PTT Mixing Studies With Saline

Joe Lamb asked, “George, I’m working on mixing study procedures for my lab. I’ve read about a saline mix and I’m looking for a reference for the technique. I’m interested in a procedure and how to interpret the results. can you recommend something for me?”

Hi, Joe. I apologize for being a little slow on this. I posted an answer earlier but the message contained some unexpected coding error that messed up the web site, so I am starting over.

I checked with Gordon Ens and Flo Newlin, who were President and Vice-president ofColorado Coagulation Consultants hemostasis reference laboratory in Aurora, Colorado. CCC is now Esoterix Coagulation. Gordon, and especially Flo, were great proponents of the saline mix, and wrote about it in the Coagulation Handbook, published by Esoterix in 2002. This was a beautiful book, but I can’t find it listed on the Esoterix site, so I guess it is no longer available–perhaps you have it on your shelf. The following is my paraphrase of the procedure on page 16 of the Coagulation Handbook:

• A platelet-free patient plasma specimen has a prolonged partial thromboplastin time (PTT) and requires follow-up mixing studies.
• First perform a thrombin clotting time (TCT) to detect the presence of therapeutic unfractionated heparin (UFH) in the plasma specimen. A typical upper limit of the TCT reference interval is ~20 seconds and UFH will prolong the TCT to 30 or 40 seconds. If UFH is present, treat the specimen with heparinase to neutralize the UFH, then proceed. If the TCT remains normal, proceed without plasma treatment.
• Mix one aliquot of the patient plasma 1:1 with normal platelet-free plasma (NP) such as CryoCheck Pooled Normal Plasma and a second aliquot with veronal buffered saline (VBS). Perform an immediate PTT on both mixtures. Also prepare a second 1:1 mix of patient plasma with NP and incubate 1 to 2 hours at 37°C.
• Immediate mix result: if the PTT result of the patient plasma-NP mix corrects to within the reference interval but the patient plasma-VBS mix result is prolonged, suspect either a coagulation factor deficiency or a specific coagulation factor inhibitor such as anti-factor VIII. Proceed to testing the incubated patient plasma-NP mix, below. Alternatively, if the patient plasma-NP mix PTT result remains prolonged (uncorrected), and the patient plasma-VBS mix corrects to within the reference interval, suspect a lupus anticoagulant and proceed to the lupus anticoagulant profile.
• Incubated mix result: if the immediate patient plasma-NP mix result was within the reference interval but the mix of patient plasma-NP is prolonged after incubation at 37ºC for 1 to 2 hours, suspect a specific coagulation factor inhibitor such as anti-factor VIII and proceed to a Bethesda titer. If the incubated mix result remains within the reference interval, the initial PTT prolongation indicates a coagulation factor deficiency. Proceed to test for individual coagulation factor activity levels.

Comments:

• Flo Newlin defines PTT correction as a result within the reference interval. Others may define correction as a PTT result that is within 10% of the NP result. This is a local decision.
• When performing the incubated mix, incubate also an aliquot of patient plasma and an two aliquots of NP. After two hours, perform a PTT on the incubated patient plasma, the NP, and a 1:1 mixture of patient plasma and NP. Prolongation may indicate coagulation factor deterioration during the incubation, and if you use the 10% rule to determine correction, use the incubated NP as the reference value.
• Patient plasma and NP must be platelet-free.
• You don’t use an incubated patient plasma-VBS mix.
• For greater sensitivity, some use a 4:1 patient plasma to NP mix.