellung einer überarbeiteten Nomenklatur und Systematik. J Lab Med 2011; 35: 261–70.

16. Mallah HS, Brown MR, Rossi TM, Block RC. Parenteral fish oil-associated Burr cell anemia. J Pediatr 2010; 156: 324–6.

17. Glader E. Hemolytic anemias in children. Clin Lab Med 1999; 19: 87–111.

18. Stiegler H, Fischer Y, Steiner S, et al. Sudden onset of EDTA-dependent pseudothrombocytopenia after therapy with the glycoprotein IIb/IIIa antagonist c7E3 Fab. Ann Hematol 2000; 79: 161–4.

19. Bizarro N, Goldschmeding R, dem Borne AE. Platelet satellism is Fc gamma RIII (CD16) receptor mediated. Am J Clin Pathol 1995; 103: 740–4.

20. Becker RC, Giuliani M, Savage RA, et al. Massive hemolysis in C. perfringens infections. J Surg Oncol 1987; 35: 13–8.

21. Baumgarten BU, Röllinghoff M, Bogdan C. Ehrlichien. Dtsch Ärztebl 2000; 97: A2456–A2461.

22. Weinberg GA. Laboratory diagnosis of Ehrlichosis and Babesiosis. Pediat Infect Dis 2001; 20: 435–7.

23. Eberhard ML, Lammie PJ. Laboratory diagnosis of filariasis. Clin Lab Med 1991; 11: 977–1010.

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 /1, 2, 3, 4/.

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 /1, 5, 7/.

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 /2, 3, 4, 5, 6/. 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 /1, 2, 3, 4, 5, 6/. 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:

• Fig. 15.15-1 – Previous FAB classification: bone marrow blast cell cutoff between AML and MDS
• Tab. 15.15-2 – Previous FAB classification of AML, can be performed as initial diagnostic step before WHO classification.

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

• Section 15.15 – Acute leukemias
• Section 15.16 – Myelodysplastic syndrome.

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 /1, 8/:

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 /7, 9/.

Refer to

• Fig. 15.15-1 – Methods for the diagnostic investigation of acute leukemia
• Tab. 15.15-5 – Laboratory findings in acute myeloid leukemia

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 /7, 10/. 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. /12, 13/.

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 /1, 2/.

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:

• Section 15.15 – Acute leukemias
• Tab. 15.16-1 – WHO classification of myelodysplastic syndrome since 2008.

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:

• Tab. 15.16-3 – International Prognostic Scoring System: prognostic classification
• Tab. 15.16-4 – Characteristic cytogenetic alterations in MDS, in comparison with AML.

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 /5, 6/. 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 /5, 9/ 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 /2, 3/.

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:

• Tab. 15.17-1 – Clinical characteristics and orienting markers in the differential diagnosis of MPN
• Tab. 15.17-2 – Methods at the time of diagnosis in chronic myeloproliferative neoplasia.

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:

• Tab. 15.17-1 – Clinical characteristics and orienting markers in the differential diagnosis of MPN.
• Tab. 15.17-2 – Methods at the time of diagnosis in chronic myeloproliferative neoplasia.

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

Polycythemia vera (PV) is one of the Philadelphia chromosome negative classical myeloproliferative neoplasms, a group of disorders that also encompasses primary myelofibrosis and essential thrombocythemia. PV is distinguished by an augmented erythocyte mass resulting from autonomous myeloproliferation. The concentration of erythropoietin is depressed.

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 /1, 2, 3/ (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 /1, 12/. 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 /2, 3, 4, 5/.

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 /1, 4, 6, 7, 8/. 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 /8, 9, 10, 11, 12, 40/.

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 /4, 7, 9, 10, 11, 40/.

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 /4, 20/.

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 /13, 14/:

• 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

Morphological 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) /1, 16, 17/. 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 /18, 19/ or of cytogenetic or molecular genetic abnormalities /1, 4/ 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 /14, 20, 21/ 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 /4, 21/:

• 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):

• Pro-B-ALL (high-risk therapy in the GMALL study)
• B-ALL (own protocol in the GMALL and ALL-BFM studies)
• T-progenitor cell ALL subtypes are treated variously (Tab. 15.18-2 – Terminology, frequency and phenotype of the immunological ALL subtypes).

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 /22, 24/. 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) /20, 36/.

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 /36, 37/.

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 /7, 27, 28, 38/.

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 /27, 38/. 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:

• Tab. 15.18-6 – Immunological marker profile of normal B cells and chronic lymphoproliferative diseases of the B cell type
• Tab. 15.18-7 – Immunological marker profile of normal T cells and T cell type lymphoproliferative diseases are not applicable in all cases. There is, at times, overlapping of immunophenotyping with various subtypes, which allows for definitive assignment only along with additional evaluation findings, such as clinical, morphological, histological and genotype data /1, 7, 11, 29, 30, 31/.

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 /33, 34/.

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.

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15.18.2 Myelodysplastic syndrome (MDS)

MDS is a heterogene myeloid clonal disease originating from hematopoietic stem cells, characterized by ineffective hematopoiesis, refractory hemocytopenia, and high risk of transformation to acute myeloid leukemia.