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No Need for Prolonged LA Incubation

From Thomas Exner, Owner, HAEMATEX RESEARCH, Sydney, Australia:
Hi George, I read your response to a question about lupus anticoagulant (LA) mixing tests in a handout at the SSC meeting in Liverpool recently. I’m a little disappointed that you still recommend prolonged incubation times for these. Most recommendations now are that LA mixes should be tested immediately and not with incubation.
There is no logical reason for time-dependent LA testing since they work by interfering with the phospholipid added in the reagent at the time of testing. Yes, you may see prolongation of an inhibitor effect on activated partial thromboplastin times (PTTs, APTTs) with some test plasmas but it is an artefact due to pH increase. I investigated this interesting effect many years ago and it was published somewhere, though not in great detail. (Exner T. Conceptions and misconceptions in testing for lupus anticoagulants. J Autoimmun 2000;15:179–83) The same problem was noted in FVIII Bethesda assays a while ago and fixed by the Nijmejen people by including a pH buffer. Uncovered unbuffered citrated plasmas drift to pH above 8.5 over an hour at 37ºC and this knocks out a lot of the factor V.
Time dependent inhibitors are more typical for FVIII antibodies and should be looked for for that purpose, not for LA. Sorry to disagree with your recommendations in this case. Best wishes from Tom Exner.
From George: Thank you to Tom for this important update. My comment suggesting that some LAs are time- and temperature-dependent appears on a handout prepared for distribution at the International Society on Thrombosis and Haemostasis Scientific Subcommittee meeting in Liverpool, June, 2012. From anecdotes, I know of several laboratories that incubate their mixing studies for the purpose of detecting LAs as well as specific inhibitors such as factor VIII inhibitors, so I appreciate the opportunity to disseminate this information, and would like to hear from others on the subject.

Comments (1)
Lupus Anticoagulant
Jul 27, 2012 9:19am

Hi George, Tom and Fritsma participants. If there is one to
Hi George, Tom and Fritsma participants. If there is one topic that has prompted more discussion on this forum than the aPTT mixing study, I am not aware of it.

The aPTT mixing study suffers from a number of well characterized weaknesses. The procedure seems simple enough, but it does not have a universally recognized standardized procedure and the method of interpretation varies widely. Few laboratories have normal reference ranges and generally use some type of interpretation that refers back to the patient’s aPTT.

The aPTT mixing study has 4 primary uses; a) to aid in the detection of a Lupus Anticoagulant(LA), b) to aid in the detection of a factor inhibitor c) to aid in the detection of a factor deficiency and d) to provide direction when working up a patient sample that unexpectedly presents a prolonged aPTT.

When a patient is known to have a Lupus Anticoagulant the aPTT mixing study is of limited utility to further define the abnormality. The aPTT mixing study is not necessarily sensitive to a LA. In our laboratory, about 50% of our LA patients show a normal aPTT and a normal aPTT mixing study. There are better tests to determine whether or not a patient has an LA.

When a patient is known to have a factor deficiency a mixing study incorporating an incubation step should be performed to rule out an inhibitor. Most often, when a sample shows a normal mixing study at immediate mix and prolongation after a 60 minute incubation at 37º a factor inhibitor is generally suspected. A normal aPTT mix before and after incubation would indicate a factor deficiency. Again, this is not very specific but is helpful and one part of a comprehensive evaluation.

More often, we see the greatest value of the aPTT mixing study in the context of helping to explain a prolonged aPTT. We are often called upon to work up a patient’s plasma that the physician wants to know why the aPTT is unexpectedly prolonged. After ruling out the presence of heparin we will perform the aPTT mixing study with a 60 minute incubation. This will drive the direction of the rest of the work up. If the mixing study is normal before and after incubation, we would work up factors. If the mixing study is abnormally prolonged before and after incubation, we would work up the specimen for LA. If the mixing study is normal before incubation and prolonged after incubation we would look for a factor inhibitor followed with a Bethesda Unit inhibitor study.

I earlier used the terms “aid” and “provide direction” for a very specific purpose. The aPTT mixing study is not 100% specific or 100% sensitive. It is one part of a complete set of tools we use in the coagulation laboratory to help us help the doctors help their patients. It is not the “end all” of all things coag. As an isolated test, on a patient with a known disorder, the aPTT mixing study is of limited utility. As part of a prolonged aPTT workup, the aPTT mixing study, with incubation, is a valuable tool to help guide the workup.

This is a basic understanding of the aPTT mixing study. There are certainly exceptions to the above discussion. We see those exceptions on a routine basis. One thing predictable about coag is its unpredictability.

Herb Crown
St. Louis University Hospital Coagulation Reference Laboratory

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