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Mixing Study Dilution

From Mahnaz Sairi, Hello, George. We use pooled normal control plasma (PNP) from George King and lately found out that mixing buffer with PNP and using that as a control is confusing our  physicians. Because they are asking why the control results are increasing? I really do not know why we are diluting the  plasma with buffer. Can you please help?

This is from our procedure manual:

  1. Run APTT on normal control plasma and patient plasma.
  2. Mix control plasma(pool plasma) 1:1 with Koller Buffer.
  3. Mix patient plasma 1:1 with control plasma (pooled plasma).
  4. Run APTT on the two mixtures.
  5. Incubate remainder of the mixtures for 2 hours, 37C in a water bath.
  6. Repeat APTT on both incubated specimens.

Hello, Mahnaz Sairi, and thank you for your question. I know of no reason for preparing a dilution of the PNP. Both the immediate and the two-hour incubated mix result should be compared with the undiluted PNP. When performing the two-hour mixing study, the PNP is incubated alongside the mix so that you can compare the mix result with the incubated PNP.

There is a modification of the mixing study called the “saline mix.” In this approach, in addition to the PNP mix, you can prepare a separate 1:1 patient plasma, buffered saline mix. Here is a quote from the 2002 Esoterix Coagulation Coagulation Handbook.

“If the PTT result obtained with the immediate PNP-patient plasma mix corrects and the saline mix result is prolonged dramatically, a factor deficiency or specific factor inhibitor is suspected. With factor deficiency, heparin, or a specific factor inhibitor, the 1:1 saline mix shows significant prolongation. If the result of the immediate PNP-patient mix shows only partial or no correction, and the result obtained with the saline mix shows near correction, then lupus anticoagulant is suspected.”

I don’t know of many who perform the saline mix, though at Esoterix Coagulation we found it helped resolve euivocal results.

Back to your question, I invite our participants to provide your mixing study experience. Do you know of instances where it makes sense to dilute the PNP? Also, have you ever used the saline mix? Thank you.

Comments (1)
Mixing Studies
Mar 10, 2016 1:26am

I am wondering here if
I am wondering here if someone has confused APTT mixing studies with factor inhibitor mixing studies? It is common to use normal pool plasma mixed with buffer 1:1 as the comparator plasma (which is then defined to have “100% residual factor level”) against the same normal pool plasma mixed 1:1 with patient plasma (or its dilution) when the patient plasma is suspected to have an inhibitor (e.g., to FVIII). If there is no inhibitor, then the patient/normal plasma mix will have close to 100% residual factor levels (or even above), but if an inhibitor is present, the residual factor level is much less than 100%. The APTT is the test that is run for most factor inhibitor assays. This may explain the confusion? Otherwise, this 1:1 mix of normal/buffer for direct APTT comparison does not make sense to me.

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