Vilas Hiremath has an LA-positive patient with recurrent pregnancy loss. She is on LMWH with a request for an anti-Xa assay. He asks about interference.
George assures Dr. Hiremath that the chromogenic anti-Xa heparin assay provides an accurate measure of unfractionated heparin, LMWH, and synthetic pentasaccharides (fondaparinux) in the present of LA.
“Guest Blogger” Dr. Emmanuel Favaloro on LA chronicity, raised by Pam Owens of TriCore Laboratories, Inc.
When an LA/APA is defined as “chronic,” does it wax and wane?
Not normally, but you have to take into consideration the lab doing the test (s) and the individual patient circumstances. In regards to lab testing, there are both false positive and false negative LA/APA test results. Patients may alternatively have transient LA/APA related to infection, etc. Patients with true LA/APA may get better and then have relapses. So, with most cases of true LA/APA, persistence and repeatability of testing can be shown. But sometimes this isn’t the case.
Or does intermittent positivity more likely signal transient innocent responses to drugs or inflammation?
See above response. It is important to retest in ~12 weeks to confirm the initial finding and check persistence. No point testing once and then rechecking every few years-how do you know the first result was a real/persistent LA/APA; could have been a false positive.
How did Dr. Pengo’s committee come up with the 12 weeks interval?
I’m pretty sure Dr. Pengo’s committee just ‘copied’ the 12-week period from the APA guidelines (reference given). The original guidelines said six weeks, but I believe that some experts on the revision committee had experience with some ‘transient’ LA/APA that ‘persisted’ for longer than 6 weeks, so they moved this interval to 12 weeks. I don’t think there’s a lot of scientific evidence around the specific value of 12 weeks, but this should be long enough to exclude transient LA/APA due to drugs/infection/etc.
Finally, from our EQA experience, we know that samples can be collected from patients with LA/APA every year or so (with their permission of course) for EQA challenges, and sometimes their levels change and at other times they are quite ‘stable'; however, although the medians from returned data are similar between collections, the inter-lab ranges are always wide-and importantly are reported as negative in some labs but positive in others. So, as I said; you have to be wary about repeatability of LA/APA; when patients are sometimes positive and sometimes negative, is this from testing in the same laboratory, and is it a good laboratory?
Pam Owens‘ response: Thanks George and Dr Favaloro! We would like to think we are a good lab and are only looking at the results we have previously generated. Our biggest bug is preanalytical. Being a reference lab, we do not get the luxury of processing all our specimens and always have this in mind when looking at inconsistent results.
Kayla Brown, Pharm D Candidate 2012, Drake University, asked if elevated HGB/HCT can affect INR. A patient’s last HGB = 20.1 mg/dL and HCT = 62%. How would these values would affect the INR?
George responds that most anticoagulation clinics employ a POC instrument such as the CoaguChek or iStat to perform PTs and compute PT/INRs. POC instruments are cleared to report PT/INR when the HCT is 25%-55%. Outside that range, a standard blue-closure venous specimen is collected and accurately assayed using a plasma-based method in the “central” laboratory. The plasma-based assay is accurate when the HCT is 65% or less. When the HCT is over 65%, the ratio of anticoagulant volume in the collection tube to plasma volume is proportionally elevated, falsely raising the PT/INR.
This message contained an error that was correct in a comment from Lauren Nutley: I think you meant to type 55%, not 65%. Lauren is correct.
“Abi” freeze-thaws platelets and aliquots them in 50 mL tubes stored at -80°C. After this he processes 2-3 rounds of freeze thaw cycles before using these platelets in MSC cultures.
1. Can we process this 2–3 rounds of freeze thaw cycle in advance, say a month prior to use, for MSC culture?
2. Will the growth factors be affected if we freeze thaw them in advance?
3. Or is it efficient to freeze thaw (2–3 rounds) before the day of the MSC culture experiment.
4. Which method do you think is more efficient for cell proliferation?
George is not an expert on preparation of platelet growth factors, and posted the question for our expert participants. He speculates that the growth factors are stable at –80°C and that the freeze-thawed preparation would be stable for at least a month.
From Pam Owens, TriCore in Albuquerque: Given that a PT is stable for 24 hrs in the original tube with the top never removed, how long is the sample good once the top has come off? If the plasma has been removed, frozen and thawed, what is the PT stability then?
George has not found stability data for fresh, recently opened specimens, but CLSI H21-A5 requires thawed specimens to be “tested immediately.” The pH of opened specimens changes rapidly, and pH profoundly affects enzyme reaction rates, so it is appropriate to require that either fresh or thawed open specimens be tested immediately.
Vanessa Chan, Sick Kids Hospital, Toronto asks, in LA testing, if the mix corrects, 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.
George responds, 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.
Herb Crown, St. Louis University, further responds. “What is the role of PTT and or PT mixing studies in identifying a lupus anticoagulant (LA) if PNP dilutes LA and make it undetectable? The PTT mixing study has limited utility in detecting LA, so if your mixing studies are negative, you should proceed with LA testing.”
Herb reviewed a study performed a number of years ago that Dr. Heinrich Joist presented: “We 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 for DRVVT and STA-CLOT LA. The standard DRVVT scheme includes screen, mix, and confirm. The screen is performed by adding reagent to a sample and waiting for the sample to clot. For the mix, the sample is mixed with NPP, reagent added timed to clot formation. The confirm step is performed when the mix step is abnormal, add DRVVT reagent rich in phospholipid to the test plasma and time it to clot.
We performed all three components asking, “is there a sample where we would see the DRVVT mix negative but the DRVVT confirm positive?” This question is important because we would otherwise stop the testing if the mix is negative. We saw seven positive patients (1.4%) who would have been reported out as DRVVT negative based on stopping the testing at the mix. Four of those seven were STA-CLOT LA (Stago) positive, which leaves three patients still being reported out as LA negative.
We concluded that 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 is that under certain criteria we may be diluting out the LA.
In a second response, Dr. Larry Brace of Edward Hospital in Naperville, IL confirms both main points of Herb’s 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.
“Begumra” asks: Deficient factor in case of prolonged PT and PTT result that corrected with the addition of NPP. All other screening tests are normal. I think it is factor V. Can you suggest that it is right?
George responds, assuming the patient is not on heparin or coumadin, and does not have liver disease or vitamin K deficiency, a prolonged PT and PTT that are corrected upon an immediate and incubated mixing study indicate a possible single factor deficiency of either prothrombin (factor II), factor V (as you suggest), or factor X. This combination of results could also indicate a severe fibrinogen deficiency, however the PT and PTT only prolong when fibrinogen is below 100 mg/dL. Most choose to perform the fibrinogen assay to confirm fibrinogen level.
Sue Bergs, Madison Area Technical College, read about monitoring heparin therapy using the chromogenic anti Xa assay instead of PTT in ASCLS Today. Is that standard practice?
In 2009 George ran a “Quick Question” to learn how many laboratory directors have switched to the chromogenic anti-Xa heparin assay. It was 16%, but hopefully, more have changed since. The anti-Xa is accurate, precise, and not prone to error related to high fibrinogen, high factor VIII, or the presence of plasma-based inhibitors.
Emily Oakley works in the special coag lab of a tertiary care hospital in West Tennessee. She has encountered problems with patients coming in on dabigatran who need percutaneous stent procedures. Typically when the cardiologist uses heparin during the catheterization he orders an ACT post-procedure looking for a target ACT of 150 seconds. Of course, we are not achieving this with dabigatran patients. The cardiologists are ordering ACTs every 4 hours to get to this target. We cannot support this type of request with personnel around the clock. We are not sure what to do next since there is no literature to help us.
George asks, “Are these emergency patients who cannot be delayed the recommended 24 hours for dabigatran to clear? If so, choices are limited.” The ACT is the only assay that accurately monitors unfractionated heparin therapy up to 5 units/mL. The test is usually performed by the anesthetist in the cardiac catheterization lab or operating suite, where cardiologists use 1-5 units/mL during the procedure. The cardiologist administers protamine sulfate at the conclusion of surgery to neutralize heparin, and monitors with the ACT to confirm the return to normal, <150 seconds. Once accomplished, the patient may continue to be monitored, if necessary, by the central laboratory using the PTT or the anti-Xa heparin assay. The ACT is seldom performed in the central laboratory, but in Emily’s case, dabigatran slows the ACT return to normal so the patient continues to be monitored for up to 24 hours.
There exists no FDA-cleared method for monitoring dabigatran, though at least three are being readied: Aniara’s dilute thrombin time and Stago’s Ecarin clotting time and Ecarin chromogenic assay. Meanwhile, many laboratory directors are using the PTT as a semiquantitative estimate of direct thrombin inhibition. My recommendation is to compare and eventually substitute the in-lab PTT for the ACT until the patient returns to normal, or alternatively to adopt one of the new methods on the research use only basis.