aPTT Mixing Studies

Plasma, frozen

2 mL

Blue-top (sodium citrate) tube

Ideally, the patient should not be on anticoagulant therapy. Avoid warfarin (Coumadin®) therapy for two weeks prior to the test and heparin, direct Xa, and thrombin inhibitor therapies for about three days prior to testing. Do not draw from an arm with a heparin lock or heparinized catheter.

Distinguish specific (ie, factor VIII) and nonspecific (lupus anticoagulant) inhibitors; determine heparin presence; detect single or multiple factor deficiencies

Lupus anticoagulant sensitive reagents are more responsive to the presence of lupus anticoagulants and are used in this panel. aPTT mixing studies will not be performed unless the aPTT is prolonged to 5 seconds or more above the upper end of the reference interval as they cannot be accurately interpreted when the aPTT is only slightly prolonged. The aPTT may not be extended in individual factor deficiencies unless the levels drop to 25% to 45% depending on the factor.⁶ Less significant individual factor deficiencies in combination (multiple factor deficiency) can extend the aPTT.⁶ The aPTT is more sensitive to intrinsic pathway factor deficiencies than common pathway factor deficiencies.⁶ Factor VIII elevations, as can occur due to acute phase reactions, can normalize a mildly extended aPTT result.⁶

To perform mixing studies, the patient specimen is mixed 1:1 with normal platelet-free plasma and with veronal buffered saline. aPTTs are performed on these mixtures. An aliquot of the normal plasma/patient plasma mixture is also incubated at 37°C for one to two hours and then an aPTT is performed to further clarify the cause of the prolongation. Normal platelet-free plasma and patient plasma are also incubated separately and then mixed after incubation as a control for possible factor degradation during incubation.

An isolated prolonged aPTT result with a normal prothrombin time implies either the presence of heparin and inhibitor (either specific factor or lupus anticoagulant) or a factor deficiency of the intrinsic system.⁷ Unless the cause for the prolonged aPTT is known, as in the case of heparin therapy, mixing studies may be necessary to determine the etiology of the prolonged result. If the result obtained with the immediate normal plasma mixture corrects to within the reference range and the saline mixture result increases dramatically, a factor deficiency or specific factor inhibitor is suspected.⁷

With factor deficiency, heparin, or a specific factor inhibitor, the 1:1 saline mix shows significant prolongation of the aPTT result. If the result obtained with the immediate normal plasma mixture shows only partial or no correction and the result obtained with the saline mixture shows near correction to the reference range or only mild prolongation, then a nonspecific inhibitor, such as lupus anticoagulant (LA), is suspected. After incubation at 37°C, an aPTT result equivalent to that of the original mixture is indicative of a factor deficiency or heparin. If the result of the aPTT on the incubated mixture demonstrates further prolongation (with the control remaining equivalent to the original result), a specific factor inhibitor is indicated. A specific factor VIII inhibitor may show initial correction of the original mixture, but will demonstrate prolongation upon incubation.

If mixing studies indicate the possibility of single or multiple factor deficiencies, a PT may be performed to differentiate between common pathway and intrinsic pathway deficiencies. Factor assays should then be performed to determine specific factors involved. If a factor inhibitor is suspected, specific factor assays and inhibitor titers may be necessary. If heparin is suspected without clinical confirmation, a thrombin time with addition of heparin neutralizer should be performed on the original specimen. Plasma suspected of containing a lupus anticoagulant (LA) should be further tested with low phospholipid tests and associated confirmatory tests. A patient with an initial positive antiphospholipid antibody or lupus anticoagulant test should be tested after 12 or more weeks to determine if the antibody is persistent.