From: Elizabeth M. Van Cott, M.D., and Michael Laposata, M.D., Ph.D., “Coagulation.” In: Jacobs DS et al, ed. The Laboratory Test Handbook, 5th Edition. Lexi-Comp, Cleveland, 2001; 327-358.
Related Information
Activated Partial Thromboplastin Time
Antiphospholipid Antibody (Lupus Anticoagulant and/or Anticardiolipin Antibody)
Factor Inhibitors
Heparin Neutralization
High-Molecular Weight Kininogen
Prekallikrein
Prothrombin Time
Thrombin Time
Synonyms Circulating Anticoagulant Screen; Inhibitor Screen
Applies to Argatroban; Danaparoid; Factor Assays; Heparin; Hirudin
Abstract Mixing studies can be performed when the PT or PTT is prolonged, to determine if the etiology of the prolongation is a factor deficiency or an inhibitor.
Specimen Plasma
Container Blue top (sodium citrate) tubes
Collection Routine venipuncture. If multiple tests are being drawn, draw blue top tubes after any red top tubes but before any lavender top (EDTA), green top (heparin), or gray top (oxalate/fluoride) tubes. Immediately invert tube gently at least 4 times to mix. Tubes must be appropriately filled. Deliver tubes immediately to the laboratory.
Storage Instructions Separate plasma from cells as soon as possible. Plasma may be stored on ice for up to 4 hours; otherwise, store frozen.
Causes for Rejection Specimen received more than 4 hours after collection, tubes not filled, clotted specimen
Turnaround Time Several hours; longer if additional follow-up tests are indicated
Special Instructions Notify the laboratory if patient is on heparin (including low-molecular-weight heparin), hirudin, danaparoid, or argatroban anticoagulation, any of which can prolong PTT and/or PT.
Reference Interval There are three types of results in the PTT mixing study:
1. If the PTT of the mixture is normal, and remains normal after prolonged (2-hour) incubation, the results indicate the presence of factor deficiency(ies). The PTT is normal in such mixtures because the normal plasma supplies the factor that is deficient in the patient plasma. There may be one or more deficient factors. Assays for factors VIII, IX, XI, and XII should then be performed to identify the specific factor deficiency(ies). If the PT is also prolonged, common pathway factor assays can also be considered.
2. If the PTT of the mixture remains prolonged, the results suggest the presence of an inhibitor, most commonly, a lupus anticoagulant. Therefore, lupus anticoagulant assays should then be performed. Heparin, hirudin, argatroban, or high-dose danaparoid, if present, will also show this type of result in a mixing study. Specific factor inhibitors against a particular coagulation factor (eg, factor IX, XI, or XII), are very rare possibilities.
3. If the PTT of the mixture is initially normal (or significantly shorter than the patient plasma’s PTT) but becomes prolonged after a 1- or 2-hour incubation, the results are characteristic of a factor VIII inhibitor (factor VIII inhibitors show an inhibitory effect only after prolonged incubation). A factor VIII assay should then be performed and, if decreased (usually to <10%), a factor VIII inhibitor assay (Bethesda assay) should be performed.
When the PT is prolonged and the PTT is normal, a PT mixing study may also be useful in determining if the etiology is a factor deficiency or a factor inhibitor, similar to that described for the PTT. However, factor inhibitors that affect only the PT and not the PTT are rare. The results of PT mixing studies in patients on warfarin are consistent with factor deficiencies, because warfarin acts as an anticoagulant by decreasing the activity of factors II, VII, IX, and X.
Methodology When the PTT is prolonged, the laboratory should first determine if the prolongation is due to heparin by treating the specimen to remove heparin (see Heparin Neutralization). Alternatively, some laboratories perform the thrombin time, which is prolonged when even a small amount of heparin is in the sample. If a prolonged PTT is not due to heparin, patient plasma is then mixed with an equal volume of normal plasma, and the PTT is repeated. The resulting PTT of this mixture indicates whether the prolongation is due to a factor deficiency or an inhibitor. Inhibitors are substances that inhibit clotting reactions. They are usually antibodies (eg, lupus anticoagulants or specific factor inhibitors) or anticoagulants such as heparin, hirudin, or argatroban. Based on the mixing study results, factor assays, lupus anticoagulant tests, or tests for factor inhibitors may be indicated. PT mixing studies can be similarly performed to evaluate PT prolongations.
References
Clyne LP, Yen Y, Kriz NS, et al, “The Lupus Anticoagulant. High Incidence of Negative Mixing Studies in a Human Immunodeficiency Virus-Positive Population,”Arch Pathol Lab Med, 1993, 117(6):595-601.
Kaczor DA, Bickford NN, and Triplett DA, “Evaluation of Different Mixing Study Reagents and Dilution Effect in Lupus Anticoagulant Testing,”Am J Clin Pathol, 1991, 95(3):408-11.
Van Cott EM and Laposata M, “Coagulation, Fibrinolysis and Hypercoagulation,”Clinical Diagnosis and Management by Laboratory Methods, 20th ed, Henry JB, ed, New York, NY: WB Saunders Co, 2001, 644-6.
