Heat Inactivation of Patient Serum or Plasma

To avoid thrombin-induced platelet activation in buffer containing physiological calcium, steps are taken to inactivate residual thrombin. Thus, plasma and serum must first be heat inactivated before use in these assays. Heating at 56°C for

30-45 min inactivates thrombin and complement. Fibrin and other precipitates are removed by high-speed centrifugation (8000 g for 5 min). More intense heating of serum (63° C for 20 min) forms platelet-activating immune complexes (Warkentin et al., 1994); thus, if a patient sample shows heparin-independent platelet activation (indeterminate result), another sample aliquot should be heat inactivated, and the HIT assay repeated. Often, this will result in disappearance of the initial artifact that presumably was caused by too intense heat inactivation. Serum is preferred for use in functional HIT assays in our laboratories, as serum contains more PF4, thereby facilitating initial formation of the antigen.

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