The dilution effect of equal volume mixing studies compromises confirmation of inhibition by lupus anticoagulants even when mixture specific reference ranges are applied.
BACKGROUND
One of the recommended criteria for the laboratory diagnosis of lupus anticoagulants (LA) is demonstration of inhibitory activity. This is confirmed by performing mixing tests with normal plasma, usually in a 1:1 ratio, and demonstrating persistence of an abnormal clotting time in the screening test with significant confirmatory test reduction. However, the mixing with normal plasma can dilute the antibodies to undetectable levels and generate apparent negative results. No guidelines or consensus exist in how to interpret mixing study results.


METHODS
The present study assessed the 1:1 mixing study results from 600 patients with a thrombotic history positive for LA demonstrated in neat plasma by individual assays, or combinations, of dilute Russell's viper venom time, dilute activated partial thromboplastin time, activated seven lupus anticoagulant assay and Taipan snake venom time, plus confirmatory tests. Mixing tests were assessed initially using locally derived neat plasma reference ranges and subsequently with mixture specific ranges.


RESULTS
The mixture specific ranges had lower upper limits. Of the total LA positive results, 32.5% were positive in the mixing studies when neat plasma reference ranges were applied, and a further 11.2% demonstrated LA activity when using the mixture specific ranges. The remaining 56.3% had been diluted such that they did not elevate the screening test above the upper limit of normal or generated minimal prolongation with an insignificant difference between the screen and confirmatory test result sufficient to confirm LA activity.


CONCLUSIONS
The significant impact of the dilution effect in 1:1 mixing studies emphasises the limitations of mixing studies as a vehicle for confirmation of inhibition by LA antibodies.
Moore
Gary W
GW
Centre for Haemostasis and Thrombosis, (Haemophilia Reference Centre), St Thomas' Hospital, Lambeth Palace Road, London, England, UK. gary.moore@gstt.nhs.uk
Savidge
Geoffrey F
GF
D001780:Blood Coagulation Tests / Q000379:methods; D006801:Humans; D016682:Lupus Coagulation Inhibitor / Q000097:blood*; D010314:Partial Thromboplastin Time; D011517:Prothrombin Time; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D016682:Lupus Coagulation Inhibitor