17.1 Thrombocyte diagnostics

Andreas Greinacher

17.1.1 Thrombocytes

Thrombocytes (platelets) are an essential component of primary hemostasis. They exert their hemostatic functions in a series of steps: adhesion to the subendothelial matrix, signal transmission, activation, release of granule contents, expression of a pro coagulant membrane phospholipid, and aggregation. Many different proteins and enzymes are also involved in these steps.

Primary disorders of hemostasis can result from a reduced thrombocyte count (thrombocytopenia) or thrombocyte dysfunction (thrombocytopathy). These disorders often present with a bleeding diathesis.

Abnormalities of thrombocyte count or function may be acquired or inherited. Acquired thrombocytopenias and thrombocytopathies are much more common than inherited disorders. Inherited disorders, however, provide important biological models to help explain thrombocyte function.

17.1.1.1 Thrombocytopenia

Thrombocytopenia can result from impaired production, increased destruction, increased consumption, or maldistribution. See Tab. 15.11-8 – Thrombocytopenia of various origins.

Impaired production

The most common cause of an acquired impairment of thrombocyte production is treatment with cytotoxic medication, followed by liver disease (often alcohol-related). Measles or other viral infections of megakaryopoiesis are commonly associated with clinically insignificant transient thrombocytopenia in childhood. Other viruses such as HIV and HCV can cause clinically significant thrombocytopenia.

Very rarely, thrombocytopenia is caused by impaired thrombocyte production in complex bone marrow disorders such as Fanconi anemia or by metabolic syndromes /1/.

Destruction

Patients who undergo massive blood transfusion without adequate thrombocyte substitution following severe hemorrhage are at risk of acquired thrombocytopenia due to increased thrombocyte destruction.

Consumption

Acquired thrombocytopenias caused by increased consumption include consumption coagulopathy (disseminated intravascular coagulation), immune thrombocytopenias, and thrombotic thrombocytopenic purpura.

Maldistribution

In patients with splenomegaly, the thrombocyte sequestration (pooling) in the spleen is increased.

Special forms

One example of a special form of thrombocytopenia is heparin-induced thrombocytopenia, which is associated with an increased risk of thromboembolic complications.

A diagnostic approach for clarification of thrombocytopenia is shown in Fig. 17.1-1 – Diagnostic approach to clarify a decreased thrombocyte count.

17.1.1.2 Thrombocytopathy

Thrombocytopathies are characterized by a bleeding diathesis that cannot be explained by a low thrombocyte count.

Drug induced

The use of nonsteroidal anti-inflammatory drugs (e.g., acetyl salicylic acid (ASA) and other anti platelet drugs such as clopidogrel is the most common cause of thrombocytopathy.

Impaired marrow production

Reduced thrombocyte function is common in myelodysplastic syndrome and leukemia. These diseases affect thrombocyte signal transmission and/or storage granules (storage pool diseases) in particular.

Pathological elevation of plasma proteins

This is responsible for the thrombocytopathies seen in amyloidosis and multiple myeloma. The plasma proteins inhibit thrombocyte function by binding to functionally important thrombocyte receptors /2/.

Essential thrombocytosis

This disorder may also be associated with thrombocytopathy. Affected patients present with a bleeding diathesis in spite of a pathologically increased thrombocyte count. The condition is caused by disorders of signal transmission or storage granules.

Immune thrombocytopathies with normal thrombocyte count

Although these disorders are rare, they may be associated with severe hemorrhagic complications as a result of antibodies blocking the function of important surface receptors on thrombocytes.

Inherited thrombocytopenias and thrombocyopathies

These disorders are characterized by familial clustering (dominant forms) and lifelong symptoms (dominant and recessive forms) /3/.

Point mutations in platelet glycoproteins

Some of these point mutations are associated with platelet alloantigens. Only larger epidemiological studies have demonstrated a mildly elevated risk of arterial thromboembolism. It is currently not possible to assess risk in individual cases.

A diagnostic approach for differentiation of suspected thrombocytopathy is shown in Fig. 17.1-2 – Differential diagnosis of suspected thrombocytopathy.

17.1.2 Indication

Decreased thrombocyte count

A decreased thrombocyte count always requires further investigation since it may be the first symptom of serious disease. Once treatable underlying diseases have been ruled out, diagnostic investigations focus on the clinical bleeding diathesis.

Thrombocytopathy

Thrombocytopathy must be ruled out in any patients with a bleeding diathesis that cannot be explained by a low thrombocyte count. It may also occur in association with thrombocytosis and moderate thrombocytopenias with thrombocyte counts of greater than 50 × 10⁹/L. Global coagulation parameters (PT, aPTT, and fibrinogen) are normal in these patients. If the patient is otherwise well, diagnostic tests should be performed in cases of chronic thrombocytopenia and thrombocytopathy. If the patient is acutely unwell, it is usually impossible to distinguish between chronic changes in the thrombocyte count and the effects of the acute illness.

Differential diagnosis

In cases of bleeding diathesis with normal global coagulation parameters, von Willebrand disease and hyperfibrinolysis (common in liver disease) must first be ruled out.

17.1.3 Method of determination

General clinical tests in suspected thrombocytopenia or thrombocytopathy include the differential, thrombocyte count, platelet volume, platelet distribution curve, and thrombocyte morphology in the blood smear.

17.1.4 Specimen

The basic parameters are determined using EDTA blood. Any abnormal findings should be checked using citrated blood. Blood smears for morphological examination should be prepared within 4 hours of blood collection.

17.1.5 Reference interval

• Thrombocyte count: depends on the hematology analyzer (150–450) × 10⁹/L (see Tab. 15.11-1 – Thrombocyte reference intervals).
• Platelet volume: depends on the methodology used, is approximately 10 fL.
• Platelet distribution width: distribution is semi-logarithmic.
• Differential blood smear, thrombocytes are individually visible, dark in color, and measure 1/3 to 1/5 the size of an erythrocyte.

17.1.6 Clinical significance

Combined evaluation of the thrombocyte count and morphology (blood smear) provides the following information:

• Thrombocyte aggregation indicates pseudo thrombocytopenia; if it is associated with a bleeding diathesis, it is suggestive of Montreal platelet syndrome
• The presence of large thrombocytes (which may be as large as erythrocytes) suggests a giant platelet syndrome such as Bernard-Soulier syndrome (recessive), MYH9-related thrombocytopenia (dominant), or type II B von Willebrand disease
• Alpha storage pool disease is characterized by pale, poorly stained thrombocytes (the most extreme form being gray platelet syndrome)
• Small, poorly stained thrombocytes are seen in Wiskott-Aldrich syndrome and a megakaryocytic thrombocytopenia
• The presence of very few thrombocytes in the blood smear despite a normal thrombocyte count as measured by the hematology analyzer suggests a glycoprotein IIb/IIIa glycoprotein deficiency (Glanzmann thrombasthenia), which inhibits thrombocyte adhesion to the glass slide.
• Large thrombocytes and Döhle bodies in granulocytes are typically seen in MYH-9 related syndromes.
• Erythrocyte anisocytosis occurs in X-linked thrombocytopenia with thalassemia /3/.

Decision-oriented clinical tests for thrombocytopenia and thrombocytopathy should be conducted in a specialist laboratory. To ensure an efficient diagnostic approach, disorders of thrombocyte function must be classified on the basis of clinical criteria and basic parameters.

The following sections describe decision-oriented clinical tests for thrombocytopenia and thrombocytopathy, in the following categories:

• Immune thrombocytopenias
• Heparin-induced thrombocytopenia
• Thrombocytopathies
• Inherited thrombocytopenia and thrombocytopathy.

17.1.7 Comments and problems

Pseudo thrombocytopenia

Pseudo thrombocytopenia can result in a falsely low thrombocyte count and is caused by:

• In vitro aggregation of thrombocytes. This occurs most commonly in EDTA blood but can also occur in citrated blood. It is most common in patients who have been treated with GPIIb/IIIa inhibitors.
• Formation of thrombocyte rosettes on leukocytes
• Abnormally large thrombocytes that are counted as erythrocytes or leukocytes by the hematology analyzer.

Pseudo thrombocytopenia is usually associated with increased platelet volume and a broad-based platelet distribution curve. It can only be reliably excluded by microscopic examination of the blood smear, which shows platelet aggregation at the feathered edge of the blood smear if pseudo thrombocytopenia is present.

Threshold values for determining the platelet volume are defined in the hematology analyzer. Thrombocytes whose volumes lie above or below these threshold values are not recorded. As a result, the platelet volume is underestimated when thrombocytes are significantly enlarged and overestimated when the thrombocyte size is significantly reduced.

Pseudo thrombocytosis

Fragmentation of red blood cells (RBC) can cause pseudo thrombocytosis. Automatic blood cell analyzers used in detecting platelet (PLT) count have a RBC/PLT channel. The channel is used for routine testing of RBC and PLT. In a reported case /4/ the true PLT number was 128 × 10⁹/L, however the measured count was 570 × 10⁹/L. Normally, the PLT peak and the RBC peak will not interfere with each other, but when the specimen has interference factors such as large PLT, aggregation or RBC fragments peaks will interfere each other.

Blood smear

Blood smears for morphological assessment should be prepared as soon as possible after blood collection. Thrombocytes can swell and become deformed following prolonged storage in EDTA. If this is suspected, tests should be repeated using citrated blood.

References

1. Kiefel V. Thrombozytenbildungs-, Abbau- und Verteilungsstörungen. Pötzsch B., Madlener K. (eds). Hämostaseologie, 2. Aufl. Heidelberg; Springer 2010: 306–315.

2. Bennett JS. Acquired platelet function defects. In: Gresele P, Page C, Fuster V, Vermylen J (eds). Platelets in thrombotic and non-thrombotic disorders. Cambridge; University Press 2002: 689–706.

3. Selleng K, Greinacher A. Thrombozytopathien. Pötzsch B., Madlener K. (eds). 2. Aufl. Heidelberg; Springer 2010: 325–334.

4. Tang W, Tang C, Zheng J, He Y, Lu F. Fragmentation of red blood cells caused pseudothrombocytosis in a patient. Clin Lab 2018; 64: 1071–4.

17.2 Autoimmune thrombocytopenia

Volker Kiefel

Anti-platelet antibodies can cause accelerated platelet destruction and thrombocytopenia. Depending on the origin of the antibodies, immune thrombocytopenia can be divided into:

• Autoimmune thrombocytopenia
• Drug-induced immune thrombocytopenia
• Alloimmune thrombocytopenia.

AITP may be idiopathic or secondary to other disorders, most commonly systemic lupus erythematosus or other autoimmune disorders and chronic lymphocytic leukemia. The simultaneous occurrence of (chronic) AITP and hemolytic anemia caused by warm-reactive antibodies is known as Evans syndrome. AITP can be categorized as follows based on its duration /1/:

• Newly diagnosed AITP (duration < months)
• Persistent AITP (duration 2–12 months)
• Chronic AITP (duration > 12 months)

Acute forms mainly occur as a result of viral infections in children and affect both sexes equally.

AITP is a diagnosis of exclusion that is often made on clinical grounds before the results of platelet antibody tests are available. The reasons are:

• Thrombocytopenia despite normal or increased megakaryopoiesis
• Absence of other (non-immune) causes such as toxic effects on hematopoietic cells or splenomegaly
• Most autoantibodies react equally frequently with monomorphic determinants on the GPIIb/IIIa and GP Ib/IX/V glycoprotein complexes.

Platelet autoantibodies have also been demonstrated in a series of female patients with cyclic thrombocytopenia, which suggests that at least some cases of this disorder have an immune basis.

In most patients, the autoantibodies induce thrombocytopenia but do not significantly affect thrombocyte function. This explains why, in younger patients in particular, the bleeding diathesis associated with low thrombocyte counts is often mild. Meanwhile, individual cases of severe, reversible thrombocytopathy caused by autoantibodies against GPIIb/IIIa have been reported (most with normal thrombocyte count). Some patients with acquired (antibody-induced) thrombasthenia alternate between phases of thrombasthenia and phases of immune thrombocytopenia.

17.2.1 Indication

Tests for anti-platelet antibodies to diagnose autoimmune thrombocytopenia should be performed in thrombocytopenic patients:

• With treatment-resistant thrombocytopenia of presumed autoimmune origin
• With suspected AITP before invasive treatment measures (splenectomy, immunosuppressant therapy)
• In association with hematological malignancy or solid tumors
• With delayed thrombocyte recovery following bone marrow or hematopoietic stem cell transplantation
• With hemolytic anemia (possible diagnosis: Evans syndrome)
• With SLE, rheumatoid arthritis, or other autoimmune disorders
• With HIV infection
• With cyclic thrombocytopenia
• In association wit acquired thrombocytic hemorrhagic diathesis following the exclusion of von Willebrand disease.

17.2.2 Method of determination

Tests are performed for:

• Circulating (free) platelet antibodies in the serum/plasma
• Antibodies bound to autologous thrombocytes

Free and cell-bound platelet antibodies can be determined by two methods:

• Tests that measure the total immunoglobulin load (mainly IgG) of thrombocytes
• Glycoprotein-specific assays that determine only those immunoglobulins that bind to the glycoprotein in question.

17.2.2.1 Membrane-bound antibodies

Tests used to determine glycoprotein platelet specific antibodies IgGs (GP-PAIgG) on autologous thrombocytes have proven to be the most reliable diagnostic method for determining membrane-bound autoantibodies. Most laboratories use in-house monoclonal antibody Immobilization of Platelet Antigen (MAIPA) assays /2, 3/. Alternatively, the acid eluates of autologous thrombocytes can be analyzed in an antiglobulin test /4/.

17.2.2.2 Free platelet-reactive antibodies

The Platelet Suspension Immunofluorescence Test (PSIFT) is used as a screening test to determine free platelet-reactive antibodies in the serum or plasma, regardless of their target antigens /5/. Because all autoantibodies detected by the PSIFT need to be distinguished from other antibodies that also react with thrombocytes, the glycoprotein-specific method is also in general use.

The diagnosis of an acquired, antibody-related thrombasthenia is substantiated by the demonstration of cell-bound and free antibodies to GPIIb/IIIa and by functional testing. To do this, the agonist-induced aggregation of antibody-coated platelets is measured using an aggregometer /6/.

17.2.3 Specimen

• Free platelet antibodies are determined in the serum and plasma. 10 mL of native blood without anticoagulant is required.
• Cell-bound antibodies. 20–30 mL of EDTA-anticoagulated blood is required for the determination. The determination is only possible if the thrombocyte count is at least 15 × 10⁹/L.

17.2.4 Reference interval

In general, qualitative assays are used to analyze free and cell-bound antibodies. Platelet autoantibodies to GPIIb/IIIa or GPIb/IX/V are not normally found in healthy individuals.

17.2.5 Clinical significance

Many patients with idiopathic AITP are diagnosed on the basis of clinical criteria alone. If a MAIPA is used, IgG determination has a diagnostic sensitivity of approximately 50% for autoimmune thrombocytopenia. In our laboratory, the assay has a diagnostic specificity of greater than 95% /3/. Comparable values for sensitivity and specificity are achieved by analyzing acid eluates of autologous thrombocytes in immunofluorescence assays using the method described in Ref. /3/. In contrast, the determination of free circulating antibodies against platelet GPIIb/IIIa and/or GPIb/IX/V has a sensitivity of only around 10%.

Autoantibodies on autologous thrombocytes indicate the presence of AITP with a high degree of certainty. However, a negative result does not rule out a diagnosis of AITP. Because determination of GPPAIgG is more conclusive, an attempt should always be made to obtain an adequate EDTA blood sample.

The demonstration of autoantibodies to the platelet receptors GP IIb/IIIa or GP Ib/IX/V in a series of female patients with cyclic thrombocytopenia supports an immunological origin for this disorder /7/.

In acquired disorders of thrombocyte function with a constellation of symptoms consistent with Glanzmann thrombasthenia, the demonstration of cell-bound and, possibly, free autoantibodies against GPIIb/IIIa provides evidence of immunological origin.

Because patients with true Glanzmann thrombasthenia (lack of an intact GPIIb/IIIa complex in the thrombocyte membrane due to a mutation) produce isoantibodies against GPIIb/IIIa following transfusion and pregnancy that react like autoantibodies against thrombocytes from any healthy individuals, and because this can sometimes affect thrombocyte function, (true) Glanzmann thrombasthenia must always be ruled out. Findings in thrombasthenia Glanzmann are: lack of intact GPIIb/IIIa complex on autologous thrombocytes; if free GPIIb/IIIa isoantibodies are present, they do not react with autologous thrombocytes.

17.2.6 Comments and problems

If free platelet-reactive antibodies are measured, it must be ensured that these are platelet autoantibodies and not alloantibodies (e.g., relatively common HLA class I antibodies that are not implicated in autoimmune thrombocytopenia).

Even if antibodies to GPIIb/IIIa or GPIb/IX/V are demonstrated, these are only confirmed as autoantibodies if they react with autologous thrombocytes (e.g., obtained at a later stage).

The detection of cell-bound autoantibodies is more clear-cut and can often help to confirm the diagnosis. Positive findings are unlikely in normal individuals or in patients with non-immune thrombocytopenia, with one important exception: the thrombocytes of patients treated with GPIIb/IIIa receptor antagonists (e.g., abciximab) are often positive for cell-bound GPIIb/IIIa-specific antibodies without autoimmune thrombocytopenia necessarily being present. When assessing the findings of autoantibody assays, therefore, it is crucial to establish whether the patient has taken abciximab or any other GPIIb/IIIa antagonists.

References

1. Rodeghiero F, Stasi R, Gernsheimer T 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.

2. Kiefel V, Santoso S, Weisheit M, Mueller-Eckhardt C. Monoclonal antibody-specific immobilization of platelet antigens (MAIPA): a new tool for the identification of platelet reactive antibodies. Blood 1987; 70: 1722–6.

3. Kiefel V. The MAIPA assay and its applications in immunohematology. Transfusion Medicine 1992; 2: 181–8.

4. Kiefel V, Freitag E, Kroll H, Santoso S, Müller-Eckhardt C. Platelet autoantibodies (IgG, IgA, IgM) against glycoproteins IIb/IIa and Ib/IX in patients with thrombocytopenia. Ann Hematol 1996; 72: 280–5.

5. von dem Borne AEGK, Verheugt FWA, Oosterhof F, von Riesz E, Brutel de la Riviere A, Engelfriet CP. A simple immunofluorescence test for the detection of platelet antibodies. British Journal of Haematology 1978; 39: 195–207.

6. Niessner H, Clementson KJ, Panzer S, Müller-Eckhardt C, Santoso S, Bettelheim P. Acquired thrombasthenia due to GP IIb/IIIa-specific autoantibodies. Blood 1986; 68: 571–6.

7. Cooper N, Ghanima W. Immune thrombocytopenia. N Engl J Med 2019; 381: 945–955.

17.3 Drug-induced immune thrombocytopenia

Volker Kiefel

Drug-induced immune thrombocytopenia is a relatively common disorder. Heparin-induced thrombocytopenia is particularly common and, unlike other forms of thrombocytopenia, is associated with an increased risk of thromboembolic complications. It is discussed in a separate section. The following classification is based on the immunological findings associated with other forms of drug-induced immune thrombocytopenia /1/:

• Drug-induced immune thrombocytopenia caused by drug-specific antibodies
• Immune thrombocytopenia mediated by autoantibodies
• Immune thrombocytopenia induced by GPIIb/IIIa inhibitors.

In cases caused by drug-specific antibodies, up to 7 days of exposure are usually required to produce thrombocytopenia in a patient given the drug for the first time. Unintentional re-exposure following previous immunization can produce thrombocytopenia within a few hours.

In most cases in which the relevant antibodies are detectable, thrombocytopenia is severe and associated with a significant bleeding diathesis. Drug-induced antibodies do not typically react with haptens; instead, they react specifically with a glycoprotein only in the presence of the triggering substance or its metabolites. The exact details of this reaction mechanism are still unknown.

Drug-dependent antibodies have similar glycoprotein specificities to autoantibodies; most react with GP IIb/IIIa or GPIb/IX/V. Unlike autoantibodies, they bind reversibly to the target antigen on the thrombocyte membrane only in the presence of the drug. When the concentration of the drug in the suspension is reduced, by washing, for example, they dissociate from the antigen. In vitro studies of the reaction of drug-specific antibodies have shown that the concentrations required are significantly higher than normal therapeutic concentrations. Drug-specific antibodies with different protein specificities are also found in cases of drug-induced immune hemolysis and immune neutropenia.

Occasionally, the serum of patients with a clinical diagnosis of drug-induced thrombocytopenia contains platelet autoantibodies that can react with thrombocytes in vitro in the absence of the drug. The ingestion of gold compounds in particular can lead to a secondary autoimmune thrombocytopenia. The antibodies found in these cases cannot be differentiated serologically from the autoantibodies produced in classical chronic AITP. A significant proportion of these gold-induced autoantibodies react with determinants on GPV /2/.

The use of GPIIb/IIIa inhibitors in cardiological interventions is becoming increasingly common. Immune thrombocytopenia is most frequently seen in association with the use of abciximab. It occurs in approximately 1% of patients following the first exposure and in 5% following re-exposure. A proportion of affected patients also develop a pseudo thrombocytopenia. Acute immune thrombocytopenias have also been observed following the use of tirofiban and eptifibatide, and delayed immune thrombocytopenias (after several days) are associated with xemilofiban and orbofiban. By now, many different substances have been linked to drug-induced immune thrombocytopenia /3/.

17.3.1 Indication

Tests for platelet antibodies should be performed in all cases of acute thrombocytopenia associated with the use of drugs.

Drug-induced immune thrombocytopenia is more likely when there is a particularly rapid decline in the thrombocyte count followed by recovery when the drug is withdrawn or where the thrombocyte count decreases again following unintentional re-exposure to the drug. In view of the often severe bleeding diathesis associated with drug-induced thrombocytopenia, intentional re-exposure does not generally occur.

In our laboratory, we demonstrated drug-specific antibodies associated with the substances listed in Tab. 17.3-1 – Drugs that can induce drug-specific antibodies and immune thrombocytopenia. In a laboratory with particular experience of these methods, an attempt to make a diagnosis would therefore seem worthwhile in these cases. However, it must always be assumed that any substance is capable of triggering immune thrombocytopenia.

When searching for possible causes of severe, etiologically unclear thrombocytopenia, it is important to remember that ingredients and additives in food and drink (quinine) may be responsible.

17.3.2 Method of determination

Antiglobulin tests are usually used to detect drug-specific serum antibodies /4/.

Because of their ability to activate complement in vitro, some drug-specific antibodies can also be detected using micro complement fixation tests.

Drug-induced autoantibodies can be detected using the same procedure as for AITP.

17.3.2.1 Detection of drug-specific antibodies

Principle: test thrombocytes from healthy blood donors are incubated in the serum sample for analysis, to which an adequate concentration of the presumed causative drug is also added. In our laboratory, we use an enzyme immunoassay with an alkaline-phosphatase-labeled goat anti-IgG to detect antibody binding (Fig. 17.3-1 – Principle underlying the detection of drug-specific antibodies in the antiglobulin test).

A control mixture without the drug (thrombocytes + patient serum) and a mixture that contains serum from a healthy individual instead of patient serum (thrombocytes + normal serum + drug) must also be run in addition to each actual reaction mixture (thrombocytes + patient serum + drug).

To confirm the presence of drug-specific antibody, the reaction mixture must be positive and both control samples must be negative.

17.3.2.2 Detection of autoantibodies and antibodies associated with the use of GPIIb/IIIa inhibitors

Autoantibodies are detected using the same methods as for autoimmune thrombocytopenia (AITP). If a glycoprotein-specific procedure is used for the investigation of gold-induced AITP, GPV should be included.

17.3.3 Specimen

• Native blood without anticoagulant: 10–20 mL
• EDTA blood: 10 mL

EDTA blood is required to simultaneously analyze drug-induced autoimmune thrombocytopenias as well as the phenomena associated with GPIIb/IIIa receptor antagonists (anti-GPIIb/IIIa antibodies on autologous thrombocytes, pseudothrombocytopenia).

17.3.4 Reference interval

Almost all of the procedures described for determining antibodies are semi-quantitative or qualitative.

17.3.5 Clinical significance

Drug-specific antibodies are regarded as specific for drug-induced immune thrombocytopenia (Tab. 17.3-1 – Drugs that can induce drug-specific antibodies and immune thrombocytopenia). The presence of autoantibodies, on the other hand, may indicate either autoimmune thrombocytopenia or drug-induced immune thrombocytopenia. A causal relationship between a particular drug and thrombocytopenia is supported by a close temporal relationship between the onset/resolution of thrombocytopenia and initiation/dis continuation of the drug.

Gold therapy can induce autoimmune thrombocytopenia and some substances that commonly induce drug-specific antibodies can also induce the production of autoantibodies.

The usefulness of determining antibodies that react with abciximab-coated thrombocytes in the diagnosis of abciximab-induced immune thrombocytopenia is debatable since these antibodies were detected in approximately 70% of healthy individuals. Because these antibodies are so prevalent, they are also unsuitable for predicting the occurrence of abciximab-induced immune thrombocytopenia.

17.3.6 Comments and problems

The detection of drug-specific antibodies is complicated by the fact that, in some cases, antibody reactions with thrombocytes are induced by metabolites of a drug rather than by the drug itself.

In the case of drugs whose metabolites are excreted renally, a urine sample from an individual who has taken the drug can be used as a metabolite preparation /5/.

In patients who have produced alloantibodies to platelet-associated antigens (e.g., HLA antibodies) the platelets used in tests that require intact platelets must be antigen-negative. If antigen-negative platelets are not available, glycoprotein-specific (MAIPA) assays must be used.

It is particularly important when determining drug-specific antibodies to perform a validation using positive and negative samples for a series of drugs.

References

1. Aster RH, Bougie DW: Drug-induced immune thrombocytopenia. N Engl J Med 2007; 357: 580–7.

2. Garner SF, Campbell K, Metcalfe P, Keidan J, Huiskes E, Dong JF, Lopez JA, Ouwehand WH. Glycoprotein V: the predominant target antigen in gold-induced autoimmune thrombocytopenia. Blood 2002; 100: 344–6.

3. George JG: Drug-Induced Thrombocytopenia – Database from Single Case Report – literature review through Oct 5, 2008. 2009; www.ouhsc.edu/platelets/DITP/Database_single/Database_single.htm.

4. Kiefel V. Differentialdiagnose der akuten Thrombozytopenie. Hämostaseologie 1999; 19: 30–41.

5. Kiefel V, Santoso S, Schmidt S, Salama A, Mueller-Eckhardt C. Metabolite-specific (IgG) and drug-specific antibodies (IgG, IgM) in two cases of trimethoprim-sulfamethoxazole-induced immune thrombocytopenia. Transfusion 1987; 27: 262–5.

17.4 Alloimmune thrombocytopenia

Volker Kiefel

Platelet membrane glycoproteins exist as genetically determined variants to which antibodies can be produced following the transfer of platelets during pregnancy or by transfusion. Platelet alloantibodies can induce clinically relevant thrombocytopenia in the following situations:

• Neonatal alloimmune thrombocytopenia
• Post-transfusion purpura
• Passive alloimmune thrombocytopenia
• Immune thrombocytopenia following transplantation.

Platelet alloantibodies can also accelerate the elimination of transfused platelets in thrombocytopenic patients, making platelet replacement more difficult.

Type I antigens (which are also expressed on blood and tissue cells other than thrombocytes) can be differentiated from type II antigens (which are specific to thrombocytes and megakaryocytes) based on their cellular and tissue distribution /1/. Type I antigens include ABH antigens and HLA class I antigens while type II antigens include immunogenic genetic variants of the glycoprotein complexes GPIIb/IIIa (α_IIbβ3 integrin), GPIb/IX, GPIa/IIa (α₂β₁ integrin), GPIV, and CD109 /1/.

Nomenclature

Newly discovered antigens are named using abbreviations derived from the surnames of immunized patients. Following comprehensive analysis, alloantigens or antigen systems are assigned a number in the HPA (human platelet antigen) nomenclature system. The most frequent allele of a system is assigned the letter “a”, while the less frequent allele is assigned the letter “b”.

17.4.1 Indication

Tests should always be performed for platelet-specific (allo)antibodies in the following situations:

• All etiologically unclear thrombocytopenias that are not simply a result of reduced thrombocytopoiesis, excessive thrombocyte sequestration (pooling) in the spleen, or complex coagulopathies (disseminated intravascular coagulation, TTP)
• Patients who develop a febrile transfusion reaction in response to platelet transfusions and in whom transfusion fails to achieve an adequate, dose-related increase in the thrombocyte count
• In neonatal thrombocytopenia for which no obvious non-immunological cause exists
• Acute post-transfusion thrombocytopenia.

17.4.2 Method of determination

Detection of specific anti platelet antibodies can be made using the indirect platelet immunofluorescence test or the monoclonal antibody immobilization platelet antigen (MAIPA) test.

17.4.2.1 Platelet immunofluorescence test

Principle

Test thrombocytes from healthy individuals are fixed in formaldehyde solution and incubated with the sera or eluates for analysis and fluorescent labeled anti-IgG (anti-IgM, anti-IgA) antibodies. Two technical variants exist; the Platelet Adhesion Immunofluorescence Test (PAIFT) /2/ and the Platelet Suspension Immunofluorescence Test (PSIFT) /3/. In the PSIFT, the quantity of platelet-bound antibody can be determined qualitatively using fluorescence microscopy or (semi-quantitatively) using flow cytometry.

17.4.2.2 Platelet ELISA

Principle

Test thrombocytes (usually in suspension) are incubated with the patient serum for analysis. Antibody binding to thrombocytes is detected following a sensitization phase by the use of enzyme-labeled anti-IgG (IgM or IgA) antibodies /4/.

17.4.2.3 MAIPA, immunobead assay

In most laboratories, in-house (MAIPA assay) or commercially available glycoprotein-specific assays are used to detect platelet antibodies.

Principle

The MAIPA /5, 6/ and immunobead assay /7/ are glycoprotein-specific methods in which the serum for analysis is incubated with intact thrombocytes. The sensitized test thrombocytes are solubilized and individual glycoproteins or glycoprotein complexes are fixed to a solid phase (e.g., the well of a micro titer tray or immunobeads) in separate batches with monoclonal antibodies. Binding of human antibody to the glycoprotein is detected by the use of enzyme-labeled anti-IgG. Currently, most laboratories use the MAIPA assay to diagnose thrombocytopenic patients.

17.4.2.4 Detection of platelet alloantigens

Principle

Platelet alloantigens are detected using molecular biological methods. This involves performing PCR with sequence-specific primers (PCR-SSP) or analyzing the restriction fragment length polymorphism of PCR products of genomic DNA (PCR-RFLP). Platelet alloantigens can still be determined immunologically in cases of doubt or for reference purposes. In this case, the alloantibody reaction is usually determined using the MAIPA assay. The basic molecular biological characteristics of the main platelet antigens are provided in Tab. 17.4-1 – Molecular biological characteristics of the main platelet alloantigens.

17.4.3 Specimen

• Determination of alloantibodies: serum 4 mL; additional sample is required in suspected post-transfusional purpura: EDTA blood for determination of antibodies to GPIIb/IIIa and the antigen (HPA 1)
• Antigen determination using molecular biological methods: approximately 1 mL of citrate or EDTA blood
• Cross match before thrombocyte transfusion in an alloimmunized patient: 5–10 mL EDTA blood from donor and serum from patient (transfusion recipient)
• Suspected case of neonatal alloimmune thrombocytopenia (NAIT): 10 mL maternal EDTA blood and 10 mL maternal native blood without anticoagulant (antibody detection, antigen determination); 10 mL paternal EDTA blood (cross match, antigen determination); and 0.5–1 mL EDTA blood from the neonate (antigen determination, thrombocyte count).

In screening tests of serum and plasma samples that analyze the non-antigen-specific binding of platelet-reactive antibodies to intact thrombocytes (antiglobulin tests with intact thrombocytes: PIFT, platelet ELISA), common alloantibodies against HLA class I antigens in particular interfere with the determination of the specificity of platelet-specific antibodies (e.g. anti-HPA-1b). In cases where the reaction pattern on the cell panel does not provide a clear result, a glycoprotein-specific test (MAIPA) must be used for further analysis.

Antiglobulin tests with intact thrombocytes can be used to check immunological compatibility before platelet transfusions. If a cross match test is positive, the serological specificity of the platelet antibodies must be determined in order to select compatible platelet donors.

17.4.4 Reference interval

Almost all of the procedures described for determining antibodies to alloantigens are semi-quantitative or qualitative. Alloantibodies are not usually found in individuals who have never received a blood transfusion or women who have never been pregnant.

17.4.5 Clinical significance

Diseases and clinical constellations associated with thrombocytopenia in which platelet-specific antibodies can be detected are listed in Tab. 17.4-2 – Platelet-specific alloantibodies in thrombocytopenias.

17.4.5.1 Fetomaternal alloimmune thrombocytopenia (FMAIT)

FMAIT occurs when a women becomes alloimmunized against fetal platelet antigens inherited from the fetus’s father (which are absent on maternal platelets), leading to fetal thrombocytopenia ( below 150 × 10⁹/L) /8/. FMAIT is triggered by a maternal platelet-specific alloantibody, most frequently anti-HPA-1, less commonly anti-HPA-5b, and rarely by other antibodies (Tab. 17.4-3 – Phenotype frequencies of the main platelet alloantigens). Although HLA antibodies (produced by around 20–30% of pregnant women) also react with thrombocytes, they are less likely to induce FMAIT. To rule out the presence of an antibody to a low-frequency alloantigen (Tab. 17.4-4 – Low-frequency alloantigens) in suspected FMAIT, a serological cross match of maternal serum against paternal platelets must be performed using a MAIPA assay.

FMAIT is a relatively rare condition with a prevalence of 0.9% in unselected populations. FMAIT is the most common cause of severe thrombocytopenia in the newborn accounting 3% of all fetal and neonatal thrombocytopenia and 27% of severe cases (below 50 × 10⁹/L or intracranial hemorrhage). Most FMAIT cases are mild, with evidence of widespread petechiae and/or skin lesions /8/.

17.4.5.2 Post-transfusion purpura

In all cases of post-transfusion purpura a highly reactive antibody against an alloantigen on GPIIb/IIIa, most commonly anti-HPA-1a, can be detected in the serum of the patient (Tab. 17.4-2 – Platelet-specific alloantibodies in thrombocytopenias). GPIIb/IIIa on the (alloantigen-negative) autologous thrombocytes is typically loaded with IgG. When these antibodies are removed (eluted), anti-HPA-1a can be detected in the eluate. If the thrombocyte count decreases immediately following transfusion of a plasma-containing blood product, a suspected diagnosis of passive alloimmune thrombocytopenia (Tab. 17.4-2) can be confirmed by demonstrating platelet-specific antibodies in the plasma of the blood product or the blood of the donor.

17.4.6 Comments and problems

In screening tests of serum and plasma samples that analyze the non-antigen-specific binding of platelet-reactive antibodies to intact thrombocytes (antiglobulin tests with intact thrombocytes: PIFT, platelet ELISA), common alloantibodies against HLA class I antigens in particular interfere with the determination of the specificity of platelet-specific antibodies (e.g. anti-HPA-1b). In cases where the reaction pattern on the cell panel does not provide a clear result, a glycoprotein-specific test (MAIPA) must be used for further analysis.

Antiglobulin tests with intact thrombocytes can be used to check immunological compatibility before platelet transfusions. If a cross match test is positive, the serological specificity of the platelet antibodies must be determined in order to select compatible platelet donors.

Antiglobulin tests with intact thrombocytes (PIFT, platelet ELISA) cannot reliably detect antibodies to antigens of the HPA-5 system due to the low antigen density. The use of glycoprotein-specific assays that use monoclonal antibodies to analyze serum can produce false positive results due to the reaction of antibodies in the serum of the patient with mouse immunoglobulins. This can be avoided in the MAIPA assay by configuring the assay appropriately /5/. Occasionally, serological typing of alloantigens on GPIIb/IIIa identifies an individual as homozygous while DNA typing indicates that the individual is heterozygous. This is presumably due to non-expression of a glycoprotein caused by a gene mutation. In this constellation, only the result of serological typing should be used to assess the immunological situation.

References

1. Kiefel V, Santoso S: Alloantigene auf Thrombozyten; in Kiefel V (ed.): Transfusionsmedizin und Immunhämatologie. 4. ed. Berlin, Springer 2010; 177–87.

2. von dem Borne AEGK, Verheugt FWA, Oosterhof F, von Riesz E, Brutel de la Riviere A, Engelfriet CP. A simple immunofluorescence test for the detection of platelet antibodies. British Journal of Haematology 1978; 39: 195–207.

3. Schneider W, Schnaidt M. The platelet adhesion immunofluorescence test: a modification of the platelet suspension immunofluorescence test. Blut 1981; 43: 389–92.

4. Kiefel V, Santoso S. Nachweis von thrombozytären Antigenen und Antikörpern. In: Mueller-Eckhardt C (ed). Transfusionsmedizin. Grundlagen, Therapie, Methodik. Berlin; Springer 1996: 597–602.

5. Kiefel V. The MAIPA assay and its applications in immunohematology. Transfusion Medicine 1992; 2: 181–8.

6. Kiefel V, Santoso S, Weisheit M, Mueller-Eckhardt C. Monoclonal antibody-specific immobilization of platelet antigens (MAIPA): a new tool for the identification of platelet reactive antibodies. Blood 1987; 70: 1722–6.

7. McMillan R, Tani P, Millard F, Berchtold P, Renshaw L, Woods VL. Platelet-associated and plasma anti-glycoprotein autoantibodies in chronic ITP. Blood 1987; 70: 1040–5.

8. Espinoza JP, Caradeux J, Norwitz ER, Illanes SE. Fetal and neonatal alloimmune thrombocytopenia. Rev Obstet Gynecol 2013; 6: e15–e21.

17.5 Heparin-induced thrombocytopenia

Andreas Greinacher

Heparin-induced thrombocytopenia (HIT, also known as type II HIT) is a prothrombotic condition caused by anticoagulants that lead to intravascular activation of thrombocytes by heparin-induced antibodies. This results in thrombocytopenia and increased thrombin formation. Affected patients are at increased risk of new venous and arterial occlusions /1/.

HIT can be viewed as a clinicopathologic syndrome with clinical symptoms and antibody production. It typically occurs between days 5 and 14 of beginning heparin treatment, by which time the immune system has produced sufficient quantities of antibodies, but can sometimes occur earlier if the patient has been treated with heparin within the previous three months.

The thrombocyte count usually falls abruptly by more than 50%. Following surgery (major surgery in particular), a reactive thrombocytosis develops from postoperative day 4–5 and reaches a peak between days 10 and 14. This is why the highest thrombocyte count after the start of heparin therapy rather than the pre-treatment thrombocyte count should be used to assess the relative decrease in the thrombocyte count in postoperative patients.

The thrombocyte count usually decreases to 40–80 × 10⁹/L; however, in around 10% of patients it does not fall below 150 × 10⁹/L; in fewer than 10% of cases, it falls to below 20 × 10⁹/L (a consumption coagulopathy is usually also present in these cases).

Paradoxically, hemorrhage is rare in HIT; vascular occlusion, however, is common, occurring in some 50–75 percent of cases. If this is then treated by increasing the heparin dose, antibody-dependent thrombocyte activation is intensified and serious thromboembolic complications may result. Early diagnosis and new treatment options have significantly reduced the complication rate associated with HIT (mortality 6–7%, amputation rate 5–6%).

Delayed-onset HIT is a special case. Patients with delayed-onset HIT typically present with acute thromboembolic complications and thrombocytopenia several days after discharge from hospital and cessation of heparin therapy. High titers of heparin-induced autoantibodies are present /2/. Therefore, to identify possible delayed-onset HIT, the thrombocyte count should be determined in all patients who present with acute thromboembolic events within 14 days of receiving heparin.

Platelet factor 4 (PF4) is the main target antigen for HIT antibodies /3/. Heparin binding to PF4 leads to the exposure of crypt antigens or autoantigens. IgG-type antibodies are the most common antibodies seen in symptomatic HIT patients. Anti-PF4/heparin antibodies of type IgM or IgA are unlikely to be clinically significant.

17.5.1 Indication

The best way to ensure that HIT is recognized at an early stage is to determine the thrombocyte count regularly, especially during treatment with unfractionated heparin (UFH).

The screening of asymptomatic patients for HIT antibodies is not indicated. The main purpose of the in vitro detection of HIT antibodies is to rule out a provisional clinical diagnosis of HIT. In around 85% of patients with suspected HIT, a negative antibody test excludes the diagnosis. This should always be carried out since it also has implications for future treatment. Reliable detection of HIT antibodies is only possible for a period of a few weeks. Laboratory diagnostics must therefore be carried out as soon as possible after a decline in the thrombocyte count is observed.

17.5.2 Method of determination

Functional assays

Functional assays include the serotonin release assay and heparin-induced platelet activation assay (HIPA). These assays detect HIT antibodies (IgG only) against a range of antigens.

Principle: washed thrombocytes from healthy donors are incubated with serum from the patient at low (0.2 IU/mL) and high (100 IU/mL) heparin concentrations. In HIT, thrombocytes are typically only activated at low (but not at high) heparin concentrations. This activation is measured by the release of radioactive serotonin (serotonin release assay) or determined visually (HIPA assay).

Assays that use platelet-rich plasma (aggregometry) have low detection limit and should not be used to detect HIT antibodies /5/

Antigen assays

These assays detect the binding of antibody (IgG, IgA, IgM) in serum from patients to PF4-heparin complexes or polyvinyl sulfate complexes, but not to other antigens. Several ELISA assays as well as an assay that detects agglutinated colored PF4/heparin coated beads in a gel card system are available commercially /5/.

17.5.3 Specimen

HIT antibodies should be determined in serum (5–10 mL native blood). Pseudo thrombocytopenia must always be ruled out (EDTA blood).

17.5.4 Reference interval

After starting heparin therapy a decline in the thrombocyte count below 50% indicates HIT. Because heparin-specific antibodies can also be detected in patients without clinical HIT, clinical information must always be incorporated into the assessment of laboratory findings.

17.5.5 Clinical significance

The simplest way to detect HIT at an early stage is to determine the thrombocyte count. The required frequency of testing during heparin treatment depends on the clinical situation (Tab. 17.5-1 – Monitoring of thrombocyte count patients with risk of developing heparin-induced thrombocytopenia) /4/.

Functional assays and antigen assays for HIT antibodies are well standardized. Although functional assays and antigen assays have similarly high diagnostic sensitivity for clinically manifest HIT, antigen tests have a lower detection limit for measuring HIT antibodies in asymptomatic patients. Therefore, functional assays have a higher positive predictive value for clinically manifest HIT. To achieve an optimal accuracy, an antigen assay should be combined with a functional assay for confirmation. This minimizes the risk of false negative findings to below 5%.

Because of the need to combine different types of assays in unclear cases and to exclude other differential diagnoses, it makes sense for hospitals to rule out suspected HIT rapidly using an antigen assay, to define a treatment concept for HIT in the event of a positive screening test, and to carry out confirmatory diagnostics in collaboration with a reference laboratory.

Reference laboratories should be able to offer at least one sensitive functional assay and one antigen assay.

The pathogenesis of HIT is shown in Fig. 17.5-1 – Pathogenesis of HIT.

17.5.6 Comments and problems

Functional assays demand a high level of experience from users. Their sensitivity depends on the platelet donors selected. Insensitive thrombocytes or technically unsatisfactory thrombocyte washing methods can produce false negative findings.

If serum from the patient has not been adequately inactivated, thrombin can produce false positive results.

If the inactivation temperature is too high, immune complexes can produce false positive reactions.

In assays that use platelet-rich plasma, sera from critically ill patients can trigger false positive reactions. In a ring trial, only highly experienced laboratories achieved acceptable results in functional assays while even non-specialist laboratories demonstrated comparable results in antigen assays /6/.

Weak or moderate reactions in sensitive antigen assays do not prove the diagnosis of HIT.

References

1. Warkentin TE. Clinical picture of heparin-induced thrombocytopenia. In: Warkentin TE, Greinacher A (eds). Heparin-induced thrombocytopenia, 4th ed. New York; Informa Health Care 2007: 21–66.

2. Warkentin TE, Kelton JG. Delayed-onset heparin-induced thrombocytopenia and thrombosis. Ann Intern Med 2001; 135: 502–6.

3. Greinacher A, Poetzsch B, Amiral J, Dummel V, Eichner A, Mueller-Eckhardt. Heparin-associated thrombocytopenia: isolation of the antibody and characterization of a multimolecular PF4-heparin complex as the major antigen. Thromb Haemost 1994; 71: 247–51.

4. Greinacher A, Lubenow N, Hinz P, Ekkernkamp A. Die Heparin-induzierte Thrombozytopenie. Deutsches Ärzteblatt 2003; 100: A2220–9.

5. Warkentin TE, Greinacher A. Laboratory testing for heparin-induced thrombocytopenia. In: Warkentin TE, Greinacher A (eds). Heparin-induced thrombocytopenia, 4th ed. Informa Health Care 2007: 227–260.

6. Eichler P, Budde U, Haas S, et al. First workshop for detection of heparin-induced antibodies: validation of the heparin-induced platelet-activation test (HIPA) in comparison with a PF4/heparin ELISA. Thromb Haemost 1999; 81: 625–9.

17.6 Thrombocytopathy

Andreas Greinacher

Thrombocytopathies are characterized by a bleeding diathesis with normal coagulation parameters (PT, aPTT, fibrinogen) due to impaired thrombocyte function. Typical clinical symptoms include mucocutaneous hematomas, increased bleeding following dental extractions, and hemorrhagic postoperative complications. Menorrhagia, often leading to iron-deficiency anemia, is a cardinal symptom in women.

17.6.1 Indication

Advanced thrombocyte function tests should be performed in patients with chronic bleeding diathesis and normal global coagulation parameters, especially if there is familial clustering of symptoms.

Extended diagnostics should not be performed during an acute (perioperative) hemorrhage. Symptomatic treatment of bleeding takes priority in this situation.

If thrombocyte morphology is normal on the blood smear, drug effects, von Willebrand disease, and F XIII deficiency should be ruled out first. If thrombocyte aggregation is also normal, hyper fibrinolysis should be ruled out /1/.

17.6.2 Specimen

With the exception of the bleeding time, functional and morphological investigations are performed using recently collected citrated blood. For quantitative glycoprotein determinations in the flow cytometer, EDTA blood or citrated blood can be used. EDTA blood is used for genetic investigations. It is now also possible to examine platelet proteins in the blood smear.

17.6.3 Tests

For the evaluation of platelet-related hemostasis different tests are used /2, 3/.

17.6.3.1 Bleeding time

Principle: the Ivy method is the traditional method for determining the bleeding time:

• The skin on the underside of the forearm is first cleaned and disinfected
• Once the disinfectant is completely dry, a blood pressure cuff is placed on the upper arm and inflated to exactly 40 mmHg. This pressure should be maintained throughout the procedure.
• A spring-loaded lancet with two 0.5 cm blades is used to make two 1 mm deep incisions
• Every 30 seconds, filter paper is used to draw off the blood without touching the edge of the wound. The time until bleeding stops is measured. The reference interval for this bleeding time is 4.5–8 minutes.

Clinical significance

Bleeding time measurement has been criticized for its poor reproducibility. It is also highly subjective and is not used routinely.

17.6.3.2 In vitro bleeding time

Principle

In the platelet function analyzer (PFA 100) device, citrated blood is aspirated through a capillary tube and collagen or epinephrine-coated surface. The time until closure of the capillary tube is measured.

Clinical significance

The capillary tube, device, and method are standardized. It is a simple procedure that is highly sensitive for detecting certain disorders of primary hemostasis but it cannot distinguish between the effects of anti-platelet drugs, von Willebrand disease, and thrombocytopathies. Furthermore, it does not detect all disorders of thrombocyte function. Therefore, a thrombocytopathy cannot be ruled out on the basis of a normal PFA 100 test.

Comments and problems

The PFA 100 test is highly dependent on pre analytic factors and blood composition. A hematocrit of less than 0.35 or a thrombocyte count of less than 100 × 10⁹/L have been shown to affect the results obtained with this test. This PFA 100 test cannot be used to distinguish between drug-induced thrombocytopathy and other disorders of thrombocyte function.

17.6.3.3 Aggregometry

Principle

The study of thrombocyte aggregation using the Born method is the most widely used method for testing thrombocyte function:

• Differential centrifugation is used to obtain platelet-rich plasma from citrated blood
• Thrombocyte aggregation is measured turbidimetrically by recording changes in the light transmission of the platelet-rich plasma over time following the administration of various platelet agonists
• The increase in light transmission is directly proportional to thrombocyte aggregation.

Clinical significance

Platelet shape change at the start of measurement, speed of reaction, and maximum aggregation are assessed.

• ADP-dependent aggregation: this is impaired in ADP receptor deficiencies, severe forms of delta storage pool disease, signal transduction disorders, and as a result of taking certain drugs (ASA, clopidogrel). With the exception of the rare receptor defects, normal aggregation can be restored by the addition of high concentrations of ADP.
• Collagen-induced aggregation: at low concentrations, this is dependent on arachidonic acid metabolism, including cyclooxygenase function; at high concentrations, it is dependent on glycoproteins GPIa/IIa and GPIV.
• Ristocetin-induced thrombocyte agglutination: the agglutination does not occur in Bernard-Soulier syndrome. It is in increased in type IIb von Willebrand disease and platelet-type von Willebrand disease (ristocetin concentrations of less than 0.3 mg/mL). In functional defects of glycoprotein complex IIb/IIIa, thrombocyte aggregation does not occur or is severely reduced but agglutination can still be triggered by ristocetin.
• Epinephrine-induced aggregation: this is reduced in storage pool diseases, signal transduction disorders, and as a result of taking anti platelet drugs.

Some typical aggregometry findings are shown in Tab. 17.6-1 – Typical aggregometry findings associated with thrombocytopathies.

Comments and problems

Aggregometry is poorly standardized. ISTH guidelines should be followed /4/. The method is dependent on how the thrombocytes are obtained for analysis. High shear stress during blood collection and marked fluctuations in temperature, in particular chilling and prolonged storage, can produce analytical artifacts. The test should be performed within 4 hours of blood collection. At low thrombocyte counts, depending on the aggregometer, less than (40–80) × 10⁹/L, aggregometry is no longer meaningful.

If thrombocyte size is increased, spontaneous sedimentation rather than differential centrifugation must be used to obtain platelet-rich plasma in order to prevent large thrombocytes collecting in the erythrocyte fraction. A control sample with normal thrombocytes must also be tested in all cases.

17.6.3.4 Impedance aggregometry and lumi-aggregometry

Principle

The electrical impedance of whole blood is analyzed turbidimetrically following the administration of platelet agonists. See Section 17.6.3.3– Aggregometry. The change in impedance measured at a conductor in the sample is directly proportional to thrombocyte aggregation. Two methods are available: the Multi plate system and the lumi-aggregometer. In a lumi-aggregometer assay, the blood sample is incubated with a luciferin-luciferase reagent. ATP released by the dense granules of thrombocytes oxidizes this reagent to produce a chemiluminescent reaction. The intensity of the chemilumiscence is proportional to the quantity of dense granules (e.g., in delta storage pool disease).

Clinical significance

This method bypasses the need to isolate platelet-rich plasma and is better standardized than aggregometry. Because, unlike in the PFA 100 test, any agonists can be used, this method is more accessible and flexible. The release of the contents of dense granules can be analyzed using lumi-aggregometry.

Comments and problems

This method can only be performed by specialist laboratories.

17.6.3.5 Verify Now

Principle

Verify Now is a point of care test that measures thrombocyte activation in citrated whole blood that is inserted into a special cartridge. The system was originally developed to assess thrombocyte function in the cardiac catheterization laboratory. The Verify Now^R device is validated to measure ADP-dependent thrombocyte activation. It can be used to measure the effects of P2Y12 (e.g., clopidogrel, prasugrel) on ADP receptors.

Clinical significance

The device is easy to use and provides an initial assessment of the effects of anti platelet drugs on in vitro activation. The clinical relevance of these results is currently the subject of a number of prospective studies.

Comments and problems

The therapeutic implications of the test results have not yet been established.

17.6.3.6 Immunofluorescence

Principle

Thrombocytes fixed on glass or suspended in solution are incubated with directly (or indirectly) labeled monoclonal antibodies. After washing, the immunofluorescence is assessed microscopically.

Clinical significance

Immunofluorescence is a simple and sensitive method for assessing the expression and distribution of membrane-bound and intracellular platelet proteins. Immunofluorescence is particularly useful for detecting granulocyte inclusion bodies in MYH 9-related thrombocytopenia.

The major advantages of this method are the small blood samples required and the stability of the blood smears, which can be stored at room temperature for several days and sent by mail, for example.

MYH-9 related platelet disorders belong to the group of inherited giant platelet disorders. The MYH-9 gene encodes the non-muscular myosin heavy chain IIA (NMMHCIIA), a cytoskeletal contractile protein. Several mutations in the MYH-9 gene lead to macro thrombocytopenia, and cytoplasmic inclusion bodies within leukocytes, while the number of megakaryocytes in the bone marrow is normal /6/.

Comments and problems

Thrombocytes that are fixed to a glass surface are always activated. In order to stain intracellular structures, the thrombocyte membrane must be permeabilized. The method for doing this is not standardized and can only be performed by specialist laboratories at present.

17.6.3.7 Flow cytometry

Principle

Whole blood or platelet-rich plasma is incubated with fluorescent labeled monoclonal antibodies to platelet proteins and the signal strength is measured in the flow cytometer. Fixed thrombocytes are used for quantitative determination of platelet proteins. In a modified procedure, thrombocytes are incubated with mepacrine and the innate fluorescence of the mepacrine is measured. The fluorescence is directly proportional to the amount of mepacrine stored in the dense granules. Flow cytometry can also be used to test thrombocyte function. In this case, the expression of activation markers following incubation with thrombocyte activators is measured over time.

Clinical significance

Flow cytometry is a suitable method for detecting significant quantitative deficiencies in thrombocyte membrane proteins and delta storage pool disease. It can be used to measure thrombocyte function using relatively small blood samples, even in children.

Comments and problems

The detection of minor reductions in platelet glycoproteins and the assessment of thrombocyte function and activation are highly dependent on pre analytic factors related to sample preparation and can only be performed by specialist laboratories. The presence of a glycoprotein does not rule out a functional defect of the protein /5/.

17.6.3.8 Release of thrombocyte contents and metabolites

Principle

Activated and non-activated thrombocytes are centrifuged and the concentrations of the released contents and metabolites are measured. This can be done using ELISA (PF4, β-thromboglobulin) or the luciferin-luciferase method (ADP, ATP), or by adding radio labeled substances before the reaction and measuring their release (serotonin) or their incorporation into metabolites (thromboxanes).

Clinical significance

These methods are poorly standardized and can only be performed by specialist laboratories /5/. A more straightforward approach involves measuring thromboxane A₂ metabolites (e.g., TXB₂) in the urine. Commercial assay systems are available for this purpose.

17.6.3.9 Genetic investigation of mutations in platelet proteins

Principle

These investigations use standard molecular biology methods. Sequence-specific amplification (SSP-PCR) or restriction enzyme digestion (RFLP-PCR) are used to detect single nucleotide polymorphisms. These methods are commercially available, standardized, and can be automated.

Clinical significance

Molecular biological investigations of thrombocyte genes are used:

• In the diagnosis of alloimmune thrombocytopenias
• To determine compatible donors for immunized patients.

The investigation of individual platelet glycoprotein polymorphisms is not suitable for estimating an individual’s risk of cardiovascular thromboembolic complications.

Hereditary thrombocytopenias and thrombocytopathies are caused by a range of different mutations of the respective genes. It is usually only possible to identify the genetic defect by sequencing the entire gene in question. Although demonstration of the genetic defect confirms the diagnosis, molecular biological investigations still have only a secondary role in the diagnostic workup of thrombocytopenia and thrombocytopathy.

17.6.3.10 Acute intracranial bleeding

Each year millions of people suffer acute intracranial hemorrhage that originates from various causes such as traumatic brain injury, spontaneous intracerebral bleeding, subarachnoid hemorrhage, and isolated intraventricular hemorrhage. About 29% of patients had clinically relevant platelet dysfunction /7/. Of those 60% were treated with antiaggregatory medication at the time of study inclusion. Platelet function analysis used impedance aggregometry and lumi-aggregometry.

References

1. Greaves M, Preston FE. Approach to the bleeding patient. In: Gresele P, Page C, Fuster V, Vermylen J (eds). Platelets in thrombotic and non-thrombotic disorders. Cambridge; Cambridge University Press 2002: 783–94.

2. Thiagarjon P, Wu K. In vitro assays for evaluating platelet function. In: Gresele P, Page C, Fuster V, Vermylen J (eds). Platelets in thrombotic and non-thrombotic disorders. Cambridge; Cambridge University Press 2002: 459–70.

3. Karon BS, Tolan NV, Koch CD, Wockenfus AM, Miller RS, Lingineni RK, et al. Precision and reliability of 5 platelet function tests in healthy volunteers and donors on daily antiplatelet agent therapy. Clin Chem 2014; 60: 1524–31.

4. Cattaneo M, Hayward CP, Moffat KA, Pugliano MT, Liu Y, Michelson AD. Results of a worldwide survey on the assessment of platelet function by light transmission aggregometry: a report from the platelet physiology subcommittee of the SSC of the ISTH. J Thromb Haemost 2009; 6: 1029.

5. Shantsila E, Watson T, Lip GYH. Laboratory investigation of platelets. In: Gresele P, Page C, Fuster V, Lopez JA, Page CP, Vermylen J (eds). Platelets in hematologic and cardiovascular disorders. Cambridge; Cambridge University Press 2008: 124–46.

6. Althaus K, Greinacher A. MYH-9 related platelet disorders: strategies for management and diagnosis. Transfus Med Hemother 2010; 37: 260–7.

7. Warmuth H, Zacharowski K, Seifert V, Konczalla J, Weber CF, Mutlak H. Reduced platelet activity in patients with acute intracranial hemorrhage. Clin Lab 2021; 67: 1846–52.

17.7 Inherited platelet-based bleeding disorders

Andreas Greinacher

Mutations in the genes for functionally important thrombocyte structures can lead to thrombocyte dysfunction. Some of these mutations have been clarified at a genetic level but many apparently genetic disorders of thrombocyte function have only been described phenotypically to date.

Inherited disorders of thrombocyte function are classified into /1, 2/:

1. Inherited thrombocytopenias:

• Disorders of megakaryocytopoiesis
• Disorders of thrombocyte production with normal megakaryocyte count.

2. Inherited thrombocytopathies:

• Disorders of receptors for adhesive proteins
• Disorders of receptors for soluble agonists
• Storage granule disorders
• Signal transduction disorders
• Disorders of pro coagulant phospholipids
• Miscellaneous disorders.

Since platelet proteins affect both thrombocyte morphology and function, it is not always possible to distinguish clearly between thrombocytopenia and thrombocytopathy.

Inherited thrombocytopenias are relatively rare but probably under diagnosed /1/. An overview of inherited thrombocytopenias and thrombocytopathies is shown in Tab. 17.7-1 – Hereditary thrombocytopenias and thrombocytopathies. Inherited thrombocyte abnormalities are associated with a bleeding diathesis. Patients typically have an increased tendency to hematoma formation. They are also at risk of severe hemorrhagic complications during and following surgery. The severity of the clinical symptoms depends on the particular functional abnormality /3/.

The autosomal dominant thrombocytopenias with giant thrombocytes are a special group. The associated bleeding diathesis is mild and patients are often misdiagnosed with autoimmune thrombocytopenia. It is important, however, to identify these patients to avoid unnecessarily intensive investigation and the use of ineffective treatments with significant side effects (steroids, splenectomy) /1, 3/.

17.7.1 Indication

Tests for inherited thrombocytopenias or thrombocytopathies should be requested if symptoms have been present since childhood or several members of a family are affected.

17.7.2 Tests

The choice of test depends on the suspected clinical diagnosis.

17.7.3 Clinical significance

Inherited disorders of thrombocyte function are uncommon and recessive forms are particularly rare. Storage granule disorders are the most common inherited thrombocytopathies. They account for 10–20% of congenital disorders of thrombocyte function /5, 6/. Tab. 17.7-1 – Hereditary thrombocytopenias and thrombocytopathies summarizes the characteristic laboratory findings.

The most common inherited thrombocytopenias are dominantly inherited macro thrombocytopenias. The exact incidence of these disorders is unknown.

An inherited platelet-based bleeding disorder should never be diagnosed based on the results of a single test; the diagnosis should be confirmed by testing a second, separately obtained blood sample. The diagnosis can also be confirmed by confirming the presence of the same abnormalities in a number of family members or by confirming one abnormality using a number of methods.

References

1. Selleng K, Greinacher A. Thrombozytopathien. In Pötzsch B., Madlener K. (eds). Hämostaseologie. 2. Aufl. Heidelberg; Springer 2010: 325–34.

2. Cattaneao M. Inherited platelet-based bleeding disorders. J Thromb Haemost 2003; 1: 1628–36.

3. Balduini CL, Iolascon A, Savoia A. Inherited thrombocytopenias: from genes to therapy. Haematologica 2002; 87: 860–80.

4. van Geet C, Freeson K, Devos R, Vermylen J. Hereditary thrombocytopenias. In Gresele P, Page C, Fuster V, Vermylen J (eds). Platelets in thrombotic and non-thrombotic disorders. Cambridge; Cambridge University Press 2002: 517–27.

5. Nurden AT, Nurden P. Inherited thrombocytopenias: history, advancesand perspectives. Haematologica 2020; 105(8): 2004-19.

6. Kremer Hovinga JA, George JM. Hereditaty thrombotic thrombocytopenic Purpura. N Engl J Med 2019, 381:(17): 1653–62.