aPTT Mixing Studies

An activated partial thromboplastin time (aPTT) mixing study is employed to investigate the cause for a prolonged aPTT.^1-9 In the standard 1:1 Mixing Study, an aPTT is performed using patient plasma that has been mixed with an equal volume of normal pooled plasma (NPP). Correction of the test result into the reference range by addition of NPP suggests that cause of the prolonged patient result is likely a deficiency of one or more coagulation factors. This interpretation is based on the presumption that the NPP supplies enough of the missing factor or factors to facilitate normal clotting in the test system. Noncorrection or incomplete correction suggests the presence of an inhibitor in the patient’s plasma. This interpretation is based on the presumption that inhibitor from the patient sample will also affect coagulation factors provided by the NPP. 

In a 1:1 mixing study, one-part patient sample and one-part NPP are mixed to produce the test sample. The aPTT is performed on the test sample immediately following admixture of the patient plasma and NPP. The laboratory uses NPP that has been shown to have approximately 100% of all factors, as measured by individual factor assays and to be devoid of a lupus anticoagulant. Factor levels greater than 50% should yield an aPTT value within the reference range. A 1:1 mix of patient plasma and NPP should contain at least 50% of all factors and should produce a normal result, if the cause of the abnormal result is simply a factor deficiency. 

Most inhibitors in hemostasis, including anticoagulants that inhibit coagulation factors, are immediately acting, and show type 1 kinetics on “Immediate Mix”.^6,7 This means that when test plasma containing such inhibitors are diluted with normal plasma in equal volume, about 50% of the inhibitory effect remains evident, and so the clotting time of the test mixture will be about halfway between that of the test plasma and that of the NPP. Some inhibitors, however, notably those that develop against FVIII, are time- and temperature-dependent,^6,7,10 meaning that inhibitory effects are not immediately evident and only become fully expressed after some time and at temperatures higher than ambient room temperature. Thus, when immediate mixing tests are performed with such samples, the mixtures may show a “false” correction, since the inhibitors are not yet able to inhibit the factors from the NPP. If a FVIII (or other time and temperature) inhibitor is present, or thought to be possible, mixing studies would need incubation at 37°C (one hour) in order to fully express the inhibitor, and to show a non-correction in a mixture. This is referred to as the “Incubated Mix.” 

In order to ensure that the extension of the aPTT in the Incubated Mix is due to inhibitor activation and not due to loss of labile factors V and VIII during the incubation period, the patient sample and NPP are incubated separately under the same conditions and then mixed immediately prior to testing (the “Incubated Control”). Some FVIII inhibitors are fast acting and demonstrate inhibition with the Immediate Mix. In these cases, there may be no or little additional prolongation with incubation relative to correction that occurs with the immediate mix.

The results of the patient incubated mix are always compared to the results of the incubated control. When the incubated patient mix aPTT clot time is shorter than the aPTT clot time for the incubated control, the presence of a time- and temperature-dependent inhibitor, such as a factor VIII inhibitor, must be considered. Some lupus anticoagulants (LA) demonstrate time and temperature dependence, although this is thought to be an artifact of pH rather than a true characteristic of the LA. EDTA samples, as well as samples containing direct Xa and thrombin inhibitor anticoagulants, will also demonstrate time and temperature dependent prolongation of the aPTT.

In some cases, the Immediate Mix aPTT value exceeds the baseline aPTT. This is referred to as the "lupus cofactor effect."⁸ While this pattern is not pathognomonic for a lupus anticoagulant but often reflects the presence of this type of antibody, this pattern can also occur with a factor VIII inhibitor or in patients receiving direct Xa or IIa inhibitor anticoagulant therapy. Factor VIII inhibitors may present with a significant hemorrhagic diathesis.