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Test for LA When Mixing Study Corrects?

From Vanessa ChanSick Kids Hospital, Toronto:

Hi George, What is your opinion on the algorithm of tests when testing for lupus anticoagulant? If the mixing tests are normal, should we continue with a confirmatory test? The recent BJH guidelines say, “Mixing tests are a criterion for LA and improve the specificity. However, they introduce a dilution factor and may make weak LA samples appear negative. In the absence of any other causes of prolonged clotting times, such samples should be considered LA positive if the screen and confirmatory tests on undiluted plasma give positive results. Whenever possible, this should be confirmed by testing a fresh sample.” This indicates that a confirmatory test is done even when mixing studies correct (appear negative). Responses from other labs and clinicians are most welcome!

Hi, Vanessa, and thank you for your question. When a patient specimen has a prolonged partial thromboplastin time (PTT) and you have ruled out heparin, you first mix 1 part patient platelet-free-plasma with 1 part platelet-free pooled normal plasma (PNP) and performing an immediate PTT on the mix. If the result “corrects” to within 10% of the PNP PTT, incubate 1–2 hours at 37°C and repeat, comparing the result to the result of incubated PNP. Some LAs are time- and temperature-dependent, so you may detect an LA upon incubation.

If the immediate and incubated mixing study results both correct, but there is high suspicion of LA, some lab operators repeat both immediate and incubated PTT mixes using 4 parts patient plasma to 1 part PNP, which provides greater sensitivity for the LA. If the 4:1 mix corrects in both phases, LA may be ruled out and it is not necessary to continue with the LA screen and confirm test. In all cases, ensure the patient specimen and the reagent PNP are platelet-free.

I hope this helps, and I invite comments, as labs all tend to approach this question in different ways.

Comments (3)
Lupus Anticoagulant
May 10, 2012 8:15am

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 the case presented by Dr Cambereri.

First, the 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 an 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 a 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 (+3 SD) 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 the 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,

Mar 25, 2012 2:30am

Hi, Herb, and thank you for your comment. I had a conversati
Hi, Herb, and thank you for your comment. I had a conversation Thursday with Dr. Larry Brace of Edward Hospital in Naperville, IL. Larry confirms both main points of your statement, that if there is suspicion of an LA you should definitely proceed with the screen and confirm test, and you should always use at least two detection systems. The two most often used in the USA are a PTT-based assay such as Stago’s Sta-Clot LA and a DRVVT such as Precision Biologic’s Cryocheck LA-Check and LA-Sure. Other detection systems are the dilute thromboplastin time and the kaolin clotting time.

Mar 22, 2012 7:26am

Hi George, and thank you Vanessa for your question. This is
Hi George, and thank you Vanessa for your question. This is an interesting question that can be interpreted in a couple of ways. The first interpretation could be, “what is the role of aPTT and or PT mixing studies in identifying a lupus anticoagulant (LA)?” George does a good job of answering that question and the only addition I would make is the aPTT mixing study has limited utility in detecting LA and if your mixing studies are negative, you should proceed with LA testing.

The second interpretation of Vanessa’s question is this; in requiring LA testing to be performed using normal pooled plasma (NPP) as a diluent causing us to dilute out the LA and thereby make it undetectable. This question has been bothering me for a number of months and prompted me to review a study we performed a number of years ago and Dr. Heinrich Joist presented at an ASH Meeting in New Orleans.

We did not directly ask whether diluting the patient plasma with NPP causes us to not identify a LA, but we have data that we can look at that will answer the question. Our LA study asked, what is the most advantageous strategy to detect a LA considering costs, time, sensitivity and specificity. We used a number of test kits and lab developed tests (LDT) and tested 485 samples from our sample bank.

I decided to look at our data from the dilute Russell Viper Venom test (dRVVT). Traditionally the dRVVT testing scheme includes 3 components; screen, mix and confirm. The screen component is performed by taking a patient sample and adding reagent and waiting for the sample to clot. The mixing component is performed when the screen is abnormal by mixing the sample with NPP and adding reagent and timing it to clot. The confirm step is performed when the mix step is abnormal, and is performed by adding dRVVT reagent rich in phospholipid to the test plasma and timing to clot.

For our LA study, we performed all three components of the dRVVT concurrently rather than sequential. This actually gave me the data to answer partially, Vanessa’s question. I reviewed the data asking the following question; “is there a sample where we would see the dRVVT Mix negative but the dRVVT Confirm positive?” This question is important because traditionally we would stop the testing if the dRVVT Mix is negative. After reviewing the data from 485 patients I can tell you we saw 7 patients (1.4%) that would have been reported out as dRVVT negative based on stopping the testing at the mix. (As an additional caveat, 4 of those 7 patients were STA-CLOT LA (Stago) positive which leaves 3 patients still being reported out as LA negative.)

This discussion leads us to draw 2 conclusions. The first is for proper detection of LA the laboratory must use a multitude of tests as there is not a “one shot” miracle test. The second conclusion, to Vanessa’s question, is under certain criteria we may be diluting out the lupus anticoagulant.

Herb Crown
St. Louis University Hospital Coagulation Reference Lab

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