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.
Index of Tests
Mixing Studies [CO006100]
Synonyms Circulating Anticoagulant Screen; Inhibitor Screen
Applies to Argatroban; Danaparoid; Factor Assays; Heparin;
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.
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,
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
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
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.
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.