A chromogenic substrate method for detecting and titrating anti-factor VIII antibodies in the presence of lupus anticoagulant

Abstract

Background and objectives: The development of neutralizing anti-factor VIII antibodies (a-fVIII) is a major clinical complication. Lupus anticoagulant (LA) might affect detection of a-fVIII, since both inhibitors may act on the same coagulation pathway. Our aim was to accomplish unequivocal detection and titration of a-fVIII even in the presence of LA.

Design and methods: We evaluated a-fVIII activity by a chromogenic substrate (CS) method in samples with a-fVIII (n=6), LA (n=12) and presumably both LA+a-fVIII (n=5). The inhibition index before (Ii) and after incubation at 37 C (Ii(37)) was estimated. We also performed factor VIII assays (one-stage and CS) and titration methods (Bethesda and CS) in parallel.

Results: Inhibition in the a-fVIII group (Ii=5-3200) was potentiated by incubation (Ii(37)=27-5200) as it was in LA+a-fVIII (Ii=9-21; Ii(37)=50-903). LA samples showed no or meaningless inhibitory effect (Ii=0-7; Ii(37)=0-4) or a-fVIII activity (0.00-0.06 CSU/ml) by the CS method; on the contrary, very low to moderate (0.52-7.00 BU/ml) a-fVIII activity was recorded by the Bethesda method. The two titration methods did not correlate (p>0.100) in the presence of LA, or LA+a-fVIII. Differences between factor VIII:C and factor VIIIcs were significant only in LA samples (p=0.005); however, patients with residual factor VIII activity from the LA+a-fVIII group also showed higher factor VIIIcs values than factor VIII:C ones.

Interpretation and conclusions: Results indicate the possibility of detecting and titrating a-fVIII without interference of LA by the CS method. This marks a difference with respect to the Bethesda method, in which a measurable effect can be expected in the presence of a strong LA.