What do you report for patients without LAC whose factor level increases with dilution? What CV do you use? Which number would you report for this?
Example: ECAT 25.46 FXII
Conc Dilution FXII
100% 1:10 21.60%
50% 1:20 28.60%
25% 1:40 36.30%
% CV of CR triggers further dilutions (high curve):
Conc Dilution FXII
12.50% 1:60 32.60%
6.25% 1:180 33.70%
First curve:
Mean CR: 32.2%
% CV of CR: 17.63
Mean CR 100% 28.7%
% CV of CR 100%: 25.5%
High curve:
Mean CR: 33.2%
% CV of CR: 2.325
Mean CR 100% 27.9%
% CV of CR 100: 33%
We reported >33%
Thank you for your help!
- Do the dilutions differentially affect interactions with PK and HMWK?
- Do the dilutions differentially affect contact activation?
4-24-2026: Expert colleague Dr. Emmanuel Favaloro recommends avoiding routine multiple-factor assay dilutions for parallelism without indications and cites his open-access article, Favaloro EJ, Pasalic L. Should multiple-factor dilutions be performed for all patient coagulation factor assays? Let the debate begin! Res Pract Thromb Haemost. 2022;6:e12689. doi: 10.1002/rth2.12689. PMID: 35308100; PMCID: PMC8918913.
4-25-2026: Expert Dr. Ali Khomami-Sadeghi replies that this question is probably best addressed directly by ECAT coordinators and Werfen technical support, as parallelism behavior could be something they want to assess in the exercise and is often assay-system specific. If this is a general issue for factor XII assay on ACL TOP analyzer, then we should find out how the abnormally low FXII QC control passes the parallelism test.
When a factor assay fails the parallelism criterion on ACL TOP (CV >20%, r² <0.98), it usually indicates a matrix/analyte difference between the sample and the calibrator used in that specific assay setup. The analyzer attempts to remove outliers and offer a better estimate, which helps in borderline situations like the case discussed here. It’s worth mentioning that non-parallelism findings may not be transferable across platforms/reagents—for instance, a silica-based aPTT might be less forgiving than ellagic acid-based reagents for certain factor inhibitors, or a lyophilized calibrator might behave differently than a non-lyophilized plasma sample for a particular factor.
That said, CLSI H48 supports using multiple dilutions (≥2) in factor assays to assess linearity and detect potential interferences, with standardized dilution approaches generally improving analytical reliability and interlaboratory comparability (as shown by some proficiency data analyses and presentations). The debate introduced by Dr. Favaloro and Pasalic raises a valid point, since in the majority of cases, minimal clinical value is added with extra dilution work—especially when inhibitor detection is not a concern. I could also add to the argument that the non-linearity of engineered recombinant factors is another upcoming issue, since most OSA calibrators contain native human factors, and non-parallelism with EHL recombinant factors (PEGylated, fused albumin, Fc-conjugated factor proteins) at low levels shouldn’t be a surprise when analytes in the calibrator and sample even differ slightly.
In this particular case, where parallelism failed for 3 serial dilutions but passed after outlier removal, I would report the ECAT Mean of CR (>32.2%, as it increases with further dilution) rather than the Mean CR 100% (average of 3 dilutions which failed by CV), with a note about the non-parallelism observation including assay details (reagent, calibrator, diluent). Interestingly, your further dilutions to reduce sample-calibrator differences also support a better estimate achieved via post-outlier removal:
25% (1:40) = 36.3%
12.5% (1:80) = 32.6%
6.25% (1:160) = 33.7%
Average: 34.2% (CV 6%)
Questions from colleague Bob Gosselin, 4-26-2026
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These are Werfen/IL reagents being used on the TOP instrument?
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This is only seen with factors XI and XII?
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Are there no other factor inhibitors present (i.e., strong inhibitors to factors V, VIII, etc)?
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Why are these factors being tested?
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Are there other results you can share on these samples to help interpret the “artwork” that is coagulation testing?
Hello, just an update. This has been frustrating. Others also have issues distinguishing FXI deficiency from LAC. Here is one report PMID: 38358905. It seems we have a patient population enriched for patients with LAC and a question of FXI deficiency. There is a direct-to-consumer genetic testing for pregnant women that looks at “carrier status” for FXI, so maybe that’s why I am seeing a lot of this. I can’t find a good reason to do higher dilutions for FXI and FXII, so when the CV is high, reporting the 1:10 is one solution. That way the error is not propagated by multiplication.
But I was thinking, has anyone tried substituting a different silica-phospholipid reagent for the PTT reagent in FXI and XII tests? As an alternative to purchasing an instrument or doing a send-out to Siemens whenever we encounter this? The SCT confirm in particular comes to mind. It looks like just a PTT with extra phospholipid, which is exactly what would help.
Correction: the target was 25% (CV 17%)
Bob:Re Werfen reagents: This is the same specimen using the Werfen setup and Werfen reagents-
100% (1:10) : 24.6% repeat 23.6%
50% (1:20): 27.7%
25% (1:40): 29.3%
12.5% (1:80): 33.0%
6.25 (1:160): 33.7%
With no extra dilution trigger, we would have reported either 28.5% or 24.6%, and the ECAT value was 25.0%. So we would have passed.
The difference may be that we are going out to higher dilutions when we shouldn’t be. Is anyone willing to share their criteria for going to 1:60 and 1:180? Maybe those dilutions are best avoided. It’s true that the Werfen reagent with the Werfen test has a tighter CV than the PB, and so we may have not done the dilutions at all.
I just need to either come up with a protocol for how to handle NON LUPUS increasing with dilution specimens or change the CV flags OR switch to Werfen factor deficient plasma.
Now that I think about it, there will be variation lot-to-lot in the amount of phospholipids in factor-deficient plasma, so just switching to Werfen may not solve this.
I think you guys cracked it–thanks!
Also, for those who don’t know, CAP’s software can’t handle a > sign. Until recently, ECAT did not either. That can result in a lab getting it wrong–really wrong–and still having it look green or “acceptable for all analytes” per cap. Recently, this happened for D-dimer at NCH, and it’s also the reason I missed the creep of the dilutions. Sacha.
Key questions:
Q1: did your abnormally low QC control passed parallelism test using your assay configuration? If yes, then your assay working fine and non-parallelism might be related to ECAT or samples. If no, then there is a problem associated with your assay. You need to swap APTT, calibrator, F-def and QC controls till you find out the culprit.
Q2: you mentioned target was 17% FXII, is this total participant average in the report or the average reported by SynthAsil users?
Thank you both so much;
We reported >33%. The target was 17.1%.
Our Werfen rep suggested this was because we are using PB factor deficient plasma, rather than Werfen. I don’t understand how that would make a difference.
Although I used an ECAT example, it’s happened with several patients. In two FXI-deficient patients, we reported a level that was too high.
I think that is because it’s those specimens that lead to the 1:80 and 1:160 dilutions, and that compounds any error.
I was thinking maybe the PB and Werfen factor deficient plasmas have different amounts of contact factors and phospholipids, and that may be important at very high dilutions.
We did one single test–the ECAT one I show here–after using the Werfen deficient plasma and the werfen set-up–it’s not as bad, but it still goes >30% at higher dilutions.