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Acquired Hemophilia, rpVIII, and Bethesda Titer

Here is a 12-2-23 email from Hrvoje Melinšćak MD, Consultant in Haematology, the Warren Alpert Medical School of Brown University, Rhode Island Hospital/The Miriam Hospital.

I would enquire regarding rpFVIII’s impact on inhibitor titre assessment through the Bethesda assay in a case regarding acquired haemophilia A. Does significant background rpFVIII (Obizur) concentrate lend to significant underestimation of a FVIII inhibitor when administered in acquired haemophilia A? Heat inactivation and the Nijmegen modification are not employed at our institution. The ambient FVIII activity was 1.54 IU/mL (154%) at the time of sample draw. The high ambient FVIII activity, due to rpFVIII replacement product, likely, if not necessarily, precludes meaningful interpretation. I was wondering how one might go about assessing Bethesda titres in this instance, short of holding further rpFVIII doses to washout and assaying thence?

I [Geo] responded on 12-4-23: Hello, Dr. Hrvoje Melinšćak, and thank you for your email. I consulted with Nijmegen-Bethesda titer expert Connie Miller, PhD, US Centers for Disease Control. Dr. Miller recommends you heat treat the plasma at 56 degrees centigrade for 30 minutes to destroy the rpFVIII concentrate and then proceed with the Bethesda titer using your laboratory’s validated approach. Without heat treatment, the residual rpFVIII is likely to cause an underestimation of the inhibitor level. She cited references to two helpful articles, one of which indicates that prolonged heating over 30 minutes will reduce the antibody level.

Here is a 12-11-23 response from Dr. Melinšćak: Thank you kindly for responding. The articles were quite helpful. The trouble with monitoring inhibitors whilst administering rpFVIII in acquired haemophilia A is interference. Another approach to utilise is one of allowing rpFVIII to wash out and then assaying the inhibitor, granted the haemorrhage has to be under some reasonable state of control. The next conundrum is employing emicizumab, as it has fallen into vogue, in acquired haemophilia A and the assaying of background native FVIII activity and Bethesda titres to boot. The bovine chromogenic assay is available to us and a chromogenic Bethesda titre was validated in the literature if I recall correctly.

In response to Dr. Melinšćak’s comment, here is a poster, Emicizumab Impact on Factor VIII Inhibitor Determination in Plasma Samples from Persons with Hemophilia A (PwHA) Using a New Kit for Modified Nijmegen-Bethesda Assay (MNBA). and here are the two article citations provided by Dr. Miller:

  • Boylan B, Miller CH. Effects of pre-analytical heat treatment in factor VIII (FVIII) inhibitor assays on FVIII antibody levels. Haemophilia. 2018;24:487–91. doi: 10.1111/hae.13435.
  • Batty P, Platton S, Bowles L, et al. Pre-analytical heat treatment and a FVIII ELISA improve Factor VIII antibody detection in acquired haemophilia A. Br J Haematol. 2014;166:953–6. doi: 10.1111/bjh.12923.
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