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Thank you for attending THSNA 2026. The virtual meeting is now closed.
Presentation Details
| Diagnostic limitation of mixing studies in coagulation assay Maryam Pourabdollah. University of Ottawa, OTTAWA, ON, Canada |
Abstract
Mixing study is a commonly used coagulation test that is usually perform along with the basic clotting assays when there is an unexplained prolongation of PT/PTT to distinguish between factor deficiency and inhibitors; however, there is no standardized version for its methodology or interpretation. The mixing study is based on the fact that at least 50% of most coagulation factors are needed to provide a timely clotting. The logic of doing a mixing study is to add extra coagulation factors to replace the deficient one and correct the clotting time.
Generally, if the clotting time corrects with mixing, it is in confirmation of factor deficiency. In contrast, in the presence of inhibitor, there is no correction. However, there are some caveats in this regard; pre-analytical factors, multiple factor deficiencies, or inhibitors make things more complex. Inhibitors could be specific (antibodies directed against a specific coagulation factor), nonspecific (e.g. lupus anticoagulant) or medications such as heparin, direct thrombin inhibitors or anti-Xa medications.Some of the specific inhibitors like the one against factor VIII are notorious to react differently, they are time/temperature-dependent; it means on immediate mix, in spite of having inhibitors, they might not be active; they need to be incubated in proper temperature for enough time while mixing to be able to bind to specific antigen; thus, we have to confirm if by doing mix incubation phase. There are different ways to determine correction; we can either validate the normal range in our lab and accept it as corrected if the result is within normal range of PT/PTT, or we can accept up to 5 seconds difference from the normal (NPP) result performed at the same time, as corrected. Since definition of correction is based on achieving the normal range, it might be troublesome in occasional situations; for example, a weak or low titer inhibitor which minimally prolonged PT or aPTT may be corrected to normal in a 1:1 mix. In contrast, factor deficiencies with a markedly prolonged PT or aPTT may not be corrected to normal in a 1:1 mix, when dealing with a strong/high titer inhibitor. In case of minimally prolonged APTT/PT, 1:1 mixing study has limited value; 4:1 mixing study is a suggested test in this condition; 4 unit of patient plasma and one unit of NPP to improve the sensitivity for detection of factor inhibitors or APLs. However, the 4:1 mixing study might not be sensitive enough to detect coagulation-factor deficiency.
For interpretation of mixing study, knowing about clinical history including bleeding vs thrombosis history is crucial. Important to consider the fact that anticoagulant medications might interfere the clot-based assays. Following mixing study, additional test might be required including intrinsic factor assay when dealing with isolated prolonged PTT, extrinsic factor assay if having isolated prolonged PT, or common coagulation pathway assay when facing with both PT and PTT prolongation like liver disease. Other tests might also be required including Bethesda inhibitor assay, lupus anticoagulant assay, etc.
No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.
Generally, if the clotting time corrects with mixing, it is in confirmation of factor deficiency. In contrast, in the presence of inhibitor, there is no correction. However, there are some caveats in this regard; pre-analytical factors, multiple factor deficiencies, or inhibitors make things more complex. Inhibitors could be specific (antibodies directed against a specific coagulation factor), nonspecific (e.g. lupus anticoagulant) or medications such as heparin, direct thrombin inhibitors or anti-Xa medications.Some of the specific inhibitors like the one against factor VIII are notorious to react differently, they are time/temperature-dependent; it means on immediate mix, in spite of having inhibitors, they might not be active; they need to be incubated in proper temperature for enough time while mixing to be able to bind to specific antigen; thus, we have to confirm if by doing mix incubation phase. There are different ways to determine correction; we can either validate the normal range in our lab and accept it as corrected if the result is within normal range of PT/PTT, or we can accept up to 5 seconds difference from the normal (NPP) result performed at the same time, as corrected. Since definition of correction is based on achieving the normal range, it might be troublesome in occasional situations; for example, a weak or low titer inhibitor which minimally prolonged PT or aPTT may be corrected to normal in a 1:1 mix. In contrast, factor deficiencies with a markedly prolonged PT or aPTT may not be corrected to normal in a 1:1 mix, when dealing with a strong/high titer inhibitor. In case of minimally prolonged APTT/PT, 1:1 mixing study has limited value; 4:1 mixing study is a suggested test in this condition; 4 unit of patient plasma and one unit of NPP to improve the sensitivity for detection of factor inhibitors or APLs. However, the 4:1 mixing study might not be sensitive enough to detect coagulation-factor deficiency.
For interpretation of mixing study, knowing about clinical history including bleeding vs thrombosis history is crucial. Important to consider the fact that anticoagulant medications might interfere the clot-based assays. Following mixing study, additional test might be required including intrinsic factor assay when dealing with isolated prolonged PTT, extrinsic factor assay if having isolated prolonged PT, or common coagulation pathway assay when facing with both PT and PTT prolongation like liver disease. Other tests might also be required including Bethesda inhibitor assay, lupus anticoagulant assay, etc.
No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.