Laboratory diagnosis of heparin‐associated thrombocytopenia and comparison of platelet aggregation test, heparin‐induced platelet activation test, and platelet factor 4/heparin enzyme‐linked immunosorbent assay

Abstract

As clinical diagnosis of heparin-associated thrombocytopenia (HAT) is often difficult, confirmation by sensitive laboratory assays is desirable. The sensitivity of the heparin-induced platelet activation (HIPA) test and the platelet aggregation test (PAT) was prospectively compared by using the sera of 209 patients with the putative diagnosis of HAT. Both assays were performed concomitantly with platelets of the same four donors using a different combination of donors from day to day. Further, all sera were assessed with a platelet factor 4 (PF4)/heparin enzyme-linked immunosorbent assay (ELISA). Positive results were obtained with 33 percent of sera in the PF4/heparin ELISA, with 33.5 percent of sera in the HIPA test, and with 11.5 percent of sera in the PAT. The PF4/heparin ELISA and the HIPA test showed no difference in sensitivity (p = 0.27 by McNemar's test) and were more sensitive than PAT (p < 10(-8) by McNemar's test). However, they recognized different patient cohorts. Nine HIPA-indeterminate and 12 HIPA-negative sera were positive in the PF4/heparin ELISA. Eight of the nine indeterminate sera caused platelet activation with high heparin concentrations in the HIPA test. Eleven of the 12 negative sera contained no IgG, but 9 contained IgM and 2 contained IgA HAT antibodies. Four sera that were indeterminate in the PF4/heparin ELISA and 18 sera that were negative were positive in the HIPA test. None of the sera that were positive in the PAT was missed in the HIPA test, but two of those were negative in the PF4/heparin ELISA. All sera were assessed with four low-molecular-weight heparins and a low-molecular-weight heparinoid in the HIPA test with platelets from the same four donors. Low-molecular-weight heparin caused platelet activation with positive sera in 98 percent of tests, and the heparinoid did so in 10 percent; in a further 12.8 percent, crossreactivity to the low-molecular-weight heparinoid could not be excluded. The majority of HAT antibodies react with a PF4/heparin complex, but there is strong evidence that other antigens are involved in some patients. The HIPA test and the PF4/heparin ELISA are sensitive for diagnosing HAT, and they complement one another.