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Spectrophotometr
Spectrophotometric Method for the Assay of Human BloodCoagulation Factor VIII
G. van Dieijena, M.P. van Dieijen-Visserb, J. Franssena, H.C. Hemker"uDepartment of Biochemistry, University of Limburg, Maastricht;bDepartment of clinical chemistry, De wever Hospital, Heerlen. The Netherlands
Key words. Factor vIII, spectrophotometric assay . clotting assay, l-stage .Chromogenic substrateAbstract. A spectrophotometric method for the assay of human blood coagulation factorvIII in plasma is presented. The chromogenic assay for factor VIII:C in plasma is performedin 3 steps: (l) activation of factor VIII by thrombin; (2) activation of factor X in a mixture offactor X, factor IXa, phospholipids/Ca2t and plasma containing activated factor VIII, and(3) determination of the rate of factor Xa formation with the chromogenic substrate 52337.Within-assay variation was between 5 and 6.90/o for factor vIII:C activities between 20 and1500/0. Clotting and chromogenic factor vIII:C actiyities were compared in plasma of 50normal healthy donors (coefficient of correlation r: 0.g3).
IntroductionFactor VIII or anrihemophilia globulin Ais a plasma protein that is essential for nor-mal hemostasis (see [l] for review). Its con-genital absence is by far the most commoncause of congenital bleeding tendency. Theprocoagulant protein (factor VIII:C) occursin plasma tightly bound to the von Wille-brand factor (factor VIII:vWF). FactorVIII:C and vWF together form the factorVIII complex. Factor VIII:C appears respon-sible for normal thrombin formation,whereas factor VIII:vWF is required forplatelet-vessel wall interaction. (For reviewson factor VIII complex and vWF see [2, 3].)
If factor VIII:C is congenitally deficient, fac-tor VIII:vWF is normal. If factor VIII:vWFis deficient, factor VIII:C is to a varyingdegree also.A widely used method to determine fac-tor VIII in plasma is the l-stage clottingassay introduced in 1962 by Hardisty et al.[a]. This assay is based on the property offactor VIII to correct the prolonged clottingtime of hemophiliac plasma to a degree de-termined by the amount of factor VIIIpresent.A new assay method for the determina-tion of factor VIII using a chromogenic sub-strate for factor Xa appeared feasible afterkinetic studies on the role of factor VIII in
Haemostasis 17: 14-24 (1987)
@ 1987 S. Karger AG, Basel030t -0t 47 / 87 lO t',l 2-O0 | 452.',l 5 / O
the intrinsic facIn this paper,metric assay fo;and compare tlthe assay withMaterials ar52337, BzJlelide and 52238, Hobtained from Kacholine (egg-PC)brain-phospbatidlSt. Louis, Mo., USBasel, Switzerlancand soybean trypSigma. Factor-defiGeorge King BirUSA. The batch n837-314; high molD30; factor XII, (Nl9; prekallikrei1501-625; factorbrand, GK 1403from General Diznumbers: factor VVII,4J759. Facrcfactor-IX-deficienpatrents. Kaolin (thin was from AsUSA.ProteinsBovine factorfactor IXa and thas published beforing of 40 0,6 brain-sonication at 0"r175mM NaCl, pIHuman DonotBlood was obt:informed consenl40 mm needle, drswas mixed with Iplastic tube and cThe plasma was as,centrifugation at 4
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