Bethesda Titer Method Summary
Make serial dilutions of the patient plasma suspected of having a factor VIII inhibitor. Mix an aliquot of each dilution 1:1 with pooled normal plasma (PNP). The PNP provides 100% (100 U/dL) factor VIII activity. Also prepare a control by mixing an aliquot of factor VIII-depleted plasma 1:1 with an aliquot of the PNP. Incubate all tubes two hours and perform factor VIII assays on each. The reciprocal of the dilution that yields a factor VIII activity level 50% (40–60%) of the control is the inhibitor activity in Bethesda units. For instance, if the control computes to 44% factor VIII and the 1:16 dilution 22%, the Bethesda titer is 16 Bethesda units.

Residual Factor VIII
The above principle assumes there is no factor VIII in the patient’s plasma, logical, as factor VIII would be neutralized in vivo by the inhibitor. In most instances, factor VIII inhibitors are alloantibodies that rise in response to factor VIII concentrate therapy for severe hemophilia, so the assumption may be valid. However, in acquired hemophilia the autoantibody possesses second order kinetics and may leave some factor VIII behind.
Most of us select a factor VIII activity level, such as 30 or 40%, above which we reject the Bethesda titer, reasoning no inhibitor can be present. Patti Tichenor and Laura Taylor affirmed this is the policy at the University of Alabama Hospital laboratory, and their comments are found in Kelly's first post.

Melissa Bethel at Esoterix Coagulation writes:
Hi Kelly,
We won't perform an inhibitor assay with an activity level above 40%. We have heat treated samples at 56°C for 10 min. for some clinical trial testing though.

A second respondent writes:
Kelly,

• We do not perform the assay unless the factor VIII is less than 1% except on a non-hemophilic.
• It is unusual for a non-hemophilic to have that low a level; there is usually some residual FVIII around. Therefore on non-hemophilics, we do the inhibitor assay unless the factor VIII is 50%.
• We do not treat the sample---do others do this before they set up the assay?
  Hope this helps, I'm curious about the answers.

However, another correspondent writes:

Hi Kelly,
Factor VIII inhibitor testing has become a hot topic in our lab for the past six months or so. Our hemophilia program has many patients on prophylaxis, often with measurable FVIII activity levels, and more and more patients on immune tolerance induction therapy whose physicians order inhibitors weekly or monthly while giving regular infusions of FVIII. Some of these patients have both measurable factor activity and positive inhibitors, so our “rules” for inhibitor testing have been gradually changing for both hemophilia A and acquired inhibitor patients:

• FVIII activity must be done on all inhibitor samples.
• If FVIII activity is ≥50 IU/dL, inhibitor is cancelled.
• If FVIII activity is >3 but <50 IU/dL, we use a spread sheet to add the activity into the inhibitor calculation.
• Because the patient’s FVIII also degrades during the incubation, we incubate the patient plasma alone to get a FVIII value to add to the calculation.

Our long term goal is to validate heat treating the patient plasma to remove FVIII and then assay the inhibitor. Good luck with this topic!

Lastly, here is a paraphrase of Steve Kitchens’ recommendations in the manual we cited above:
Quantitative inhibitor assays are most frequently performed on test plasmas from patients with severe haemophilia. These samples contain little or no measurable factor VIII:C. If the test plasma contains more than 5 U/dL factor VIII, this must be taken into account during the calculation of inhibitor titre. This can be done in two ways:

• By adding more factor to the control mixture than to the test mixture to compensate for the factor VIII in the test mixture. For example, if the test plasma contains 20 U/dL factor VIII, then the control mixture is made from 120 uL normal plasma and 80 ul 0% FVIII. Both test and control mixtures then contain approximately 60 U/dL factor VIII at the start of the incubation phase.
• By taking the starting factor VIII level in the test plasma into account during the calculation. In this case the assay mixtures are constructed in the normal way.

In conclusion, I am not certain the usual clinical situation calls for the level of Bethesda titer accuracy we can provide through technical or mathematical manipulations of this sort, but we are challenged to develop a common approach by the number of patients with acquired hemophilia or hemophilics on prophylaxis or immunoreduction therapy.

I’m indebted to Kelly Townsend for this thorough and thought-provoking discussion. Geo.