Chromogenic Factor IX Assay for Clinical Use

A chromogenic factor IX assay is developed which requires only two timedependent steps. Diluted plasma is mixed with a reagent containing factors VIII and X. The reaction is started by addition of a reagent containing factor XI", thrombin, CaCl2, and phospholipids. Then factor XIu activates factor IX if present, thrombin activates factor VIII, and subsequently the complete factor X activating complex (factor IXu, factor VIII1, Ca ions, and phospholipids) rapidly activates factor X. Finally, ethylenediaminetetraacetic acid plus a chromogenic substrate are added to stop the reaction and to measure formed factor Xu. Factor X" formation is proportional to the plasma factor IX concentration (from 0 to 1400/o). The two reagents needed for the assay are stable at room temperature during a whole working day and for 3 h at 37 'C. A new isolation procedure for factor VIII is described. Factor VIII is purified from bovine plasma in a few steps with a yield of 20o/o and a 8,000-fold purifrca-