A tentative diagnosis can be made by following the stepwise process of evaluation outlined in Figs. 116–1 and 116–2. However, further testing usually is required to establish a definitive diagnosis.
When the laboratory reports an abnormally low platelet count, looking at the blood film to exclude pseudothrombocytopenia as a result of anticoagulant-induced platelet clumping (e.g., induced by ethylenediaminetetraacetic acid [EDTA]) is essential.14 Examination of the blood film also can reveal the presence of giant platelets, as in some inherited thrombocytopenias; giant platelets and Döhle bodies in leukocytes, as in May-Hegglin and other MYH9 platelet syndromes; moderately enlarged platelets, as in immune thrombocytopenia or other conditions associated with shortened platelet survival; small platelets, as in Wiskott-Aldrich syndrome; schistocytes and burr cells, as in the hemolytic uremic syndrome and thrombotic thrombocytopenic purpura, and occasionally in DIC; rouleaux formation, as in monoclonal gammopathies; macrocytosis and/or hypersegmentation, as in vitaminB12 or folic acid deficiency; and abnormal white blood cells, as in leukemias and myeloproliferative disorders. Chapter 117 further discusses the evaluation and differential diagnosis of the thrombocytopenias.
Coagulation factors usually are assayed by measuring their clotting activity. The most common assays analyze the ability of dilutions of the patient’s plasma to correct the clotting time of a plasma known to be deficient in the factor being measured (substrate plasma). The results are compared to the ability of dilutions of a normal reference plasma to correct the abnormality in the substrate plasma. The activities of factors II, V, VII, and X usually are determined in PT-based assays, whereas the activities of factors VIII, IX, XI, and XII, prekallikrein, and high-molecular-weight kininogen are measured in aPTT-based assays. The plasma level of fibrinogen most commonly is measured by assessing the time required for thrombin to clot the patient’s diluted plasma (Clauss method).15 Several assays of transglutaminase activity are available for measuring factor XIII activity,16 but a simple qualitative test based on dissolving a fibrin clot in 5 M urea usually is sufficient (Chap. 124). The RCF function of von Willebrand factor can be measured by the ability of the patient’s plasma to support the agglutination of a suspension of formaldehyde-fixed normal platelets by ristocetin.17 This activity is defined as RCF activity. As with the coagulation factor assays, the results using patient plasma are compared to the results obtained with a normal reference plasma.
To determine whether a coagulation factor activity deficiency results from a quantitative decrease in protein or a qualitative abnormality in the protein, immunologic assays can be performed using specific polyclonal or monoclonal antibodies to assess the presence of the protein, independent of its function. Electroimmunoassays, enzyme-linked immunosorbent assays (ELISAs), and immunoradiometric assays all have been used successfully. Crossed immunoelectrophoresis measures both the immunologic reactivity and the mobility of the protein in an electric field; thus, it can detect protein abnormalities that affect electrophoretic migration. The abnormalities include the presence of antibody–antigen complexes that migrate differently from the protein itself, such as antiprothrombin–prothrombin complexes in patients with systemic lupus erythematosus or antiphospholipid syndrome. Diagnosis of the specific type of von Willebrand disease requires additional tests of the multimeric structure of plasma and, perhaps, platelet von Willebrand factor.
INHIBITORS TO COAGULATION FACTORS
If an inhibitor is suspected as a result of a prolonged PT or aPTT performed on a 1:1 mixture of the patient’s plasma and normal plasma, further studies can help define the nature of the inhibitor and its titer. Among inhibitors that do not require incubation (i.e., immediate-type), perhaps the most common cause is the presence of heparin in the sample. This cause can be verified by finding a prolonged thrombin time on a test of the patient’s plasma that is corrected with toluidine blue or other agents that neutralize heparin. The lupus anticoagulant also does not require incubation, and several methods for its detection are available (Chap. 131). However, with lupus anticoagulant, the PT usually is less prolonged than is the aPTT, and aPTT reagents have markedly different sensitivity to lupus-type anticoagulant depending on the amount of phosphatidyl serine present in each reagent.
Immunoglobulin inhibitors to specific coagulation factors may develop either after factor replacement therapy in patients with inherited deficiencies of coagulation factors (Chaps. 123 and 124) or spontaneously in patients without factor deficiencies (Chap. 127). Antibodies that neutralize factor activity frequently can be detected by incubating the patient’s plasma with normal plasma, usually for 2 hours at 37°C, and then assaying the specific factor. The Bethesda assay originally was designed to quantify factor VIII inhibitors but can be modified to detect other inhibitors of coagulation factors (Chap. 123).18 Some inhibitors do not directly neutralize clotting activity; instead they reduce factor levels by forming complexes with coagulation factors, which then are rapidly cleared from the circulation. Such plasmas do not produce prolonged clotting times when mixed 1:1 with normal plasma and thus may be confused with inherited deficiency states. More elaborate assays are required to identify this type of inhibitor, which may, for example, produce severe deficiency of prothrombin in some patients with the antiphospholipid syndrome (Chap. 131) and deficiency of von Willebrand factor in some acquired forms of von Willebrand disease (Chap. 126).19
PLATELET FUNCTION DISORDERS
Some laboratories nowadays routinely use an automated PFA to detect qualitative defects in primary hemostasis. Use of the RCF activity assay, platelet aggregation, and/or clot retraction are useful for assessing whether the patient has von Willebrand disease or a platelet function disorder (see Fig. 116–2). Chapter 120 contains a flow diagram of the steps required to diagnose the different qualitative disorders of platelet function. Additional platelet function assays and glycoprotein analysis may be required to establish the diagnosis.