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More on Mixing Studies: Dr. Ortel

We’ve had three posts and several comments regarding lupus anticoagulant (LA) mixing studies since March. These are…

In these posts several experts held that it is necessary to continue with LA detection studies even when the initial mixing study demonstrates correction. Their rationale is that a weak LA is “washed out” or neutralized in a 1:1 mix, and thus may go undetected. The LA may yet be detected and confirmed using the dilute Russell viper venom time (DRVVT) “screen and confirm” and the partial thromboplastin time (PTT)-based hex-phase phospholipid neutralization method.

On Friday, May 4, 2012, Thomas Ortel, MD, PhD, Director, Anticoagulation Management Service and, Duke Clinical Coagulation and Platelet Immunology Laboratories, Duke University Medical Center, Durham, NC, spoke on LA profiling at the Thrombosis and Hemostasis Summit of North America in Chicago. His position parallels those of the experts who contributed to the previous three posts. He contends that it is necessary to perform the LA detection assays even when the mixing study corrects, given the potential for a weak LA. He further recommends that the reagent pooled normal plasma mixing study always be included as part of the profile. This is because the original test order is most likely based upon some abnormal finding and the mixing study may detect and identify some additional, otherwise undetected abnormality. The addition of mixing study results to the DRVVT and PTT screen/confirm results provides a complete picture and helps develop the final findings. Dr. Ortel added in a private follow-up that in most cases, he sees mixing study results whose “correction” is borderline or equivocal.

Comments (1)
Lupus Anticoagulant
May 11, 2012 6:11am

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, his patient was on Lovenox (LMWH). Neutralization of anticoagulant activity of metabolized LMWH in the DRVVT assay is not reliable and sometimes not reproducible, partially because of a short incubation step (~3 min at 37 C) and poor performance of most heparin neutralizers used in reagent formulations. This could explain why after dilution with pooled normal plasma and lowering the titer of Lovenox a correction was observed. Second, the above case was at acute thromboembolic event, which is warned about clearly by ISTH (An Update of 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 the clotting time. So LA-mixing is different from a routine mixing study in respect to platelet-debris content.
Is the 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 (+3 SD) higher than normal plasma samples (TH 1991, 320) to be considered LA-positive. By the way, there are exceptional situations in which people claimed that 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 decent mixing study miss LA-effect? It depends…
To answer some concerns raised by Herb Crown around the dilution of LA-antibodies due to mixing step with PPPNP, I have to say everything depends on sensitivity of the method (instrument/reagent/cutoff calculation). If 2X 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, PrecisionBioLogic

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