A message from Rose Markarian, Providence Health & Services: What is the purpose of incubating pooled normal plasma (NP) separately and the patient’s plasma separately for 60 minutes? Then after 60 minutes of incubation you mix them together and run to get a PTT and this is labeled as control tube. Please explain!
Thank you for your question, Ms. Markarian. The typical approach to the 1:1 PTT mixing study begins with patient plasma that has a prolonged PTT. Mix 1:1 with NP and repeat the PTT on the mixture. If the PTT result of the mixture is shortened to within a selected limit, the result indicates “correction.” It then becomes necessary to perform an incubated mixing study. For the incubated mixing study, a common approach is to incubate a aliquot of the 1:1 mix and an aliquot of NP, and repeat the PTT on both. If the result of the mix is shortened to within the selected limit, the mix is “corrected,” leading to the conclusion that the original prolongation was caused by a coagulation factor deficiency. If uncorrected, the operator continues testing, looking for an inhibitor such as anti-factor VIII.
The key to your question is, “how do we set the limit?” Many use a fixed PTT such as the upper limit of the reference interval or another arbitrarily selected limit. For those who choose to employ a fixed limit, the incubated NP is unecessary. Others prefer a limit based on the NP, reasoning that the mix result is relative to the NP PTT, not to the reference interval. In this case, many select an arbitrary value such as 10% longer than the NP. Performed this way, it is necessary to inclubate the NP along with the 1:1 mixture, because it’s PTT prolongs as a function of factor VIII deterioration. Assuming the factor VIII deterioration is proportional in each of the two tubes, the new mix is logically compared to a limit base upon the new incubated NP value, which is always prolonged.
Many of us choose a slightly more elaborate approach, the “Rosner” index, based on the formula: Rosner Index = (1:1 mix PTT–NP PTT)/patient PTT X 100. Others use the Chang index, where % Correction = (Patient PTT–1:1 Mix PTT)/(Patient PTT–NP PTT X 100. In either case, the patient PTT becomes part of the computation, and the patient plasma must be inclubated along with the NP, presumably so the level of patient factor VIII deterioration is accounted for. There are no standards that dictate a fixed limit, percentage limit, Rosner Index, or Chang percentage, however it is necessary to choose a consistent approach in your laboratory.
I hope this answers your question. I avoid using the term “control” when discussing mixing studies, because by strict definition, NP is not a control. By avoiding “control” I also hope to reduce mixing study confusion, as we are only comparing the mix result to the NP result and not to a control. It is conceivable that a laboratory director would insist on assaying a true control alongside a patient specimen, indeed, this approach is now being applied to Nijmegen-Bethesda titers, however, for now, no one is doing that (and I hope we never will).
Also, I chose not to address the incubation period. Some insist on 60 minutes, and some 120 minutes. The CDC established 120 minutes for the Nijmegen Bethesda assay, so they advocate for a 120-minute mixing study incubation. Of course, 120 minutes also introduces the additional variable of aliquot evaporation. There is also no firm guideline for incubation time.
If you will pardon some self-promotion, I’m presenting a talk on mixing studies at the Northeast Laboratory Conference in Portland, Maine on Tuesday, October 17, 10–11:30, please plan to attend!