From Tom Exner, Haematek: Hope you had a good 4th of July…
Thanks very much for including me sometimes in your interesting online discussions. This time I have an issue on which I would appreciate your advice and possible comments from others if you think it might be suitable for your online interest group.
Regarding significance of weak dRVVT LA test results.
The dRVVT test is favoured by many labs as being simple and reliable for LA. Most labs seem to find 10–20% of “LA positive” results with this test (using current reagent suppliers) in a hospital patient setting based just on “Screen/Confirm” or “LA-1/LA-2″ ratios. This is a much higher incidence than I used to find when I was working at a lab bench. Many such cases display only weak LA and are often on warfarin or other anticoagulants at the time of testing. The combination of thrombotic episode plus LA leads to a diagnosis of anti-phospholipid syndrome according to current classification guidelines.
I would like to know if these weaker LA are “true” LA or if they have been induced somehow by the effect of anticoagulant treatment on the dRVVT. Of course DOACs are well known to mimic LA with the most popular dRVVT reagents, (but not with some newer ones). I’m mainly concerned about the effect of vitamin K antagonists (VKA).
How many such patients with weaker LA whilst on VKA still show abnormal dRVVT Screen/Confirm ratios indicative of LA when the VKA is discontinued or when the INR normalizes. (Note that INR and “Confirm” results are also affected by VK deficiency, and not just VKA).
I would be grateful for any case reports of patients losing their weak LA status and consequently ADS diagnosis when taken off AVK.
Hi George and Tom,
Hi George and Tom,
Very interesting discussion regarding weak LA positivity. Establishment of reference interval and cut-off points were briefly alluded to in the discussion but I think an important issue that determines the rate of positivity and was not specifically addressed in the current discussion, is how the reference interval was established(+/- 2 or +/-3 standard deviations?) in the papers cited.
Dear Dr. George Fritsma,
Dear Dr. George Fritsma,
According to the Devreese KM 2012 slide presentation at the 20th Annual BSTH Meeting in Antwerp about LAC testing and acute phase reaction:
Analytical interference of C-reactive protein: imitates aPL binding of CRP to negatively charged phospholipids (PL) and can influence test results for LAC, causing false positive results:
• Type of PL in the reagent is important
– PTT-LA and Staclot-LA (PTT) are sensitive
– No interference observed for dRVVT reagents
• Interference increases with increasing CRP (positive at >8.6 mg/dL)
Another recent paper from Ruinemans-Koerts J, et al. When to screen for lupus anticoagulant? Influence of testing during acute phase and consequences for clinical practise. Lupus. 2015; 24: 1233–5. suggests that almost all specimens with positive APTT-LA only (negative dRVVT) had CRP>80 mg/L and all of them became negative in >12 weeks with concomitant CRP level normalization.
Direct comparison of two dRVVT reagents performance among patients positive with LA or treated with VKA/DOAC was also recently published:
Depreter B, Devreese KM. Dilute Russell’s viper venom time reagents in lupus anticoagulant testing: a well-considered choice. Clin Chem Lab Med. 2016 Jun 22. [Epub ahead of print]
http://www.ncbi.nlm.nih.gov/pubmed/27331311
Thanks also to Ali for a
Thanks also to Ali for a comprehensive discussion of this issue. I wonder if the weak positives we detect using LAC assays are in reality cross-reacting inflammatory proteins such as C-reactive protein. See a recent publication: Just SA, Nybo M, Laustrup H, Hansen IJ, Junker P, Vinholt PJ. Single test isolated lupus anticoagulant positivity is associated with increased plasma levels of inflammatory markers and dyslipidemia. Lupus. 2016; 25: 24–-7. See also: Schouwers SM, Delanghe JR, Devreese KM. Lupus anticoagulant (LAC) testing in patients with inflammatory status: does C-reactive protein interfere with LAC test results? Thromb Res. 2010; 125: 102–4.
George posts this comment by
George posts this comment by Ali Sadegh-Khomami, PhD, lead scientist at Precision BioLogic Inc.
Hi everyone,
Here are my comments on Tom’s concern about the rate of weak LA positives detected by dRVVT and potential factor deficiency interferences:
1) LA positive tests need to be repeated after 12 weeks for ultimate assay interpretation. This is written in the guideline (JTH-2009-1737) for a good reason but perhaps it was not explained explicitly why. This is because “to get rid of transient or lupus-like antibodies in plasma samples,” which are typically detected as weak positive results.
2) According to the ISTH guideline, samples for LA testing preferably need to be obtained in the absence of anticoagulant therapy (e.g. Coumadin, heparin and DOACs). Unfortunately this is not a quite practical recommendation to follow; reference labs usually have no information regarding patient medication history or stopping anticoagulant therapy is not always a risk free option for patient. In a small comparative study using various commercial dRVVT products (un-published data), I have noticed different susceptibility to anticoagulant interferences by different products. This could explain why the committee has recommended “not to test plasma samples from patient on anticoagulant therapies.” However, I have not seen any study on patients with vitamin K deficiency (potential bleeders) as questioned by Tom.
3) Does a persistent weak lupus anticoagulant really exist? I have seen article showing that a fraction of patients with weak LA results ended up with thrombotic events. Also I have seen articles not believing in weak LA at all; over time these samples either turn to normal or a distinct LA positive. Well, in the absence of a gold standard it is hard to say who is right but results are really depend on investigation set-up (inclusion/exclusion criteria) or analytical sensitivity of reagent used in each study.
4) A true diagnosis is not really applicable to LA assays because LA is only a risk factor. Association between LA positivity and future thrombotic events could only be confirmed by prospective cohort clinical studies. Lupus anticoagulants are non-specific antibodies, so they are not really defined yet. Despite tremendous research efforts on lupus anticoagulants, our molecular mechanistic knowledge about them is very poor. So we have a long way ahead of us in order to find out the truth. For these reasons, we should not expect to see a gold standard assay for LA testing very soon.
5) In addition to reagent and method of LA testing, prevalence of LA positive results also depends on attributes of the population that plasma samples were collected from. For instance, in-patient samples vs. out-patient samples, samples analyzed in a cancer center, veterans’ hospitals, fertility centers, etc. A quick literature survey indicates that LA prevalence reported as 5% in Germany (Miesbach W. et al. 2006), 6–12% in Italy (Tripodi A. et al. 2013, Pengo V. et al. 2005, Mameli A. et al. 2011), 10% in USA (Proven A. et all. 2004), up to 19% in Japan (Habe K. et al. 2013). I have no comment on justification for LA testing requested in those publications but it is interesting to mention that even 2–3% LA positive results are obtained in 106 healthy individuals (false positives). Although an international survey needs to be consulted for better estimation, it seems inclusion criteria is the key to avoid false results.
6) Application of inappropriate decision making cut-off has an impact on the rate of samples with borderline results (weak LA positive and false negative results).
7) If LA positive prevalence of 10–20% by dRVVT is suspiciously high, then we need to really watch out for Staclot-LA with LA positive findings of around 30–35%. Regards, -Ali
Thank you to Robert and Dave
Thank you to Robert and Dave for your comments, I look forward to seeing others’ responses. I echo Dave’s position, we should not be performing DRVVTs on patients who are on VKAs. Likewise, we should not be performing LAC testing on inpatients.
George posts this comment on
George posts this comment on behalf of Robert Gosselin at the University of California at Davis:
I have been in conversation with TE and he was surprised at our “positivity rate,” but had that number confirmed by others at the SSC meeting in Montpellier.
Here are my anecdotal observations (as I shan’t be encumbered by facts):
1. There are more weak positive LAs by DRVVT with traditional ratios of 1.21–1.29 in the inpatient population.
2. In this group, my personal feeling is that mixing studies have limited value, given the 1:1 ratio of sample to normal pooled plasma (NPP).
3. As the lyophilized reagent ages, I see a trending of more of these increased ratios, which makes me wonder about the screening method’s reagent stability.
4. I use both fresh and frozen prepared reagent (can freeze and thaw once) to determine my reference range–not sure if that is good or bad, but mimics clinical practice.
5. Many of the weak positive results as inpatients, tend to go away upon repeat. So are we seeing some other drug effect other than anticoagulation? Perhaps we are seeing stuff in the inpatient population (alas, we shouldn’t test as inpatient).
6. We rarely have patients with PERSISTENTLY slightly positive LAs in the range between 1.21– 1.29.
7. We’ve had fairly robust positive LAs (~1.30–1.40) that met the criteria (mix, prolonged APTT, etc) but was negative using other methods. So are those negative or positive or unequivocal?
8. We hedge all of our weak positive LA results with a “could be,” but also with comment about potential interferences associated with anticoagulants, antibiotics, etc.
Lastly, what is truth? Without standards, or a real confirmation method, it is difficult to determine an LA. Is there a difference between transient LAs and the real deal? I asked Jacob Rand about whether he tested transient LAs and whether there was a difference between transient LAs and “true” LAs in the formation of macromolecular processes/disruption of the annexin V shield as he described at the last NASCOLA meeting. It would be nice if we had something better to ferret out weak LAs versus test interferences. By the way, JR had not tested transient LAs.
But this is just my humble opinion as a lab mule…
Always a pleasure to hear
Always a pleasure to hear from Dr. Exner. My first question is why test for the presence of an LA when a subject is on any anticoagulant that could possibly compromise the test results? Years ago we researched the effect that coumadin (VKA) had on the platelet-neutralization procedure. We discovered a high incidence of false positives with that procedure. Since the VKA also affect the levels of FX and FII, the DRVVT screen/confirm assay outcomes could be compromised due to low factor levels with the INR in the therapeutic range. Further, the low molecular weight heparinoids and the unfractionated heparin meds levels depending on the dosing may not have enough polybrene reagent to over-ride the heparin affect. Therefore, any testing on anticoagulated patients should be repeated once the subject is off of the medication.
Some information regarding
Some information regarding incidence and follow up of patients with LA-positive testing (where positive dRVVT was in 20 of 53 or 37.7%) but negative aPTT or dRVVT mixing studies are presented on the link below: Alkayed K, Kottke-Marchant K. Indeterminate lupus anticoagulant results: Prevalence and clinical significance. Korean J Hematol. 2011; 46: 239–43.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3259515/