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More on LA Mixing Studies

I received this message from Dr. Rich Cambereri on March 8, and have been holding it pending additional data, but given our March 18, 2012 post from Vanessa Chan in Toronto, and subsequent responses from Herb Crown and Dr. Larry Brace, I’ve decided to use Dr. Cambereri to start a discussion about the use of mixing studies in the lupus anticoagulant (LA) profile:
Hi George, I do heme-onc and have a new cluster of “alleged” LA patients that has prompted me to revisit the dilute Russell viper venom time (DRVVT). One 48 y/o man has had DVTs, PEs, an IVC filter,  etc. He has been on low molecular weight heparin (LMWH, Lovenox). The DRVVT done at Mayo, Scottsdale had the initial mix normal (corrected) after the screen was prolonged. This, as I understand it, is incompatible with an inhibitor as normal plasma corrects the prolongation. They proceeded with the confirmatory step of phospholipid adsorption and the ratio compared to control was 1.6, consistent with LA.
1. What’s the story? If the initial mix corrects, a “deficiency”should only apply to factor V and X?
2. Why would one proceed to the confirmatory step if the mix was not still prolonged?
Thanks, George. PS: I’ve just repeated the DRVVT and the Staclot LA hexagonal study; pending. Patient still has clotting issues.

Given Herb’s and Larry’s comments on Ms. Chan’s post, I’m inclined to conclude that, because the effect of a weak LA can be diluted by the normal plasma in a mixing study, it may be necessary to continue to the confirmatory high phospholipid reagent step. This, of course, contradicts ISTH guidelines, which imply you can discontinue the profile if the mixing study corrects. It also raises the question, “Why do the mixing study at all?” I’ve distributed this question to a few experts, watch for more discussion here. Meanwhile, Dr. Cambereri, based on the discussion, it appears Mayo, Scottsdale is doing the right thing!

Comments (3)
Lupus Anticoagulant
May 11, 2012 6:10am

Hi George, I found this discussion very interesting. Before
Hi George, I found this discussion very interesting. Before we could make any judgment regarding the value of mixing study in LA-detection, I like to clarify a few things in case presented by Dr Cambereri.

First, patient was on Lovenox (LMWH). Neutralization of anticoagulant activity of metabolized LMWH in dRVV assay is not reliable and sometimes reproducible, partially because of a short incubation step (~3 min at 37 C) and poor performance of most heparin neutralizer used in formulations. This could explain why after dilution with pooled normal plasma and lowering titer of Lovenox a correction was observed. Second, the above case was at acute thromboembolic event, which is warned clearly by ISTH (an update guidelines for LA-detection, JTH 1737, 2009) “Caution should be exercised in interpretation of the results of tests performed close to thromboembolic event as patients may be treated with UFH and/or VKA”. Third, application of platelet poor pooled normal plasma (PPPNP) is critical for LA-mixing study because any phospholipid released from platelet could correct clotting time. So LA-mixing is different from routine-mixing study in respect to platelet-debris content.

Is mixing study important in detection of LA? Yes
It is absolutely necessary. Since, it will help to overrule all factor deficiencies or possibility of vitamin K antagonist therapies as the cause of prolongation of LA-screen tests. Don’t forget, result of mixing should be significantly (+3SD) higher than normal plasma samples (TH 1991, 320) to be considered LA+. By the way, there are exceptional situations that people claimed LA-cofactor proteins need to be added to patient plasma for maximum prolongation of clot time by LA-antibodies (controversy, but I recall a report/question on this blog that even more prolongation was observed after mixing study in LA-detection). Obviously it makes sense to run PPPNP mixing prior to running more costly tests but sequence of testing is not crucial for interpretation of result, for instance Staclot-LA is an integrated (screen/confirm) mixing test.

Could a descent mixing study miss LA-effect? It depends ……
To answer some concerns raised by Herb around dilution of LA-antibodies due to mixing step with PPPNP, I have to say everything depends on sensitivity of method (instrument / reagent / cutoff calculation). If two times dilution of patient plasma brings titer of antibodies below limit of quantification of test then obviously we have a problem and result of our PPPNP-mixing assay would be a false-negative. Therefore, if you are using a kit with a poor LA-sensitivity which is not optimized for mixing study or lacking a validation study including an adjusted cutoff to support its performance under mixing condition, this is a wrong choice. However, in practice discovery of a weak LA has a very limited clinical value because most of the times it indicates transient situations (infection, inflammation, acute phase reactions, drug interactions, or even not fully blown LA). This is why repeating LA-test after a few weeks (12 weeks according to ISTH guideline) is required to clarify presence or absence of real LA antibodies.

Dr. Ali Sadeghi-Khomami,

Apr 20, 2012 5:51am

Hi Scott, your comment is interesting and I can definitely s
Hi Scott, your comment is interesting and I can definitely see your point. In addition to questioning the 50/50 mix component of the DRVVT, we also include a 50/50 mix as an integral part of the popular hexagonal phospholipid LA test system. That seems to me to automatically introduce, by default, a dilution of the antibody.

This is just me thinking out loud, but it would seem to me that performing the hexagonal LA study withOUT the 50/50 mixing component would be a more valid study. In this scenario, since an alleged factor deficency would be present in both the buffer tube and the hexagonal tube to the same extent, the NPP adds nothing to the study (other than polybrene which could be added to a different component of the test system).

Herb Crown
St. Louis Coagulation Reference Laboratory

Apr 6, 2012 8:07am

George, I have always wondered at the utility of a mixing st
George, I have always wondered at the utility of a mixing study as part of an LA screening. These comments over the last few weeks here only strengthened my opinion. If the accuracy of a DRVVT-based screen/confirm is in question for some reason, it seems like one would should simply go directly to a hexagonal study. How old are the ISTH guidelines? Are there any recent studies to support using one set of tests over another? Scott Miller, St. Mary’s of Michigan.

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