A new method for identification of false-negative testing for lupus anticoagulant due to contamination of test plasma by platelet-derived micropatricles

Abstract number: P1499

Gosavi S., Grosso L. E., Chance D., Kister J. L., Joist J. H.

St. Louis University School of Medicine, USA

Inappropriate blood specimen centrifugation may lead to contamination of plasma by platelets. When such plasmas are frozen and sent to a reference laboratory for lupus anticoagulant (LA) testing, they may contain substantial amounts of highly procoagulant platelet-derived microparticles (PMP), which may result in false-negative LA testing. We determined whether PMP in plasma can be quantitated by performing a dilute Russel Viper Venom test (dRVVT) before and after removal of PMP from plasma (delta dRVVT) as a means to identify plasmas likely to be tested false-negative for LA. PMP were removed from freshly obtained normal and LA-positive platelet-free plasmas (PFP) (centrifugation of platelet-poor plasma at 12 000 × g for 5 min) by either ultra-centrifugation at 100 000 g for 60 min at 4 °C or 90 000 × g for 15 min at room temperature (Beckman Airfuge) or filtration using a 0.22-mm disposable filter, with similar efficiency as verified by flow cytometric analysis using anti-CD 61 antibody. When dRVVT was determined on mixtures of frozen LA-positive (STACLOT LA, Diagnostica Stago; LA Screen/Confirm, Gradipore; dilute PT-Index with Innovin, Dade Behring), citrated plasmas and normal citrated platelet-rich plasmas (final platelet concentration 10 000, 20 000, 30 000, 40 000, 50 000 mL^-1) delta dRVVT decreased progressively with increasing concentration of platelets, with dRVVT of some mixtures becoming normal (LA-negative screen) at platelet concentrations >15 000 mL^-1. Although the relatively simple method of plasma filtration was associated with a 30% loss of factor VIII activity (no significant loss of other coagulation factors) this did not affect the dRVVT. Delta dRVVT using filtration of frozen plasmas sent for LA testing varied substantially and were generally greater than the upper limit of the delta dRVVT reference range obtained with platelet-poor (<10 000 mL^-1) plasmas from normal subjects. The findings indicate that this relatively simple clotting test may be used to identify referred frozen plasmas likely to yield false-negative tests for LA. A prospective clinical validation study using both delta dRVVT and PMP quantitation by flow cytometry is in progress.