A NEW LUPUS ANTICOAGULANT NEUTRALIZATION TEST BASED ON PLATELET-DERIVED VESICLES

We here present an easily standardizable and reproducible procedure which clearly separates lupus anticoagulants (LA) from coagulation factor inhibitors. This new LA neutralization test makes use of platelet-derived microvesicles which were prepared as follows: gel-filtered platelets (4 x 10(5)/mu-l) were incubated with 60-mu-M of the calcium ionophore A23187 for 20 min at 37-degrees-C. The vesicles were separated from the platelet aggregates by centrifugation at 1000 g for 10 min. The vesicle containing supernatant was then spun down at 15 000 g for 15 min, lyophilized and stored at -20-degrees-C until used. The vesicles were resuspended in plasma from normal individuals, from patients with LA activity, from patients with factor VIII inhibitors, from patients with congenital factor deficiencies and from patients receiving oral anticoagulants or intravenous heparin. A kaolin clotting time was performed in the absence (KCT) or presence of these vesicles (KCT(ves)) and the ratios of these times to their respective mean normal times were calculated. Segregation of LA patients from all remaining patients except heparinized ones could be made with a high degree of accuracy. A thrombin time was needed to separate LA from heparinized patients. The method was highly reproducible and only minor (negligible) differences in potencies were observed between different vesicle preparations. Both the intra-batch and the inter-batch coefficients of variations on the KCT(ves) were lower than 6%.