A new lupus anticoagulant neutralization test based on platelet‐derived vesicles

Abstract

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)/microliters) were incubated with 60 microM 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 (KCTves) 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 KCTves were lower than 6%.

Guidelines On The Investigation and Management Of The Antiphospholipid Syndrome

“…A neutralization test based on platelet‐derived microvesicles has also been described (Arnout et al , 1992) and may be useful. An alternative method for the confirmation of LA uses a modified APTT reagent containing hexagonal phase lipids, which specifically bind LA and remove its effect on the APTT reagent.…”

Sensitivity of Sixteen APTT Reagents for the Presence of Lupus Anticoagulants

“…A variety of tests have been introduced to detect and confirm LAs [3,[11][12][13][14][15], but the heterogeneity of these LAs might perhaps ex plain the lack of a common reference proce dure for their detection. However, the acti vated partial thromboplastin time (APTT) is still the most common assay used to screen for LA.…”

Conceptions and Misconceptions in Testing for Lupus Anticoagulants

“…Thus, they are known to correct the defects due to heparin and factor V inhibitors in addition to lupus anticoagulants. The degree of platelet washing may affect the amount of residual factor V, but no amount of washing, even in the improved method of Arnout [9], appears to reduce their heparin neutralizing characteristic. The PNP is therefore well known to give false-positive results in heparin patients and those with weak factor V inhibitors [e.g.…”