Introduction

Disorders of platelet number are frequently caused by immune-mediated mechanisms.¹ Antibody-coated platelets can be destroyed intravascularly, via comple-ment-mediated cytolysis, or can be removed by the Fc receptor-bearing reticuloendothelial (RE) cells in the spleen and liver. The production of platelet-reactive antibodies may be triggered by exposure to foreign platelet antigens [e.g. post transfusion purpura (PTP), or neonatal alloimmune purpura (NAIT)], by viral illness [e.g. childhood type idiopathic thrombocytopenic purpura (ITP)] or by exposure to certain drugs

(e.g. quinine/quinidine). However in many instancesthe triggering event leading to autoantibody production cannot be identified. This is the case of ITP. Forty years ago, Harrington was first to demonstrate the immunological basis for ITP, showing that normal platelets are destroyed when exposed to plasma from patients with ITP. ² The nature of the process governing antibody generation in ITP remains unknown.

Platelets bear a variety of antigens which are capable of inducing serologic reactivity.^3,4 Table 1 summarizes several of the most important platelet antigens and their involvement in platelet consumptive processes. Some antigens are not specific to platelets, being shared either with erythrocytes (the antigens of the blood groups systems) or with other mononuclear cells (the HLA class I antigens). The carbohydrate ABH-group antigens are passively absorbed from plasma onto the platelet surface. They are often responsible for the shortened lifespan of transfused platelets in the setting of ABO-mismatched platelet transfusion. On the other hand, the HLA class I protein-antigens present on platelet surface (mainly types a and b) are involved in acquired platelet transfusion refractoriness. Platelet-specific antigens are membrane glycoproteins, which are largely responsible for the interaction between platelets and the endovascular wall components.⁵ Among those, integrin aIIbb3 mediates the interaction of platelets with fibrinogen, whereas integrin a2b1 mediates attachment to collagen. The glycoprotein (GP) Ib binds non-covalently to factor IX (GP-Ibb) and V (GP-Iba) forming the complex GPIb-IX-V. The receptor for von Willebrand factor is located in the amino terminal region of the GPIba chain.⁵

Alloantibodies to some of these glycoproteins are responsible for thrombocytopenia encountered after HPA1-incompatible transfusions or observed in pregnancies associated with HPA feto-maternal incompat-

Table 1: Platelet-related Antigens and Their Role in Generation of Alloantibodies

Idiopathic Thrombocytopenic Purpura (ITP)

Two distinct clinical pictures associated with ITP can be discerned. On the one hand, the childhood-type ITP, which is an acute, self-limiting disorder, is often preceded by a viral infection. On the other hand, there is the chronic, relentless and often refractory ITP which is encountered in adults.¹⁸ The pathogenesis is similar, yet the evolution in time and prognosis remain discrepant. No differences in the type of autoantibodies associated with these two different presentations of ITP are known.^19,20 Predicting the long-term outcome relies on clinical observation. In fact the diagnosis of ITP is merely a diagnosis of exclusion. One rarely confirms characteristic diagnostic platelet autoantibodies. \"Antigen-capture\" assays can be performed using monoclonal antibodies fixed to a solid phase (MAIPA) with the goal of identifying platelet-directed antibodies. In every day practice these assays are not required to make the diagnosis of ITP.²¹ The blood smear examination demonstrates few platelets, which are usually large (so-called \"giant platelets\"). They represent megakaryocytic fragments, which suggest an increase in platelet production and turnover.

Table 2 summarizes causes for secondary immune-mediated thrombocytopenia and tests required to exclude these entities. Performed routinely in patients with ITP are immunological assays to exclude SLE, the anti-phospholipid antibody syndrome, HIV infection and other hematological disorders.

Treatment of ITP is directed towards preventing serious bleeding.^22,23 Traditionally corticosteroids are considered the cornerstone of therapy. Steroid therapy can prevent bleeding, even before a rise in the platelet count is recorded, by sealing off the small fenestrations within the capillary wall. IVIG and more recently anti-D immunoglobulin (WinRho) are capable of inducing a pharmacological blockade of the Fc receptors in RE cells of the spleen, precluding

Heparin-induced Thrombocytopenia (HIT)

Characteristically HIT is defined by thrombocytopenia induced by exposure to heparin. An interval of at least 5 days precedes the occurrence of thrombocytopenia after initial exposure to heparin. Arterial and venous thrombotic episodes can occur in 30-50% of patients with HIT.^42,43 In one series, the odds ratio for developing thrombosis for HIT patients when compared to patients without HIT was 36.9.⁴⁴ All patients in this study received prophylactic heparin during the post-surgical period. The use of unfractionated heparin (UFH) is associated with a greater risk of HIT than low molecular weight heparin (LMWH) (2.7% versus 0).⁴⁴ Once the syndrome has developed, the substitution of UFH with LMWH is not recommended as cross-reactivity can occur.⁴⁵ The mechanism of HIT has been elucidated. IgG antibodies generated in response to heparin-platelet factor 4 (PF4) complexes trigger HIT.⁴⁶ Initial exposure to heparin leads to release of PF4 from platelet a granules. PF4 forms a complex with heparin and pathogenic antibodies are produced in response to these complexes which bind via their Fc portion to the platelets.^47,48 Platelets become later activated releasing microparticles that trigger the generation of thrombin.⁴⁹ This cycle is responsible for inducing both thrombocytopenia as well as thrombotic phenomena. Antibodies against complexes between heparin and chemokines (interleukin-8 and neutrophil activating factor-2) have been recently reported ⁵⁰ and may be the cause of rare false-negative results for ELISA assays in patients with HIT.

Two types of assays can prove the diagnosis of HIT.^51-53 On the one hand, the antibodies against the heparin-PF4 complex can be detected via an ELISA technique. On the other hand, heparin-induced platelet activation can be demonstrated through a variety of functional assays (platelet serotonin release, platelet aggregation, microparticle generation). The concordance between the antigen-based and the functional assays varies between 80% to 90%. Both types of assays should be performed if the diagnosis is uncertain, as the \"HIT\" label could have serious implications in planning treatment and various surgical procedures (such as heparin-requiring cardiopulmonary bypass).

Treatment relies on discontinuation of the offending drug (heparin). If anticoagulation is needed, a non-cross-reacting agent should be used, such as a direct thrombin inhibitor (lepirudin or argatroban) with close PTT monitoring. The use of argatroban is favored over lepirudin in patients with impaired renal function, whereas lepirudin is preferred in the setting of hepatic dysfunction. No clear guidelines for anticoagulation in the absence of thrombosis in patients with HIT are currently in use. However given the significant pro-thrombotic tendency that these patients have, use of a thrombin inhibitor in high-risk situations may be justified.

Post-transfusion Purpura (PTP)

PTP is a rare cause of severe thrombocytopenia that usually occurs 1 week after a transfusion of blood-derived products. The typical patient is a multiparous woman or a patient with history of previous transfusions. Thrombocytopenia is usually severe with platelet counts below 10,000 and can be associated with significant hemorrhage.^54,55The mortality can be as high as 10-20% and is due in part to failure to recognize this entity in a timely manner. The mechanism leading to platelet destruction remains an unsolved enigma. For HPA-1b homozygous patients, alloimmunization to HPA-1a can occur either as a consequence of pregnancy (with a HPA-1a fetus) or of a previous transfusion. Upon re-exposure to HPA-1aplatelet antigen, platelet consumption is triggered, hypothetically due to cross-reactivity of the alloantibodies against the subject\'s antigen-negative platelets.⁵⁶ The process cannot be reproduced in vitro and remains in large part unexplained.⁵⁷ Exposure to antibodies directed against different platelets antigens (HPA-2, 3, 4 or 5) has been reported less frequently and a clear association with HLA DR3 and DRw52a has been documented.^58-62 The diagnosis is based on clinical criteria. Anti HPA-1a platelet antibodies can be demonstrated by platelet immunoflourescence or by immobilization of the platelets antigens with monoclonal antibodies (MAIPA assay).

Treatment consists of prompt initiation of IVIG at a dose of 2 g/kg over 2 or 5 days, in split fractions. Steroids can be used in conjunction and plasma exchange has minimal, if any role. Transfusion of HPA-1a negative platelets can be used in life threatening hemorrhagic circumstances.⁶³

Neonatal Alloimmune Thrombocytopenia (NAIT)

NAIT is the platelet analogue of the hemolytic disorder of the newborn induced by feto-maternal alloimmunization. It affects 1 in 1500 pregnancies and can cause severe thrombocytopenia of the fetus or the newborn leading to intracranial hemorrhage.^64,65 Unlike hemolytic disease of the newborn, as many as half of the reported cases of NAIT occur during first pregnancies. Recurrence of thrombocytopenia during subsequent pregnancies is common and a high concordance of clinical features between successive pregnancies is the rule.

NAIT is typically associated with anti-HPA-1a antibodies generated in response to fetal HPA-1a platelets in a mother homozygous for the HPA-1b phenotype. Less frequently, alloimmunization due to HPA-3, 4 and 5 allelic variants has been reported.⁶⁶ Maternal antibodies can be detected as early as week 14, and the titer in the last trimester is a reliable predictor for the severity of thrombocytopenia in the newborn.⁶⁷ Association with specific HLA subgroups (specifically the DRB3 type) is characteristic.⁶⁷ Fetuses and newborns affected by NAIT have a risk of intracerebral bleeding and hydrocephalus that approaches 10%. In one series, one serious/fatal bleeding event was predicted for 123 pregnancies that occur in HPA 1a-negative, DRB3-positive mothers.⁶⁷

Treatment consists of maternal platelet transfusion (HPA1a-negative) to the severely thrombocytopenic newborns. IVIG can be administered to the neonate, if thrombocytopenia is life-threatening and persistent. Of note, newborn thrombocytopenia sometimes persists for weeks, until all maternal antibodies have been cleared. Strategies for antenatal management of these cases are less clearly defined. High titers of anti-HPA-1a antibodies during the last trimester of pregnancy, HLA-DRB3 genotype and previously affected fetuses predict fetal thrombocytopenia. In one series of 10 patients, administration of IVIG to the mother during the last trimester prevented serious fetal hemorrhagic complications.⁶⁸ Intrauterine platelet transfusions could be performed, but carry a small risk of fetal loss. For severely thrombocytopenic fetuses, delivery via a Cesarean section is associated with decreased hemorrhagic risk.

Drug-induced Thrombocytopenia

A diagnosis of drug-related thrombocytopenia requires that a causal relation between the administration of the suspected drug and thrombocytopenia be established. This is not easily accomplished in patients receiving multiple medications which potentially can be linked to thrombocytopenia. Resolution of the process following discontinuation of the medication strongly supports a diagnosis of drug-related thrombocytopenia.^69,70 A prototypical mechanism is quinidine-induced thrombocytopenia. In 1991, quinine/quinidine-induced antibodies were identified as being directed against the GP Ib-IX complex and aIIbb3 integrin.^71-73 Similarly, sulfon-amide-induced antibodies are directed against epitopes on the aIIbb3 glycoprotein.⁷⁴ A potential mechanism implicated in this process is that the offending drug induces platelet neoantigen presentation, which triggers autoantibody generation. Antibody-coated platelets are then removed by the RE cells or destroyed in the circulation via complement activation. Discontinuation of the suspected medication leads to resolution of the process.

Thrombocytopenia Associated with Lupus Anticoagulant

Up to one third of patients with lupus anticoagulant (LA) have concomitant thrombocytopenia. This is due to immune-mediated destruction of platelets triggered by platelet related autoantibodies.⁷⁵ In a series of 146 patients with systemic lupus erythematosus (SLE), of which 65 had a positive LA test and/or anti-cardiolipin antibodies, thrombocytopenia was strongly correlated with positive LA and high-titer anticardiolipin antibodies.⁷⁶ An association between thrombocytopenia and arterial thrombosis was noted in this study. This could be explained by platelet activation triggered by the lupus-related antibodies. Platelet activation was noted in thrombocytopenic patients with LA by flow cytometry measuring the expression of an activation marker CD62.⁷⁷ Decreased platelet count in patients with LA should be taken into account when assessing the thrombophilic potential of such patients.

Conclusion

In summary, a variety of immune mediated disorders are associated with thrombocytopenia. Table 3 summarizes the various syndromes discussed here. Distinct pathogenetic mechanisms have been identified and characteristic clinical and laboratory features are helpful in distinguishing these entities. Treatment is tailored accordingly, with the ultimate goal of preventing serious bleeding.