Novel method to dissociate platelet clumps in EDTA-dependent pseudothrombocytopenia based on the pathophysiological mechanism

Background: EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) is an in vitro phenomenon of platelet clumping that leads to spuriously low platelet counts by automatic hematology analyzers. The mechanism is not clearly defined, but is known as an immunologically mediated phenomenon due to the presence of EDTA-dependent antiplatelet autoantibodies that induce platelet clumping. The purpose of this study was to identify antiplatelet antibodies in EDTA-PTCP samples and to design a method to dissociate platelet clumps based on the pathophysiological mechanism. Methods: The antiplatelet antibody was investigated using direct and indirect immunofluorescent flow cytometric methods in 23 EDTA-anticoagulated whole blood (WB) samples and 12 serum samples of EDTA-PTCP patients, respectively. A novel mixture containing 9 mmol/L CaCl ₂ and 0.1 unit/L sodium heparin, that provides calcium replacement while curbing coagulation, was designed to dissociate platelet clumps. The effect on dissociation was demonstrated in 26 samples of EDTA-PTCP and compared with the established method of kanamycin supplementation. Results: The direct test was positive for IgM and IgG antiplatelet antibody in 60.9% and 4.4% of patients, respectively [mean median fluorescence intensity (MFI) of 223.9 and 128.4, respectively]. The indirect test was positive for IgM antiplatelet antibody in 58.3% of patients (mean MFI of 123.4). The novel method dissociated the platelet clumps with a mean increased platelet count of 242.3% and was equivalent in efficiency to kanamycin supplementation. Conclusions: The novel method is an easily applicable and efficient measure that allows dissociation of platelet clumps, based on the pathophysiological mechanism of EDTA-PTCP.