Development of a sensitive and rapid chromogenic factor IX assay for clinical use.
A chromogenic factor IX assay is developed which requires only two time-dependent steps. Diluted plasma is mixed with a reagent containing factors VIII and X. The reaction is started by addition of a reagent containing factor XIa, thrombin, CaCl2, and phospholipids. Then factor XIa activates factor IX if present, thrombin activates factor VIII, and subsequently the complete factor X activating complex (factor IXa, factor VIIIa, 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 Xa. Factor Xa formation is proportional to the plasma factor IX concentration (from 0 to 140%). The two reagents needed for the assay are stable at room temperature during a whole working day and for 3 h at 37 degrees 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 20% and a 8,000-fold purification.
Wagenvoord
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R
Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
Hendrix
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H
Tran
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Hemker
H C
HC
D000818:Animals; D002417:Cattle; D002863:Chromogenic Compounds; D005164:Factor IX / Q000032:analysis*; D005169:Factor VIII / Q000032:analysis / Q000302:isolation & purification; D006801:Humans; D010743:Phospholipids / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013997:Time Factors
D002863:Chromogenic Compounds; D010743:Phospholipids; D005169:Factor VIII; D005164:Factor IX