Mixing test

Mixing studies are used to distinguish factor deficiencies from factor inhibitors (lupus anticoagulants or specific factor inhibitors such as antibodies directed against factor VIII). Mixing studies take advantage of the fact that factor levels of 50% of normal should give a normal PT or PTT result. If the problem is a simple factor deficiency, mixing the patient plasma 1:1 with plasma that contains 100% of the normal factor level results in a level ≥50% in the mixture (say the patient has an activity of 0%; the average of 100% + 0% = 50%). The PT or PTT will be normal (the mixing study shows correction). However, if there is an inhibitor that inactivates the added clotting factor, the resulting factor level will be low and the clotting test will be prolonged (fails to correct). Therefore, correction with mixing indicates factor deficiency; failure to correct indicates an inhibitor. Unfortunately, it is not as simple as that. Some inhibitors are time dependent. In other words, it takes time for the antibody to react with and inactivate the added clotting factor. The clotting test performed immediately after the specimens are mixed may show correction because the antibody has not had time to inactivate the added factor. A test performed after the mixture is incubated for 2 hours at 37ºC will show prolongation.Nonspecific inhibitors like the lupus anticoagulant usually are not time dependent; the immediate mixture will show prolongation. Many specific factor inhibitors are time dependent, and the inhibitor will not be detected unless the test is repeated after incubation (factor VIII inhibitors are notorious for this).

INVESTIGATING AN ABNORMAL COAGULATION TEST (PT ORPTT) RESULT

A common problem is an unexplained increase in the PT and/or PTT . The first thing to do is get a fresh sample andrerun the test. Another consideraton is heparin. It is possible that theblood sample was mistakenly drawn though a running line. Interference byheparin can be detected by absorbing the heparin with a resin (“Heparsorb”)or by using an enzyme to digest the heparin (“Hepzyme”). Also,check the history: Is the patient on any anticoagulants? Does the patient haveliver disease? Provided that the abnormal result is reproduced on a goodspecimen and there is no obvious explanation from the history, the nextthing to do is a mixing study. If the mixing study shows correction and noprolongation with incubation, you will need to do factor assays to look forfactor deficiency: start with VIII and IX since they are the most commondeficiencies. It is useful to do a few vitamin K–dependent and a few nonvitaminK–dependent factors to be sure that the problem is not accidentalor surreptitious warfarin ingestion.

If the mixing study fails to correct, then you need to think about aninhibitor. The most common inhibitor is a nonspecific inhibitor such as alupus anticoagulant. Perform a test to demonstrate a phospholipid-dependentantibody, such as a platelet neutralization procedure. Spontaneous specificinhibitors against clotting factors occur (ie, not in hemophiliacs), mostoften against factor VIII. This can occur in patients with systemic lupus,monoclonal gammopathies, other malignancies, during pregnancy, and forno apparent reason (idiopathic). These patients can have devastating bleeding.The thing to do is identify the specific factor involved and find out howhigh the titer is. If the patient has a low titer inhibitor, try to overwhelm itwith high doses of the factor. If the patient has a high titer antibody againstfactor VIII, try porcine factor VIII or prothrombin complex concentrates tostop the bleeding. Prednisone will often lower the titer over time. Intravenousimmunoglobulin has been reported to also help (however, IVIGdoes not seem to work for hemophiliacs with an inhibitor).