Development of a high-pressure liquid chromatography method for diagnosis of heparin-induced thrombocytopenia.

Abstract

Owing to the disadvantage of radioactivity of the carbon 14 serotonin release assay and the time-consuming procedure of the enzyme immunoassay, we developed a high-pressure liquid chromatography (HPLC) method to detect serotonin released from donor platelets in the presence of heparins and serum samples from patients with heparin-induced thrombocytopenia (HIT). Samples were analyzed from 60 healthy control subjects, 19 patients with HIT, and 20 patients without HIT after incubation with heparin, low-molecular-weight heparin (LMWH), and danaparoid. Serotonin release was measured from platelets, 300 x 10(3)/microL, by HPLC. Serotonin eluted as a single peak from the HPLC column. Serum samples from patients with HIT released 5.5 to 352.5 and 6.6 to 1,533.3 ng/mL of serotonin from platelets in the presence of 0.2 IU/mL of heparin and LMWH, respectively. In the presence of 0 IU/mL of heparin, LMWH, danaparoid, and control samples, less than 2.5 ng/mL of serotonin were released. The HPLC method permits a rapid, sensitive, and quantitative determination of serotonin released from donor platelets for laboratory confirmation of HIT.