Factor IX Inhibitor

Plasma, frozen

4 mL (2 mL in each of two tubes)

Blue-top (sodium citrate) tubes

The patient should not be anticoagulated. Do not draw from an arm with a heparin lock or heparinized catheter.

Confirmation and characterization of factor IX inhibitor

If there is residual coagulation factor activity, it could falsely lower the Bethesda titer result. Lupus anticoagulant activity and antithrombotic agents that function as inhibitors should be ruled out prior to assaying for factor inhibitors in order to avoid factitiously positive results. Some inhibitors may fully neutralize factors in less than two hours. Autoimmune inhibitors that demonstrate second order kinetics cannot be accurately measured in the Bethesda titer system.⁶

The factor IX inhibitor (Bethesda titer) assay is performed using an activated partial thromboplastin time (aPTT)-based system.⁶ Serial dilutions are made of patient plasma with veronal buffered saline, then mixed with normal plasma containing close to 100% factor IX activity and are then incubated for two hours. An aPTT-based factor IX assay using factor IX-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 IX inhibitor that neutralized 0.5 IU of factor IX in this system. The number of serial dilutions tested is based on the anticipated level of the inhibitor.

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 while non-neutralizing antibodies react with the protein somewhere other than the functional epitopes. 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. Factor IX inhibitors arise as alloantibodies in 3% to 5% of hemophilia B (Christmas disease) patients in response to factor IX concentrate, prothrombin complex concentrate, or fresh frozen plasma therapy. Inhibitors of other coagulation factors are extremely rare, as deficiencies of these factors are rare. Autoimmune antifactor IX inhibitors have been described.