Mixing Study Dilution

Mixing Study Dilution
Mar 6, 2016 7:40am

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.

1 Comment

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.

By Dr Emmanuel Favaloro
Mar 10, 2016 2:26am
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.

Leave A Comment

You must be logged in to Comment - Sign In