Tests for Glanzmann Thrombasthenia

Laboratory Features

Table 121–3 provides characteristic laboratory data in patients with Glanzmann thrombasthenia. Patients have normal platelet counts and morphology, prolonged bleeding times, decreased or absent clot retraction, and abnormal platelet aggregation responses to physiologic stimuli. Platelets of patients with Glanzmann thrombasthenia have a normal (or near-normal) initial slope of high-dose ristocetin-induced aggregation, reflecting the normal levels of plasma von Willebrand factor and the normal platelet GPIb/IX content; at lower doses of ristocetin, however, where GPIb/IX-mediated activation of IIb3 (see Chap. 114) normally contributes to the aggregation response, patient’s have decreased second-wave aggregation.104 The interesting cyclical aggregation observed at high doses of ristocetin105 probably reflects a complex interaction between ristocetin-induced binding of von Willebrand factor to GPIb/IX and inhibition of this interaction by released ADP and collagen.106 Glanzmann thrombasthenia platelets undergo normal shape change in response to ADP and thrombin, demonstrating their ability to undergo metabolic and cytoskeletal changes in response to these agents. Similarly, high doses of thrombin and collagen produce normal release of dense body and -granule contents6,8,107; the release reaction abnormalities observed with lower doses of these agents reflect the lack of augmentation of the release reaction normally produced by platelet aggregation.6,104,108–110

Table 121–3. Laboratory Features of Glanzmann Thrombasthenia
I. Platelet Count: Normal
II. Bleeding Time: Markedly Prolonged
III. Tests of platelet function
A. Platelet aggregation
1. Epinephrine—no observable response
2. Adenosine diphosphate (ADP) and thrombin—shape change, but no aggregation
3. Collagen—shape change followed by variable increase in light transmission due most likely to progressive adhesion to collagen fibers (pseudoaggregation)
4. Ristocetin—normal initial slope of aggregation; at low doses, inhibition of second wave; at high doses, cyclical aggregation–disaggregation
B. Aperture closure time (PFA-100): Prolonged
C. Clot retraction: Absent or reduced
D. Platelet release reaction: Decreased with epinephrine and low dose ADP, thrombin and collagen; normal with high dose thrombin and collagen
E. Interaction with glass (platelet retention test): Absent or reduced
F. Platelet coagulant activity: Variably abnormal
G. Microparticle formation: Variably abnormal
H. Ex vivo interaction with deendothelialized blood vessels in flow chambers: Marked abnormality in platelet thrombus formation and defective platelet spreading. Decreased platelet adhesion at high shear rates
IV. Tests of IIb3 and V3 Receptors: Number and Functional Integrity
A. IIb3 content: Reduced or absent, except in variants
B. V3 content: Reduced or absent in patients with 3 defects; normal or increased in patients with IIb defects
C. Platelet binding of fibrinogen and other adhesive glycoproteins to IIb3: Reduced or absent
D. Platelet fibrinogen content: Markedly reduced, except in some variants
V. Platelet Coagulant Activity: Normal or Reduced

Platelets in whole blood or platelet-rich plasma adhere to glass because fibrinogen first becomes deposited on the glass and the platelets then adhere to the immobilized fibrinogen.111,112 Platelets from patients with Glanzmann thrombasthenia fail to adhere to glass,6,8,111 and this forms the basis of their abnormality in the glass bead retention assay.113 Platelet coagulant activity has been variably reported as normal or abnormal,6–9,114–116 probably as a result of variations in the assays used to assess this activity or individual patient differences. A defect in platelet microparticle formation and support of thrombin generation has been identified in some patients,115–118 but not all patients appear to share this abnormality.119 IIb3 and V3 have been shown to bind prothrombin, probably accounting for some of the abnormalities identified.120,121

In flow chamber studies, thrombasthenic platelets adhere normally to deendothelialized blood vessels at low and intermediate shear rates, but do not spread normally or form platelet thrombi.122–124 A defect in adhesion occurs at higher shear rates. A paradoxical increase in fibrin formation on these surfaces has been observed with thrombasthenic platelets, but the explanation for this phenomenon remains unknown.125 In contrast to normal blood, blood from nearly all patients with Glanzmann thrombasthenia fails to occlude a 150-m aperture in collagen-coated membranes under high sheer, either in the presence of ADP or epinephrine (PFA-100).126,127

Platelet IIb3 and V3 can be quantitated by any one of several techniques, including, monoclonal antibody binding (using flow cytometry or radiolabeled binding), immunoblotting, and surface-labeling followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Based on the results of such studies, patients with Glanzmann thrombasthenia have been subcategorized by IIb3 content into those with less than 5 percent of normal IIb3 (type I), 5 to 20 percent (type II), or 50 percent or more (variants).10,128 In one review of 64 patients, 78 percent were type I, 14 percent were type II, and 8 percent were variants.10 The subtyping of Glanzmann thrombasthenia into type I, type II, and variants predated the identification of IIb3 abnormalities as the cause of Glanzmann thrombasthenia and was based on functional data. With current methods of more precise laboratory analysis and the recognition of the diverse clinical and functional abnormalities present in Glanzmann thrombasthenia, this categorization provides only limited information.

Measuring V3 content is technically more demanding than measuring IIb3 because there are so few V3 receptors per platelet.53 The V3 level is very useful, however, in making a preliminary assessment of whether the patient has a defect in IIb or 3, since, in general, patients who lack V3 receptors have a defect in 3 rather than IIb.129 A 3 missense mutation (H280P) that differentially affected IIb3 more than V3 has, however, been described.60

Fibrinogen binding studies assess the function of the IIb3 complex.13,14 The method used for early studies was to add radiolabeled fibrinogen to platelets suspended in buffer (prepared either by washing or gel-filtration) and then measure the binding of radioactivity when the platelets were stimulated with ADP13,14 or a similar agonist. Fibrinogen can also be labeled with a fluorescent molecule and then flow cytometry can be used to measure fibrinogen binding. These techniques are most useful in detecting qualitative abnormalities of IIb3 in patients with variant Glanzmann thrombasthenia. The binding of a monoclonal antibody (PAC1) to platelets gives similar information because the antibody only binds to the activated form of IIb3.130

Carriers of Glanzmann thrombasthenia have essentially normal platelet function.22 Their platelets, however, only contain approximately 60 percent of the normal number of IIb3 receptors; the overlap in values between normals and carriers, however, doesn’t permit for unequivocal diagnosis of carriers by this technique.131 Carrier detection is most accurately performed by DNA analysis when the defect is known, and advances in polymerase chain reaction technology allows this to be performed even with DNA obtained from cells in random urine samples.132

Platelet fibrinogen is reduced to approximately 10 percent of normal in patients with marked reductions in IIb3,6,9,32,33 but is variably reduced in patients with significant amounts of IIb3.128,133,134 Its presence may provide insights into the nature of the functional defect.

Differential Diagnosis

A history of mucocutaneous hemorrhage, as opposed to hemarthroses and muscle hemorrhage, helps to differentiate disorders of platelet function (including von Willebrand disease and afibrinogenemia) from the hemophilias and related disorders. The symptoms of qualitative platelet function disorders and thrombocytopenia are essentially identical, so their differentiation depends on laboratory studies, most importantly the platelet count. Similarly, the symptoms of von Willebrand disease, afibrinogenemia, and the different qualitative platelet disorders are often indistinguishable, and again laboratory tests are required to make the definitive diagnosis. Hereditary disorders, such as Glanzmann thrombasthenia, are usually present at birth or have their onset in early childhood. Thus, the history can be helpful in distinguishing inherited from acquired abnormalities. Figure 121–1 is a flow diagram that depicts a logical series of steps one may take in evaluating patients with mucocutaneous hemorrhage.

Autoantibodies to IIb3 may produce the phenotype of Glanzmann disease and many of the characteristic laboratory abnormalities.135–143 Mixing studies using patient plasma and normal platelets should identify these acquired autoimmune disorders.