mixing study mixing study mixing studies are tests performed on blood plasma of patients or test subjects to distinguish factor deficiencies from factor inhibitors, such as lupus anticoagulant, or specific factor inhibitors, such as antibodies directed against factor viii. the basic purpose of these tests is to determine the cause of prolongation of prothrombin time (pt), partial thromboplastin time, or sometimes of thrombin time (tt). 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. factor deficient plasmas (adsorbed plasma & aged plasma, etc.) are used in mixing studies. plasma with known factor deficiencies are commercially available but are very expensive, so they are often prepared in the laboratory and can then be used for mixing experiments. test method fresh normal plasma has all the blood coagulation factors with normal levels. plasma from patients on oral anticoagulants (warfarin etc.) for 48–72 hours is deficient in factor vii. adsorbed plasma or plasma from patients on oral anticoagulants (warfarin etc.) for a week or more is deficient in factor ii, factor vii, factor ix, and factor x. aged plasma is deficient in factor v & factor viiic. serum is deficient in factors i, v & viiic. 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. correction of prothrombin time prothrombin time (pt) may be corrected as follows: factor deficiency/abnormality prothrombin time corrected by mixing with normal plasma adsorbed plasma aged plasma coumarin plasma factor i yes yes yes yes factor ii yes partial yes yes factor v yes yes no yes factor vii yes no yes no factor x yes no yes yes anticoagulants no no no no correction of partial thromboplastin time partial thromboplastin time (ptt) may be corrected as follows: factor deficiency/abnormality partial thromboplastin time corrected by mixing with normal plasma adsorbed plasma aged plasma factor viiic yes yes no factor ix yes no yes factor xi yes yes yes 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. if this is observed, the test should be repeated with a fresh sample. 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, especially with regard to anticoagulant use or liver disease. provided that the abnormal result is reproduced on a fresh specimen and there is no obvious explanation from the history, a mixing study should be performed. if the mixing study shows correction and no prolongation with incubation, factor deficiency should be looked for, starting with viii and ix. vitamin k-dependent and nonvitamin k–dependent factors should be considered to rule out accidental or surreptitious warfarin ingestion. inhibitor if the mixing study fails to correct, then an inhibitor should be added. 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 erythematosus, 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. in order to provide specific cutoffs to distinguish an inhibitor defect from a factor deficiency, the rosner index and/or the chang percentage can be used: r o s n e r i n d e x = ( a p t t o f 1 : 1 m i x ) − ( a p t t o f n o r m a l p o o l e d p l a s m a ) a p t t o f n o n m i x e d p a t i e n t p l a s m a x 100 x100} results are: ≤10 is classified as correction, ≥15 indicates presence of an inhibitor, and 11-15 is classified as "indeterminate". c h a n g p e r c e n t a g e = ( a p t t o f n o n m i x e d p a t i e n t p l a s m a ) − ( a p t t o f 1 : 1 m i x ) ( a p t t o f n o n m i x e d p a t i e n t p l a s m a ) − ( a p t t o f n o r m a l p o o l e d p l a s m a ) x 100 x100} results are classified as follows: <58% as inhibitor and >70% as correction.> in case of a corrected mixing test, a lower dose of normal pooled plasma is often used.