College of American Pathologists
Printable Version

  Q & A





cap today

October 2004

Richard A. Savage, MD, Editor

Q.  At our clinical laboratory, the mixing study on prolonged activated partial thromboplastin time, or aPTT, is done by mixing an aliquot of the patient's plasma with an aliquot of normal plasma. The test is immediately repeated on this mixture. If the aPTT doesn't correct to normal, the mixture is then incubated for one hour at 37°C, and an aPTT is again performed on the incubated mix.

From my experience, the mixing study that did not correct immediately has never corrected after incubation. References recommend incubating only prolonged aPTT that corrects to the reference range immediately. What is the correct procedure for performing an aPTT mixing study?

A.  The mixing study is a common coagulation test used to distinguish between a coagulation factor deficiency, such as factor VIII deficiency, and a factor inhibitor, such as a specific factor VIII inhibitor or lupus anticoagulant. The test is performed when a patient has an unexplained prolongation of a coagulation screening assay, such as the activated partial thromboplastin time. The mixing study is usually done by mixing equal volumes of patient plasma and pooled normal plasma and then repeating the aPTT on the mixture.1 The basic principle is that the normal plasma contributes a sufficient concentration of clotting factors to "correct" for a factor deficiency. A mixing study that corrects the aPTT is characteristic of factor deficiency, whereas a mixing study that does not correct the aPTT indicates a factor inhibitor.

There are two types of mixing studies: the immediate mix and the incubated mix. In the immediate mix, the aPTT is performed immediately after mixing the patient plasma and normal plasma, without further incubation. Specimens with fast-reacting factor inhibitors will not correct the immediate mix.

In contrast, specimens with slow-reacting, or time-dependent, factor inhibitors may correct the immediate mix, giving the false impression of a factor deficiency. The majority of factor VIII inhibitors and eight percent of lupus anticoagulants are sufficiently time dependent that they correct the immediate mix.2,3 Therefore, it is essential that an incubated mix be performed to diagnose these conditions. In the incubated mix, the aPTT is performed after incubating the mixture of patient plasma and normal plasma for one to two hours at 37°. Specimens with time-dependent inhibitors will not correct the incubated mix.

In summary, the incubated mixing study is necessary for diagnosing coagulation factor inhibitor. Clinical laboratories should develop a standard policy for performing incubated mixing studies on plasma samples that have a corrected immediate mix to avoid missing the diagnosis. An incubated mix is not necessary if the immediate mix shows evidence of an inhibitor. Some laboratories may decide to set up immediate and incubated mixing studies simultaneously to simplify their testing protocol.


  1. Brandt JT, Triplett DA, Alving B, et al. Criteria for the diagnosis of lupus anticoagulant: an update on behalf of the Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the ISTH. Thromb Haemost. 1995; 74: 1185-1190.
  2. Lossing TS, Kasper CK, Feinstein DI. Detection of factor VIII inhibitors with the partial thromboplastin time. Blood. 1977; 49: 793-797.
  3. Clyne LP, White PF. Time dependency of lupuslike anticoagulants. Arch Intern Med. 1988; 148: 1060-1063.

Mark T. Cunningham, MD
Department of Pathology
University of Kansas Medical Center
Kansas City
Member, CAP Coagulation Resource Committee

Q.  Should the creatine kinase-MB/total creatine kinase ratio, or relative index, be abandoned for diagnosing myocardial infarction now that more specific tests, such as troponin I, are available? Is there a good scientific reason to continue using the relative index?

A.  Experts agree that the relative index, originally proposed because it provided better cardiac specificity than CK-MB alone, has become an anachronism with the availability of reliable troponin I and T assays. But because troponin has an extended elevation time of three days to two weeks in the blood after cardiac necrosis, most experts agree that CK-MB mass is still a useful test. In contrast to troponin, CK-MB is cleared in 24 to 36 hours and can provide information about the timing of the cardiac event.

Robert Christenson, PhD
Professor of Pathology
Director of Rapid Response Laboratories
University of Maryland