From Chad Siniard, MD, University of Alabama at Birmingham (UAB) Hospital Laboratory Medicine. George, We have a FVIII activity level that is exactly 30%. We haven’t documented an official level for triggering an inhibitor titer, though our coag lab scientist Laura Taylor customarily runs an inhibitor assay if the result is <30%. We assayed using several dilutions and the results were parallel, so they don’t indicate an inhibitory pattern. We’d like to document an official cutoff but have found no literature that offers advice. Do you know what other labs are doing in this scenario?
Hi, Dr. Siniard, and thank you for your question. My initial response is to suggest running an inhibitor titer (Bethesda titer; Nijmegen Bethesda Assay) is unnecessary if your multiple-dilution assay gives parallel results. This doesn’t answer your question, however. Here’s a response from Ali Sadeghi-Khomami, PhD, Senior Scientist, Precision BioLogic Inc.
Hi George, A while ago when we were developing FVIII inhibitor products we asked our clients about their criteria. The summary of answers was: if there is no specific order for FVIII inhibitor testing then the labs only do inhibitor testing based on their internal cutoff criteria. A range of FVIII activity cutoffs was found among the various labs for inhibitor testing, from 25–55% FVIII using the one-stage clotting assay or the chromogenic substrate assay. Regards, Ali.
Here’s a response from Emmanuel Favaloro, PhD, Institute of Clinical Pathology and Medical Research, Westmead Hospital, NSW 2145, Australia:
We don’t reflex to full inhibitor testing by Bethesda assay unless we get a specific request–we try not to make extra work for ourselves, given the Bethesda assay time sink as we are already doing several Bethesda assays per week as requested by our clinical colleagues. As a hemophilia treatment centre we also have to do assays to monitor for potential inhibitor development in hemophilia patients even where inhibitors are not suspected.
We reflex to an inhibitor screen when we get low FVIII results in a patient we have not seen before, typically if the factor level is ≤20 U/dL. In our regular patients, we first check results against past results and only progress to inhibitor screens if the result is significantly lower than established past FVIII values. We also do inhibitor screens if a clinician has specifically requested an inhibitor assay, even where the factor levels are normal. Our inhibitor screen is similar to performing only the first tube of the standard Bethesda assay; we can treat the sample to heat inactivation–56ºC for 30 min– to remove the residual factor if there are significant factor levels, and then after centrifugation do a 1:1 mix of the residual plasma supernatant with normal plasma. We run the standard control–normal plasma mixed 1:1 with sample dilution buffer–to get the 100% residual factor level. Samples are incubated at 37ºC for 1 hr (not 2 hrs as per standard Bethesda assay) to speed up test results. Factor levels are then performed. By definition, a result of 50% residual factor indicates 1 Bethesda Unit of inhibitor, a level of 25% indicates 2 BUs of inhibitor, and a level of 75% residual factor indicates ~0.5 BUs of inhibitor. Any results >80% residual FVIII are taken to mean no inhibitor is present (or is below the assay detection limit). If a result suggests of an inhibitor, we then reflex to the full Bethesda assay with dilutions.
Oh, I should also mention that we don’t run sample dilutions for our patient factor assays; reasons for this are given in our RPTH paper (attached – open access so feel free to provide to others). If factor levels are ≤20%, we repeat to confirm, and if confirmed and unexpected we run our inhibitor screen. Most factor assays we do are normal or increasingly are high since we get many requests for patients being investigated for thrombosis.
And from west coast colleague Bob Gosselin: Heat-treat the sample 56ºC for 30 minutes to get rid of the FVIII activity, then run the Bethesda assay test. We did that for any factor value >10% as the presence of factor activity does not necessarily rule out an inhibitor. Our lower level of quantification (LLOQ) for the regular Bethesda assay was 0.3 BUs using NPP/Owren veronal buffer solution as the FVIII calibrator so when we run the patient samples against that calibration curve, the extrapolated results will equal residual factor activity. However, with heat treating, we had to bump up the LLOQ to 0.5 BU.
No comments here.