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Incubated PT Mixing Studies?

From Lori PinelliGlens Falls Hospital: Hello George. Our current prothrombin time (PT) mixing time procedure only includes running the initial 1:1 mix along with the patient and the normal pooled plasma (NPP). Twice in the past year, I have been asked to perform an incubated PT mix in addition to the initial analysis. Both patients had normal activated partial thromboplastin times (aPTTs, PTTs) and elevated PTs. They were both factor VII deficient and corrected upon addition of NPP. I was asked to perform the incubated mix to rule out an inhibitor. Are there time/heat dependent Factor VII inhibitors? Should I be routinely performing the incubated mix for PT correction studies? Thank you!

Hi, Lori, and thank you for your question. I farmed it to several colleagues who have mixing study expertise, and so far have received responses from Dave McGlasson andDr. Larry Smith. I anticipate additional responses, however meanwhile, both Larry and Dave confirmed my opinion that there is no value in performing an incubated PT mixing study.

To summarize their comments, if both the PT and PTT are prolonged, perform only the PTT mixing study. If the immediate mix corrects, perform the incubated PTT mixing study to determine if a specific inhibitor, usually anti-factor VIII, is present. Anti-factor VIII is an IgG antibody that may require 37°C incubation to be detected. Additionally, some weak lupus anticoagulants (LAs) don’t show up until after incubation.

If only the PT is prolonged, the likelihood of an LA is low. The phospholipid concentration of the PT reagent exceeds the phospholipid concentration of the PTT reagent and is consequently less sensitive to LA. According to both Larry and Dave, the greatest likelihood is a factor VII deficiency, as you have found. This requires no incubation.

In the past, anti-factor II (prothrombin) and anti-factor V antibodies would be detected in patients who had been treated with bovine thrombin based fibrin glue in a prior surgical procedure. The antibodies arose as cross-reaction to the bovine thrombin and could generate severe bleeding as they neutralized most available factor V and prothrombin. We see fewer of these antibodies now as distributors have switched to human thrombin, though there may still be some around. The anti-factor II and anti-factor V antibodies could be detected in either the PTT and in the PT mixing study without incubation. Dave McGlasson told of a patient whose anti-factor V antibody was triggered by Keflex antibiotic therapy and who experienced severe hemorrhage.

Finally, Larry Smith suggests that when the PTT is normal and the PT is prolonged, avoid the PT mixing study altogether and go straight to a factor VII assay for the best and most timely results! Please watch this post, it is likely to attract additional comments. Geo.

Comments (3)
Mixing Studies
Jan 23, 2014 2:03pm

Hi Chris, I am the pathologist who gave that recent lecture.
Hi Chris, I am the pathologist who gave that recent lecture. The case I presented was that of an EDTA plasma submitted for factor V activity and inhibitor studies, including PT mixing studies. As it turned out, the entire order was one big error as the physician actually intended to order factor V Leiden mutation analysis.

Since we had the PT mixing study data, I included them because I thought the pattern of prolongation in the incubated mix as compared to the incubation control for our EDTA plasma was interesting. Factor V inhibitors have been reported to show time dependence in some cases (e.g., Br J Haematol 1975; 29:397-404; Am J Hematol 2011; 86:710-712 (with commentary and another case report in Am J Hematol 2012; 87:313-315)). In the case I presented, you would have to consider the possibility of the wrong sample type, and then you would need some basic chemistry tests (potassium, calcium) to make a diagnosis of EDTA plasma. Admittedly, this is not a typical problem in hospital laboratories dealing with primary tubes, but it does come up from time to time in reference laboratories dealing with aliquots.

Karen Moser

George Fritsma
Jan 22, 2014 12:29pm

Posted on behalf of Chris Ferrell, Special
Posted on behalf of Chris Ferrell, Special Coagulation Lead, Harborview/University of Washington: Up until this point, I haven’t incubated my protime mixes. Just recently, Stago gave a webcast from a pathologist at Esoterix.  One of the cases she presented was discovered using an incubated mix on the PT. I didn’t keep the handout because it was a very rare situation. It may have been a temperature and time dependent lupus that affected the PT and not the PTT. Maybe someone in this group may have listened in to that webcast and remembers.

As far as inhibitors I’ve seen directed at the extrinsic system, none of them have been temp/time dependent. We’ve had both bovine and human thrombin inhibitors. The thrombin inhibitor also had a very strong lupus that balanced out the bleeding and the clotting issues. I’ve also seen factor V inhibitors associated with a necrotizing insect bite. All of them have been immediate acting. I would love to see a factor VII inhibitor before I retire, but it’s unlikely. I’m told they’re extremely rare.
If someone asked me to incubate a PT mix, I would ask them what they were looking for and what literature are they reading so I could also educate myself.

Jan 21, 2014 12:00pm

Hi George,
We know a sub-population (~10-30%) of lupus anti

Hi George,
We know a sub-population (~10-30%) of lupus anticoagulant (LA) antibodies is time-dependent. It means their inhibitory effect like anti-F8 shows up after longer incubation at 37C. Also some believe that dilute PT (dPT) could discover a sub-population of LA. These inhibitors are targeting coagulation factors in the extrinsic pathway that aPTT or dRVVT assays cannot discover (i.e TF-F7-TFPI inhibitors). So, considering the heterogeneity of inhibitor antibodies, in theory at least, there is a merit to do dPT and incubated version of that. I am not sure about practicality of this approach for LA testing though, i.e. I don’t know in practice what would be the rate of success in LA diagnosis. Bear in mind, there is another valid argument against the value of prolonged incubation at 37C for LA-testing, reasoning that pH changes and its artifact on clotting time is responsible for difference in results.
Alternatively, I am wondering if this test has anything to do with emerging recombinant products to the market that affecting extrinsic pathway (e.g. NovoSeven RT, rF7a). They might want to assess no inhibitor induced by rF7a in the patient! See the link:

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