Factor II Inhibitor Profile, Comprehensive

Plasma, frozen

6 mL (2 mL in each of three tubes)

Blue-top (sodium citrate) tubes

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.

Confirmation and characterization of factor II inhibitor

If there is residual coagulation factor, it could falsely lower the result. The presence of direct Xa or thrombin inhibitor anticoagulant therapy may cause false-positive results. Lupus anticoagulant activity should be ruled out prior to assaying for factor inhibitors as this may cause false-positive titers. Individuals with lupus anticoagulant hypoprothrombinemia syndrome (LAHS) typically have low factor II levels but no measurable inhibitor titer, as the factor II antibody complex is rapidly cleared by the reticuloendothelial system. A negative factor II inhibitor profile, therefore, does not exclude a factor II inhibitor in individuals with a lupus anticoagulant. Autoimmune inhibitors cannot be accurately measured in the Bethesda titer system because of their second-order kinetics.⁶

The factor II inhibitor (Bethesda titer) assay is performed using a prothrombin time-based system, using thromboplastin reagent.⁶ Serial dilutions are made of patient plasma with veronal buffered saline, then mixed with normal plasma containing close to 100% factor II activity, and are then incubated for two hours. A PT-based factor II assay using factor II-depleted plasma substrate is then performed on these incubated mixtures. Results are compared to those of incubated normal plasma. One Bethesda unit is defined as the amount of factor II inhibitor that neutralized 0.5 IU of factor II in this system. The number of serial dilutions tested is based on the anticipated level of the inhibitor.

The development of factor II inhibitors is a rare occurrence. Antibodies to prothrombin may develop in some individuals who also develop antiphospholipid antibodies, such as the lupus anticoagulant. In this instance, the antiprothrombin antibodies bind to prothrombin and result in its clearance from the circulation. This leads to low factor II activity levels and a bleeding tendency which can be severe. Because the factor II antiprothrombin antibody complex is rapidly cleared from the circulation, neutralizing antibodies cannot be measured using a Bethesda titer. Factor inhibitors (also called circulating anticoagulants or inactivators) are endogenously produced antibodies that interfere with coagulation in vivo or in vitro.⁶ These inhibitors are frequently specific for their respective coagulation factors. Specific factor inhibitors can be classified as neutralizing or non-neutralizing. Neutralizing inhibitors interact with the functional component of the coagulant protein and may cause clinical bleeding. Non-neutralizing antibodies react with the protein somewhere other than the functional epitopes and may be clinically silent. Factor inhibitors are not normally present in plasma. When present, factor inhibitors are measured by Bethesda titer units (BU). Titers <5 BUs are classified as low responders, titers >10 BUs as high responders. Responder status influences the approach for clinical treatment. Factor inhibitor assays are ordered in response to clinical impression, and are performed periodically on individuals who have inhibitors and are receiving therapy. During therapy, periodic assays are used, both to manage the dosage and to establish whether a patient is a low or high responder. Low responders maintain titers consistently <5 BUs, while high responders may generate extremely high titers and rapid anamnestic responses to therapy. Assays must also be performed repeatedly during inhibitor suppression therapy.