Mixing Study

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The page below is a sample from the LabCE course Laboratory Evaluation of the Lupus Anticoagulant found in Antiphospholipid Syndrome (APS). Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

Learn more about Laboratory Evaluation of the Lupus Anticoagulant found in Antiphospholipid Syndrome (APS) (online CE course)
Mixing Study

Mixing studies with pooled normal plasma (PNP) can be used to demonstrate the presence of inhibitors. If an inhibitor is present, the mixing study will remain uncorrected in contrast to a patient with a factor deficiency, where the mixing study will correct. In patients suspected of LA, the aPTT will be prolonged when using an LA-sensitive reagent. The PT is usually unaffected since the PT reagent contains a high concentration of phospholipids and neutralizes the presence of an antibody.
The mixing study will mix equal parts of the patient's plasma and a pooled normal plasma and repeat the aPTT. If there is no correction, an inhibitor is suspected. The mixture is then incubated at 37°C for one hour and an aPTT is performed to determine if there is a time-dependent inhibitor. It is important to verify that the pooled normal plasma has normal factor levels to ensure there are sufficient quantities of factors capable of correcting a factor deficiency.
There are several criteria used to determine if a mixing study has corrected:
  1. Based on PT or aPTT normal range: Within limits at 2SD or 3SD
  2. Within five seconds of the 2SD upper limit of the normal range
  3. Rosner index, using the following formula:
    • Clotting time (CT) of the Index = aPTT of 1:1 mix – aPTT of PNP/aPTT of patient x 100
    • CT of patient <15 = factor deficiency; >15 = Inhibitor
  4. Pooled normal plasma (PNP) tested with mixing study and compare the result to the PNP using either a correction of within five seconds of PNP value or 10% of PNP value
Determining what constitutes a correction in a mixing study can be confusing. There are no recommendations or guidelines from the College of American Pathologists (CAP) Coagulation Resource Committee or the International Society on Thrombosis and Hemostasis. It is up to the laboratory to determine best practices and adhere to those policies. Laboratories can use the testing discussed on the following pages to demonstrate a correction.