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Incubated Mixing Studies

From Ruth Spates: Hi George, our mixing study procedure requires the patient plasma and the pooled plasma to be incubated separately, and then mixed, and the PT or PTT performed. This is in addition to the 1:1 mix being incubated and retested, and the separately incubated patient plasma and NPP to be retested. I don’t recall the reason why we do this additional mix?? What are your thoughts? Thank you!

Hello, Ruth, and thank you for the question. Operators who use either the Rosner or Chang index to establish their correction/no correction decision limit incubate the patient plasma, normal plasma, and the 1:1 mix in order to apply the formula. The Chang index provides a percentage limit as follows:

% Correction = Patient PTT – 1:1 Mix PTT / Patient PTT – Normal plasma PTT X 100. A typical limit is 75%

Chang SH, Tillema V, Scherr D. A “percent correction” formula for evaluation of mixing studies. Am J Clin Pathol 2002;117:62–73.

Here is the Rosner index, which reports as a ratio:

Rosner index = 1:1 Mix PTT – Normal plasma PTT / Patient PTT X 100. A typical limit is 11

Rosner E, Pauzner R, Lusky A, Modan M, Many A. Detection and quantitative evaluation of lupus circulating anticoagulant activity. Thromb Haemost 1987; 57: 144-147.

Many of us use a percentage difference, typically 10%, or a fixed PTT value such as the upper limit of the PTT reference interval. In this case, we incubate only the normal plasma and the mix and compare the differences.

However, I don’t know of any index or correction/no correction limit that requires testing a 1:1 mix of the incubated patient and normal plasma, perhaps one of our participants uses this approach and can provide an answer.


Comments (3)
Mixing Studies
Nov 28, 2018 4:06pm

Incubated mixing studies are
Dr. Connie Miller: Incubated mixing studies are best for detecting FVIII inhibitors. Specific FVIII inhibitors show progressive inhibition over time. The Bethesda assay uses 2 hours, based on studies by Allain and Frommel (Blood 44: 313, 1974) and others. They show time-course graphs. The slow interaction of FVIII and antibodies is thought to be due to steric hindrance by the large size of the von Willebrand factor with which FVIII is complexed.

Sep 10, 2018 1:38pm

Putting on my biochemist hat,
Putting on my biochemist hat, I always thought it mysterious why incubating for a whole hour at 37 deg C should change the results of these mixing assays. Why not 5 min? 10 min? 20 min? Simple noncovalent biochemical interactions shouldn’t take an hour to establish.
[Response from George, could this be one of those “expert opinion” principles?]

Jun 19, 2018 8:56am

We use this same procedure,
We use this same procedure, and had an excellent example of why just last week. Patients with acquired inhibitors will (hopefully) show an enhanced prolongation with incubation in their 1:1 mix. When you make another 1:1 of the incubated straight patient and PNP, that 1:1 should look more like the initial rather than the incubated time. It also shows that the enhanced prolongation is not due to factor degradation during the incubation. We do this for PTTs only–we do not incubate PT only mixes.

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