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Bethesda Titer

Cassandra McGeachy, Louisiana Coagulation, asks, I am trying to evaluate a set of FVIII inhibitor results and I am not sure what to call it. My patient + normal pool on my Bethesda assay yields a 23% FVIII and the next 4 serial dilutions bounce around between 15 to 19% instead of giving a higher value. Since this is a possible acquired FVIII inhibitor case I asked the tech to repeat the test using the Nijmegan method. This test method basically yields the same thing with the patient + pool giving 15% FVIII and the next 5 serial dilutions yielding results of 15 to 19% but no higher. What causes this and how to you report the result? Do you know of any references for this pattern demonstrated when performing the inhibitor titer?

Hello, Cassy, and thanks for your question. I’m not sure I have an answer for you, but I want to get this posted to learn if anyone has had a similar situation. I’m assuming you’ve run a lupus anticoagulant profile on the plasma and ruled out an LA as an interference? Also a thrombin time for heparin? I found a recent case study that seems to parallel your situation, Abt NB, Streiff MB, Gocke CB, Kickler TS, Lanzkron SM. Idiopathic acquired hemophilia A with undetectable factor VIII inhibitor. Case Rep Hematol. 2014;2014:484563. doi: 10.1155/2014/484563. Epub 2014 May 14. The article is a free download, but I’m not sure it actually provides an answer.



Comments (7)
Bleeding Disorders
Mar 26, 2015 8:44am

Hello folks, what an
Hello folks, what an interesting question and I’ll do my best to add a few more.
Our protocol requires that we use 10 dilutions, to 1024, for each inhibitor assay we set up. Most often, the additional dilutions are of little significance, but if necessary I can test them and result the patient without any lose of time. As mentioned additional dilutions may be helpful with this patient.

One more observation: you reported a Factor VIII of 23% on a 50/50 mix. Assuming your control tube was 50%, that would yield a residual Factor VIII of 30% which calculates to a 1.2 Bethesda Unit Titer. I, like you, am skeptical of this result (but I am a skeptical person).

That leads me to a few questions; what is the factor VIII content of your normal pooled plasma? What is the factor VIII value of your NPP/Owens buffer tube? If your normal pooled plasma is “boofed” (not sure what the writer means,edit by HC, “boofed”: slang for compromised), that can give you the exact results you are experiencing (assuming there is no inhibitor present).

Additionally, the clinical condition of the patient is a key component to this discussion. If this is indeed an acquired factor VIII inhibitor, with a Bethesda titer of >200 as suggested, this patient would be bleeding significantly.

With medical director’s approval I would be tempted to roam off the reservation. Since acquired inhibitors have “interesting kinetics” it may be helpful to reset the assay in 2 ways. The first way I would set it up is with 10 dilutions and incubate for 2 hours and test. Concurrently, I would set up 10 dilutions and and incubate for 4 hours and test. I have had to do this before and I maybe able to find the data at some later date. The pH of your NPP will be fine, we use a product that we evaluated the pH over 6 or 7 hours and noted the pH was stable.

I would love to hear how this progresses.

Herb Crown
St Louis University Hospital Coagulation Reference Lab

Mar 24, 2015 3:44pm

I will look for the articles
I will look for the articles mentioned by Dr Favaloro. I will try retesting using higher dilutions and see if I get a better pattern of results.

Mar 23, 2015 11:59pm

The serial dilutions noted: 5
The serial dilutions noted: 5 serial dilutions: means you have only taken this to 1:32; which means that your BT is >32; you probably need to go to at least another 5 serial dilution points; otherwise try 1:5 dilutions as suggested. The inibitor titre is probably between 200-1000 Bethesda units.

Mar 23, 2015 7:15pm

On behalf of Cindy Johns,
On behalf of Cindy Johns, LabCorp: I was just looking at our LabCorp SOPs to verify how far out we dilute specimens for Bethesda titers and found that Esoterix Coag will dilute out to 1:2560. So this agrees with Dr. Favaloro’s opinion. It also makes perfect sense, in addition to your suggestion to verify that there isn’t a lupus anticoagulant present, although a saline mix should help to distinguish that as well.

Mar 23, 2015 5:36pm

Sorry. In my earlier comment
Sorry. In my earlier comment I said that the residual FVIII levels did not go above 50%. That was not the calculated residual FVIII values. I meant that my sample FVIII values after a 2 hour incubation did not reach 50% of my pool value. Therefore technically speaking I did not reach a value high enough to calculate for the inhibitor titer.

Mar 23, 2015 4:40pm

This is very representative
This is very representative of an ‘atypical’ or complex high titre inhibitor. You need to dilute further. Try serial 1/5 dilutions instead of 1/2 dilutions if you want to reduce the number of dilutions you do; then if you overshoot the mark you can repeat with dilutions around the point where you are getting 50% (or between 25–75%) residual activity. We have seen this pattern in patients with inhibitor titres >200 BU. Several suggested papers talk about this: Tiede et al. Semin Thromb Hemost 2014;40:803–11. Kershaw & Favaloro. Pathology 2012;44:293–302. The Nijmegen method will not resolve the discrepancy – you just need to go further with the dilutions.

Mar 23, 2015 3:18pm

Yes, George. We did rule out
Yes, George. We did rule out Lupus anticoagulant and heparin. I had reviewed the article mentioned. The FVIII baseline in my case was decreased as was noted in the article. Technically speaking you report the Bethesda titer when you have a residual FVIII equal or higher than 50% of the pool normal. But in this case I never recover the 50% and my residual values do not change with increasing dilutions of the patient plasma. Therefore I do not know how to report a titer in this case.

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