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Mixing Studies and the Thrombin Time

This question arrived as a comment from “skierktc;” I have reproduced it as a post here:

I am preparing to update our laboratory’s procedure for prothrombin time (protime, PT) and partial thromboplastin time (PTT) mixing studies and wonder if anyone screens additionally with the thrombin clotting time (TCT, TT) or the TT plus protamine sulfate to rule out heparin in the specimen as the source of
prolongation. If yes, what is their policy for continuation of the study? We have encountered physician insistence even when heparin effect is evident.

Hello, and thank you for your question. Yes, when you encounter a prolonged PTT that is not corrected by the normal platelet free plasma in the first step of your mixing study, perform a thrombin time to determine if the patient is receiving therapeutic unfractionated (standard) heparin. Heparin will prolong the thrombin time from its normal mean, which is often around 18 or 19 seconds, to 40 seconds or longer. You can confirm using protamine sulfate, which neutralizes heparin, however you are reasonably safe to conclude that heparin is the culprit without taking this step. You may also confirm using the anti-Xa heparin assay.

Once you have concluded heparin is present, treat the specimen with Hepsorb or Hepzyme, then repeat the mixing study. The same rules may apply to a PT mixing study, though they are rarely performed and heparin has relatively little effect upon the PT.

You also asked, “What commercial controls do you use for the pathogenic control for both PT and PTT mixing studies?

Generally, no normal nor any “positive” or pathological controls are employed in mixing studies, as the results are compared directly to the original PTT prolongation.

I hope this is helpful. You may also review our Audio Modules 7 and 8 for details on mixing studies and lupus anticoagulant testing. Geo.

Comments (1)
Nov 22, 2010 1:35pm

Hi George and “skierktc”, I think I will take a stab at this
Hi George and “skierktc”, I think I will take a stab at this as well. As a reference laboratory we are often presented with plasmas for inhibitor studies. These samples are presented to us with little clinical information. More often than not, these are irretrievable specimens. Under these circumstances we use the following testing scheme: 1) perform a PT, aPTT and thrombin time (TT). If the TT is significantly abnormal we will treat the plasma with Hepzyme, repeat the aPTT and the TT. If heparin is the offending agent, the aPTT may or may not correct, but the TT will correct. We will use the treated plasma to perform the mixing study consisting of the aPTT performed immediately and after 60 minutes incubation at 37 degrees. (By the way, if you perform a mixing study on heparin contaminated plasma, if will be a false positive.)

We also have a “home-grown philosophical control” consisting of Factor VIII deficient plasma and normal pool plasma mixed 1:1 and tested at immediate time and after 60 minutes incubation at 37 degrees. (Our “control” for PT mixing studies consist of normal pooled plasma and absorded plasma mixed 1:1.)

We have established normal reference ranges for the patient results and controls at zero time and 60 minutes. (I have previously written about this and I am sure George can link up.) Under this testing protocol, we are evaluating the patients mixing study results to a reference range rather than to itself.

You do raise an interesting question about controls. While it is not our practice, I think one could certainly build an abnormal control using a lupus anticoagulant positive plasma (available commercially) mixed with normal pool plasma. This control would be your inhibitor positive pathogenic control.

A second control could be constructed using normal pooled plasma and abnormal factor plasma (available commercially) and test at zero time and 60 minutes. This would evaluate and control your test system in the presence of a factor deficiency.

A third control could be constructed using normal pooled plasma and a Factor VIII Bethesda positive plasma.

Also mentioned is physician reluctance to canceling a mixing study when heparin is the identified as the offending agent. It is important to remember that even though heparin is removed from a plasma, a mixing study may still be of value whether it is positive or negative. It is also important to remember that just because a plasma tests negative for inhibitor studies doesn’t mean an inhibitor is NOT present. And lastly, one should remember to perform a mixing study at zero time and after incubation, enabling the differentiation between a factor inhibitor and a lupus anticoagulant both of which have significant implications for the patients under our care.

Herb Crown
St. Louis University Coagulation Reference Laboratory

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