Dr. Ali Sadeghi-Khomami, Precision BioLogic Inc., provided these in-depth technical questions about the Nijmegen Bethesda Assay (NBA), which we use to quantitate specific factor inhibitors such as anti-factor VIII or anti-factor IX. George forwarded his questions to Connie Miller and Amanda Payne, US Centers for Disease Control and Prevention (CDC) who have been working on NBA accuracy and precision upgrades for several years. Here are Dr. Ali’s questions and answers provided by Drs. Miller and Payne; thanks to all for your support.
Q: What are the critical Bethesda unit (BU) values in FVIII inhibitor measurements? I assume one is the lower limit or “cut-off,” which, based on ISTH-SSC consensus for classic clotting, is equal or greater than 0.6 BU. So far, I could not find any article to describe how to arrive at BU cutoff determinations in the literature. Any hint or suggestion?
A: Because assay performance characteristics may change based on the instrumentation and reagents, we suggest using an assay-specific lower limit. There are several ways the cut-off can be established, including evaluating sensitivity and specificity of the assay at various cut-offs using known inhibitor positive and negative specimens or evaluation of the percentage of specimens positive for anti-FVIII antibodies at various cut-offs. We have illustrated this in Dr. Miller’s paper linked here (click or tap):
Miller CH, Platt SJ, Rice AS, Kelly F, Soucie JM; Hemophilia Inhibitor Research Study Investigators.Validation of Nijmegen-Bethesda assay modifications to allow inhibitor measurement during replacement therapy and facilitate inhibitor surveillance. J Thromb Haemost 2012;10:1055–61.
We have also examined the prevalence of anti-FVIII antibodies in patient specimens as an aid to setting cut-offs in a paper that has been submitted.
Q: What about the value of higher BUs? I assume that people choose to quantify BUs when they have a clinical plan in mind, such as when they triage patients therapy based on titre result, correct? So what are those clinically important higher BUs? For instance, are 5 and/or 10 BUs clinically important because by-pass therapy or immune tolerance induction (ITI) therapy has an indication at these levels?
A: Inhibitor treatments such as ITI rely on accurate quantitation of inhibitors, because treatment is started based on titer level and modified based on whether titers are going up or down and by what percentage. Other treatment decisions may be made based on the possibility of overcoming an inhibitor of a certain titer with a factor product vs a by-passing agent. Decisions regarding clinical management should not rely solely on titer value. Other factors, such as lack of response to product and changes in titer levels (e.g. increasing titers), should also be considered.
Q: What is the upper limit or maximum BU titre that the lab should report? We know from a residual FVIII activity vs BU titre curve that the maximum BUs that could be read from curve is 2 BU and the rest is mathematically determined by multiplying by the dilution factor. So how far should we go in dilution and quantification of inhibitory effect without breaking physics and analytical measurement rules? A 64-times dilution of patient plasma means 128 BU max and 128-times dilution means 256 BU max. Is there any different action plan for patient with 100 BU vs. 200 BU?
A: The laboratory should attempt to quantify the inhibitor, even if the titer is very high, by making dilutions until the diluted sample can be read from the curve. Data on inhibitor kinetics suggest that this is analytically valid, except on those rare inhibitors that do not increase with dilution (type 2 kinetics). As noted above, it is important to distinguish between titers such as 100 and 200 BU, because the physician may make treatment decisions based on the actual titer and whether it is going up or down, particularly if the patient is receiving ITI therapy. The simplest way to do that when an inhibitor is suspected or known to be present is to make a series of dilutions of the patient plasma prior to incubation in order to have one available that will be on the curve without having to repeat the whole assay.