Mixing study

Mixing studies are tests performed on blood plasma used to distinguish factor deficiencies from factor inhibitors, such as lupus anticoagulant, or specific factor inhibitors, such as antibodies directed against factor VIII. Mixing studies take advantage of the fact that factor levels that are 50 percent of normal should give a normal Prothrombin time (PT) or Partial thromboplastin time (PTT) result.^[1]

Test method

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). Correction with mixing indicates factor deficiency; failure to correct indicates an inhibitor. Performing a thrombin time on the test plasma can provide useful additional information for the interpretation of mixing tests.

Time-dependent inhibitors

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 its target factor. A test performed after the mixture is incubated for 1 to 2 hours at 37°C will show significant prolongation over the clotting time obtained after immediate mixing. 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).

Abnormal coagulation test results

A common problem is an unexplained increase in the PT and/or PTT. The first thing to do is get a fresh sample and repeat the test. Another consideration is heparin. It is possible that the blood sample was mistakenly drawn though a running line. Interference by heparin can be detected by absorbing the heparin with a resin (“Heparsorb”) or by using an enzyme to digest the heparin (“Hepzyme”). Also, the patient's history should be checked: Is the patient on any anticoagulants? Does the patient have liver disease? Provided that the abnormal result is reproduced on a good specimen and there is no obvious explanation from the history, the next thing to do is a mixing study. If the mixing study shows correction and no prolongation with incubation, factor assays will need to be done to look for factor deficiency, starting with VIII and IX since they are the most common deficiencies. It is useful to do a few vitamin K–dependent and a few nonvitamin K–dependent factors to be sure that the problem is not accidental or surreptitious warfarin ingestion.

Inhibitor

If the mixing study fails to correct, then you need to think about an inhibitor. The most common inhibitor is a nonspecific inhibitor such as a lupus anticoagulant. Perform a test to demonstrate a phospholipid-dependent antibody, such as a platelet neutralization procedure. Spontaneous specific inhibitors against clotting factors occur (i.e. not in hemophiliacs), most often against factor VIII. This can occur in patients with systemic lupus, monoclonal gammopathies, other malignancies, during pregnancy and for no apparent reason (idiopathic). These patients can have devastating bleeding. The thing to do is identify the specific factor involved and find out how high the titer is. If the patient has a low titer inhibitor, try to overwhelm it with high doses of the factor. If the patient has a high titer antibody against factor VIII, try porcine factor VIII or prothrombin complex concentrates to stop the bleeding. Prednisone will often lower the titer over time. Intravenous immunoglobulin has been reported to also help but it does not seem to work for hemophiliacs with an inhibitor.

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