Bethseda Inhibitor Assay

Purpose of the test

Factor VIII inhibitor assay:

Antibodies to the infused Factor VIII develop in about 10% of haemophilia A patients. In previously haemostatically normal subjects an antibody can develop against the patient's own Factor VIII. This is usually associated with other autoimmune conditions e.g. S.L.E., R.A., neoplasms. Anti VIII can also develop in the mother during pregnancy in response to the baby’s factor VIII.

The Bethesda assay is based on measuring residual VIIIC activity remaining after dilutions of test plasma are mixed with normal plasma and incubated for 2 hours at 37°C compared with a control mixture of normal plasma and buffer. The prolonged incubation time is necessary because Factor VIII inhibitor activity increases over time. These inhibitors are therefore known as progressive inhibitors.

The residual VIIIC activity is expressed as a percentage of the residual VIIIC in the control mixture and a reference graph is used to convert any inhibitor activity to Bethesda units.

One Bethesda unit is defined as the amount of inhibitor which will neutralise 50% of added VIIIC after 2 hours at 370C.

A Bethesda assay is performed if the inhibitor screen shows the presence of an inhibitor and Factor VIIIC assay shows reduced levels. The inhibitor can cross-react with the factor VIII of other species. Porcine Factor VIII can be used to treat bleeding episodes in patients with an inhibitor, if the anti-porcine inhibitor is much lower than the anti-human inhibitor.

Recombinant Factor VIIa (NovosevenTM) can be used to bypass Factor VIII in the treatment of inhibitor patients. A patient with Anti VIIIC is monitored at intervals to measure activity against human and possibly porcine VIIIC.

Factor IX inhibitor assay:

Patients with Haemophilia B (Christmas Disease) occasionally develop inhibitors to their Factor IX replacement therapy. Factor IX inhibitors may also rarely develop spontaneously. These inhibitors are not progressive (see above) so the incubation time in the assay can therefore be reduced to 30 minutes.

Reference Range

Normal range: Bethesda unit <1

Sample Requirements

Two coagulation citrated sample with no clots and adequately filled must be sent directly to the Haemostasis laboratory.

Storage and Transport

Centrifuge in refrigerated centrifuge for 15 minutes at 3,600 rpm and separate the plasma into plastic tubes. Place in –40°C freezer within 1 hour of collection if assay is not performed immediately.