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Interpreting Inhibitor Mixing Study in a Hemophilia A Patient

I [Geo] received a physician’s question on 1-31-24:

Dear Sir, Hope you are doing well. Thank you for creating this platform and sharing valuable resources with the world.
We present a case of Hemophilia A with recent retroperitoneal hematoma and multiple transfusions–PRBC, FFP, Factor VIII, and NovoSeven.

  • APTT of Test Plasma: 47 seconds [RI 22.2–33.1 seconds]. Most recent FVIII transfusion was 12 hours before.
  • FVIII of test plasma: 18.3%
  • APTT of control plasma: 24.7 seconds [FVIII: 88%].
    Inhibitor Screening [Mix]:
  • APTT (test + Control plasma) at Zero hour without incubation: 35.2 sec (Rosner index is 22.3, Chang 52.9%)
  • APTT (test + Control plasma) at one hour incubation: 34.8 sec
  • APTT (test + Control plasma) after 2 hour incubation: 33.8 sec (Rosner Index is 21.9, Chang is 60.2%)
  • APTT of 2 hours incubated control plasma was 25.1 seconds.
  • Lupus anticoagulant testing by DRVVT was negative.
    I have the following questions
    1. According to Rosner and Chang index, inhibitor screen is positive, although the trend in APTT doesn’t show an increase in APTT with time.
    2. Should a Bethesda assay be done in such cases?
    3. Should we treat the patient as inhibitor positive based on this evidence?
    Thank you Sir.

My response: Hello, doctor, and thank you for your email. Based on your mixing studies, it appears that your patient has an FVIII inhibitor and that a Bethesda titer or Nijmegen assay would be appropriate as you follow the patient’s treatment plan. There are some reliable chromogenic Nijmegen-Bethesda assays available.
Your immediate mix is uncorrected. While most anti-FVIII inhibitors are IgG4, which are time- and temperature-dependent, a few are detected upon immediate mix. Of course, when a mixing study is uncorrected upon immediate mix, the first possibility is a lupus anticoagulant. Although your DRVVT results are negative for LAC, you may wish to perform a PTT-based LAC assay such as the Sta-Clot LA. The Sta-Clot LA and DRVVT are not confirmatory for each other, rather their results may stand alone, as LACs have varying specificity. The presence of a LAC could account for the parallel results of your 1-hour and 2-hour incubation mixing studies and could render the Bethesda titer unnecessary.
Nevertheless, since your patient is diagnosed with Hemophilia A, do you have access to emicizumab [HemLibra] therapeutic? HemLibra bypasses the effect of an FVIII inhibitor and has a prolonged half-life, unlike NovoSeven. HemLibra is approved for use in hemophilia A worldwide with or without an inhibitor.

Response 2-1-24: We do have emicizumab and is being used in patients who can afford it. Thank you for your opinion, Sir

Dr. Emanuel Favaloro: yes, they need to do a Bethesda assay; although incubated mixes not clearly showing temperature/time dependence, the mixing tests themselves suggest an inhibitor.

Robert Gosselin: Are they sure they are dealing with isolated HA?

Some commentary on 2-1-24: From me [Geo] I’m curious about the technical protocol. If uncorrected in the immediate mix, why do incubation? Why do both a 1 and 2-hour incubation? Some skip the immediate altogether and go straight to the 2-hour on the assumption that some LACs require incubation and some FVIII inhibitors show up in the immediate mix.

Dr. Emanuel Favaloro: we don’t bother with any incubated mixes; too hard to train and maintain skills for the 200+ techs doing routine coags in our 60+lab 80+instrument network; we just do the factor levels (at select sites) and if it looks like an inhibitor, we do either an inhibitor screen (mix factor levels) or Bethesda.

Robert Gosselin: I think it is more about what is used to determine “correction” versus “no correction.” Likely there will always be outlier cases but the vast majority follow the “rules” of testing outcomes.. I had a non-neutralizing VII inhibitor in a burn patient.

Dr. Favaloro: Sure, but the APTT of control = 24.7s; APTT of patient = 47s; mix APTTs = 34-35s, so about halfway, suggestive of inhibitor; if they assess correction as return to within NR, then could almost be ‘corrected’ by definition, but clear to me that this is an inhibitor event; whether FVIII inhibitor or something else I can’t say without FVIIIs on the mix plasmas or a Bethesda assay.

Comments (1)
Mixing Studies
Ali Sadeghi-Khomami PhD
Feb 13, 2024 11:32am

Hello George, Not all FVIII-inhibitors are time/temperature dependent.So we shouldn’t always expect more inhibition after a longer incubation.The case presented here is dRVVT negative (no PL dependency of inhibitor on the common pathway) but an inhibitor positive in a mixing APTT study. I agree with others that further inhibitor testing is required. Cryocheck Hex LA is an integrated mixing study that could be used in the detection of PL-dependent inhibitors in the intrinsic pathway.If Hex-LA is negative, then specific factor inhibitors could be tested by APTT-based serial dilutions (non-parallelism) as screening tests and followed by quantitative titration (Bethesda) of inhibitor as the confirmatory test (the most common: FVIII 2h incubation, FIX 1h incubation, and even for other factors such as AT, vWF and ADAMTS13?).
One clarification about Rosner Index (ICA, Index of Circulating Anticoagulant) though. Rosner only suggested a formula for percent correction in a mixing study (Thrombosis and HAemostasis-1987-144).The initial work described an immediate mixing KCT assay requiring three testings a) patient plasma sample b) control normal plasma sample, c) 1:1 mix of control NP and patient plasma samples. Later colleagues adopted his calculation method for prolonged (1h or 2h) incubations mixing study at 37C. Those need to incubate control and patient as well as 1:1 mix plasma for the same length of incubation (like vs. like approach). If investigators are interested to the kinetic and temperature dependency of inhibition, in addition to immediate mix, then they might do an immediate mixing assay of incubated samples too. Since the long incubation could impact pH and factor activity levels of non-buffered plasma, a variable hard to control in patient plasma samples, buffered systems were incorporated in Bethesda-Nijmegan FVIII inhibitor mixing assays to mitigate this risk.

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