Test Conditions of Heparin Dependent Platelet Activation Perform Platelet Activation Studies Under Various Test Conditions

Comment. In Hamilton, sera are studied using six different reaction conditions: (1) buffer; (2) unfractionated heparin (UFH), 0.1U/mL; (3) UFH, 0.3U/mL; (4) UFH, 100 U/mL; (5) low molecular weight heparin (LMWH), enoxaparin, 0.1 U/mL; and (6) UFH, 0.3 U/mL plus a monoclonal antibody (IV.3) that inhibits platelet Fc receptor-mediated platelet activation. In Greifswald, routine testing is performed using (1) buffer; (2) LMWH (reviparin), 0.2 U/mL; (3) UFH, 100 U/mL; and (4) danaparoid, 0.2 U/mL (to assess cross-reactivity); (5) sometimes LMWH 0.2 U/ mL plus IV.3 is performed to resolve unclear results. In Greifswald, the LMWH preparation reviparin (Clivarine) is used because of its narrow molecular weight (MW) range (80% of its chains have molecular mass of 2.4-7.2 kDa, i.e., 4-12 disaccharide units) (Jeske et al., 1997); this results in more consistent formation of PF4-H complexes, enhancing sensitivity of the assay (Greinacher et al., 1994b). Platelets are incubated with various test and positive and negative control sera under these various reaction conditions for up to 30 min (Greifswald) or 60 min (Hamilton). Addition of hirudin avoids thrombin-dependent platelet activation. The order of pipetting is important in optimizing assay results (Eichler et al., 1999) (Table 3). After adding serum to the microtiter plate wells, high heparin concentrations are added to the appropriate wells: this will disrupt PF4—heparin complexes that may be present in the serum. After adding washed platelets, buffer, LMWH (low concentrations), and danaparoid (for cross-reactivity testing, if desired) are added. If inhibition by monoclonal antibody IV. 3 is tested, this reagent is added before addition of the washed platelet suspension. In Hamilton, the pipetting order for the SRA is (1) addition of buffer-heparin, (2) serum, and (3) platelets.

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