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A Summary of Mixing Studies

George: I was looking at different coagulation methods, possibly doing factor testing and mixing studies.  We are looking at various instruments, one being the ACL TOP.  I was wondering if there was a list of the specific disease states and the correction factors involved.  For example, if a specimen was corrected by the addition of normal pooled plasma, but the Russell viper venom test was abnormal, what disease state would this indicate?  It would be nice to have a chart for quick reference.  Thanks for your time.

Julie Schartiger MLT (ASCP)
Holzer Medical Center
100 Jackson Pike
Gallipolis, OH  45631

Hi, Julie. Thanks for your question. I’d like to start by linking you to our educational modules on lupus anticoagulant and on laboratory detection of thrombosis. I think most of what you need may be provided there. Also I’ll mention that the Russell viper venom time test is seldom used, although the dilute Russell viper venom time assay is an important component of the lupus anticoagulant profile.

I’ll also include a summary that helps you follow up a prolonged PTT here:

When the PTT is prolonged beyond the upper reference interval limit, first determine there is no heparin present by performing the thrombin time. A prolonged thrombin time indicates heparin, which may be neutralized using Hepsorb®.

The absorbed or heparin-free patient plasma is next mixed 1:1 with reagent normal plasma (pooled normal plasma, PNP, NP) and the PTT repeated immediately on the mixture. Reagent normal plasma is usually purchased from a distributor who prepares it employing good manufacturing practice (GMP).

If the mixture PTT corrects to within 10% of the previously determined reagent normal plasma PTT, and the patient is bleeding, suspect a coagulation factor deficiency. Proceed with factor activity assays, assaying the most likely first, often factor VIII.

If the mixture PTT fails to correct to within 13% of the reagent normal plasma PTT, and the patient is not bleeding, suspect lupus anticoagulant. Lupus anticoagulant confirmation is available from reference and specialty laboratories.

Some lupus anticoagulants and most specific factor inhibitors, particularly anti-factor VIII, are time- and temperature-dependent. If the PTT corrects to within 10% of the reagent normal plasma PTT, prepare a new mixture and incubate one to two hours at 37°C. If the incubated PTT fails to correct to within 13% of the incubated PNP PTT, an inhibitor is present. Proceed with lupus anticoagulant confirmation or detection of a specific factor inhibitor.

PTT mixing studies, as all coagulation assays, are performed using platelet-free plasma. The specimen is centrifuged so that the plasma platelet count is <10,000/uL. If a mixing study result is equivocal, meaning if it falls between 10 and 13%, ensure platelet-free plasma is being used and repeat the study.

Julie, in the spirit of blatant self-promotion, you may also want to purchase “Quick Guide to Coagulation Testing,” available from AACC Press. This is an inexpensive pocket-size handbook that has all the information you need. Geo

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