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Mixing Studies: Incubate Control Plasma?

From Louise Pleiss, MT (ASCP)Southcoast Medical Group: We recently changed our mixing study procedure to include rerunning of the normal pooled plasma (NPP) after a 2 hour incubation as one of our system controls, but we are having a difference of opinion about what should happen to the NPP. One site says that the partial thromboplastin time (aPTT, PTT) will increase to about 42 s due to heat labile factors. Our site does not agree and we think we should get nearly the same value as when we first ran the NPP before incubation. Do you have any data to support either side? Thank you.

Hello, Louise and thank you for your question. I agree with your outside site, though they may be overestimating the degree of prolongation. You should expect your normal control plasma clotting time to lengthen by two or three seconds during a 2-hour incubation at 37°C as factor VIII activity becomes reduced. Among other references, Domen RE. Labile coagulation factor activity in pooled random donor platelet concentrates. South Med J. 1988;81:418 indicates the degree of factor VIII activity loss during incubation. The best approach is to compare the PTT result of the incubated NPP-test plasma mix with the PTT result of the incubated, and not the initial NPP to determine correction. Please feel free to follow up with any questions or additional data. Geo.

Comments (3)
Mixing Studies
Herb Crown
Jan 15, 2014 1:10pm

In addition to the above excellent comments I would emphasiz
In addition to the above excellent comments I would emphasize the importance of establishing a range for your “new control”. Doing such would eliminate the “guess work” of determining what should be seen. Once your range is established, the question changes to, “did the assay perform within the established parameters?”.

Herb Crown
St. Louis University Coagulation Reference Lab

Jan 14, 2014 12:38pm

I agree with Ali comment and would like to suggest about 20%
I agree with Ali comment and would like to suggest about 20% of FVIII depletion in 2 hours at 37C based on these findings:

The percent residual factor VIII activity (FVIII:C) after incubation at 37 degrees C for 4 hr was higher in warfarinized patients than in normal subjects; 76.9 +/- 10.8% of the initial F.VIII:C in the patients versus 61.6 +/- 5.8% in normal subjects (p less than 0.001).

From Takahashi H, Hanano M, Tatewaki W, Shibata A.Factor VIII lability, protein C and other vitamin K-dependent proteins. Thromb Res. 1986 Sep 1;43(5):561-8.

Jan 14, 2014 8:29am

Hi George,
One thing very important here is also the level

Hi George,
One thing very important here is also the level of Factor-8 sensitivity of aPTT reagent they used in their observations. Without knowing the exact method and reagents they used, it is hard to speculate something accurate. Details need to cover instrument they used, how NPP has been prepared, initial factor levels in NPP, etc. If we focus on only factor-8, I recall from an article that calculated F8 deterioration rate is 5% per hour at room temperature, so at 37C after 2h the impact would be noticeable. However, as I have already said, the initial level of Factors in NPP and factor sensitivity of reagnet/instrument they used will ultimately determine the magnitude of impact.

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