Mixing Studies: Distinguishing Factor Deficiency from Inhibitors

A mixing study is used to study the cause of a prolonged screening test. This study can determine if the cause is a deficiency of one or more factors or an inhibitor.⁴ In a mixing study, platelet-free, normal plasma that is replete with all coagulation factors (near 100% activity for each) is mixed with the patient's sample. For example, in a 1:1 mix, one part patient sample is mixed with one part normal plasma, and the mixture is tested. In this case, the lowest possible concentration for any individual factor in the mixture would be approximately 50% (in the case of a patient with a factor concentration of zero and the normal pool has an activity of 100%). The mixture is tested using the same test system that produced the extended screening result. Note: When the screening assay (such as the APTT or PT) is only minimally elevated, it may not be possible to interpret the results of mixing studies in a meaningful way.

Some inhibitors are time- and/or temperature-dependent. In these cases, the sample that is tested immediately after mixing will correct, while results after the sample has been incubated for one hour at 37^o show prolongation. All plasma samples that correct upon immediate mix are retested after an incubated mix to rule out time- and/or temperature- dependent inhibitors.

Three different types of results can be observed in mixing studies:

1. No Correction with Immediate Mix
   • When the addition of normal plasma fails to correct the clotting time into the normal range, the cause of the abnormal test is likely an inhibitor.
   • In the presence of a non-specific inhibitor, such as a lupus anticoagulant, addition of normal plasma to the reaction mixture serves to dilute the inhibitor, but typically does not completely neutralize its effect.
   • In the presence of a neutralizing inhibitor, such as a factor VIII inhibitor, the factor present in the normal plasma is neutralized by the inhibitor resulting in failure of the mixture to correct into the normal range.
   • The most common cause of inhibition that is observed in the clinical laboratory is caused by inhibitor-type, anticoagulants that have been given therapeutically or have contaminated the sample at collection (ie, from a heparin line).
   • Extended PT values observed in patients on antivitamin K therapy (ie warfarin) typically correct in a mixing study because these therapeutics are not coagulation factor inhibitors. They work by reducing vitamin K-dependent factor levels.
   • When therapeutic factor inhibitors have been ruled out, further studies can be performed to differentiate specific factor inhibitors from lupus anticoagulants. (See Lupus Anticoagulant section.)
2. Correction with Immediate Mix --------------- No Prolongation After Incubation
   • This result is consistent with the presence of a time- and/or temperature-dependent inhibitor.
   • Historically, it was felt that lupus anticoagulants were exclusively immediate type inhibitors. Subsequent studies have shown that some lupus anticoagulants are time- and temperature-dependent.⁴ Specific inhibitors, particularly those to factor VIII, are characteristically time- and temperature-dependent.
3. Correction with Immediate Mix --------------- No Prolongation After Incubation
   • This result rules out the presence of immediate inhibitors as well as time- and/or temperature-dependent inhibitors. This would suggest in favor of factor(s) deficiency.
   • The normal plasma supplies the deficient factor at a concentration high enough to allow normal clotting. In the absence of inhibitors, a sample with at least 50% of all factors will produce a normal clotting time for both the PT and aPTT tests.

Note: Mixing studies performed on samples with minimally prolonged screening tests (ie, less than eight seconds) will often produce confusing results. The addition of normal plasma can sometimes dilute weak inhibitors out. This causes the mixing study to correct, a spurious result that suggests a factor deficiency.

References