The Establishment And Clinical Application Of Combined-Test Of Various Parameters Of Von Willebrand Disease

Objective:(1) To study the biological function of glycoprotein protein Iba, A cell lineexpressing recombinant active fragment rGPIbaH1-V289 was constructed and purifiedfragment was acquired to research its function. (2) To establish a new method that candetect ristocetin co-factor activity (VWF:RCO) by ELISA which can. evaluate VWFfuction accurately in the clinical diagnosis. (3) To compare the difference of quality andquantity of the yon Willebrand factor on the ABO blood groups. (4)To establish asystematic muitiple tests about VWD to ensure accurate diagnosis and subtyping in clinicalapplication.Methods:(1) The CHO cells were transfected with plasmid pCMV3 includingGPIbaH1-V289 DNA by Lipofectamine.The recombinant fragment was purified withaffinity chromatography column. (2) We describe an ELISA method in which arecombinant fragment of platelet glycoprotein Iba (rGPIba) is bound to an anti-GPIbamonoclonal antibody 2D4 immobilized onto microtiter plate wells, which capture plasmaVWF in the presence of ristocetin. Then we compare the sensitivity and repeatability ofthis new method with platelet agglutination test.(3) We detected the levels of VWF:Ag,VWF: CB,VWF: RCO and VWF: FⅧB of the healthy donors(different ABO bloodgroup), the VWD patients and the other bleeding disorders by ELISA,and the multimerassay by sodium dodecyl suphate agarose gel electrophoresis.Results: (1) The supernatant liquid included high expression of rGPIba by the cellculture, It could inhibit binding activity between plasma VWF and platelets. (2)Wedetected VWF:RCO by ELISA, there were conspicuous difference between the VWDpatients and the healthy donors (P=0.0001). But, the other bleeding disorders were similarto the healthy donors.(P＞0.05). The coefficient of variation for inner-batch and inter-batchof this new method was 5.4% and 9.4% respectively, and 17.8% and 18.3% respectively by platelet agglutination test. (3) There were conspicuous differences of VWF:Ag, VWF:RCO and VWF: CB between the O group and non-O group (P＜0.001). (4)We used PLTand APTT as screening,VWF:Ag,VWF:CB and VWF:RCO-ELISA as diagnosis, andRIPA, VWF:FⅧB, multimer assay as subtyping.Conclusion:(1) CHO cell line can express rGPIba H1-V28 stably and therecombinant fragment has high purity, immune activity and biology activity . (2) TheVWF:RCO measured by ELISA is highly sensitive, specific and reproducible, and easy tooperate. This method can provide a more accurate laboratory evidence of the clinicaldiagnosis, especially type 1, type2A and 2B, type 3 VWD. (3) The O group individualsmight have a greater risk for bleeding than non-O group, the influence of ABO blood groupto VWF quality and quantity should be taken into accounted in clinical practice. (4)Thecombined-test of various parameters is so significant that can provide an accuratediagnosis, therapy and prognosis for VWD.