The Detection And Mechanism Of The Factor Ⅷ Inhibitor With Hemophilia A

Hemophilia A(HA), a sex-linked recessive disease, is an inherited bleeding disorder due to the mutation of the gene that encodes the coagulation protein factor VIII,and the factor VIII(FVIII) replacement therapy strongly reduces HA-related mortality and disability. Recently, people attach great importance to monitoring the FVIII inhibitor for those HA patients with replacement therapy while they adopt comprehensive prevention and control measures. The patients with FVIII inhibitor are more prone to bleeding and the standard alternative approaches have limited impact, which seriously threatens their lives. An alloantibody, which has intrinsic heterogeneity, would be produced from those patients undertaking FVIII replacement therapy.Some of this kind of alloantibody, called FVIII inhibitor, can interact with the functional domain of FVIII, resulting in inhibiting or destroying its coagulation activity ,and can be detected by the methods of Bethesda or improved Nijmegen ; while others can bind the FVIII antigen epitope and form antigen-antibody complex, resulting in accelerated clearance from the circulation though they can not be easily analyzed by the above mentioned methods.Our research aim intends to explore the relationship between C domain of FVIII and its alloantibody and the relevant elements associated with FVIII inhibitor production, such as the polymorphism of TNF-αand CTLA-4, and try to find out the incidence of FVIII inhibitor using the ELISA established by ourselves in order to direct the treatment of this kind of patients.Related factors of inhibitor development in patients with hemophilia ATo analyze the clinical features of hemophilia A(HA) and to investigate the elements associated with the factor VIII(FVIII) inhibitor development in these patients. 113 patients with HA were recruited in this retrospective study, among whom, 85 patients were undertaken with replacement therapy with FVIII, and correspondingly, the FVIII inhibitor levels and some elements associated with the inhibitor development were investigated in these patients. 74, 27 and 12 patients were found in the severe,moderate and mild conditions respectively..The low-titer inhibitor levels run out of the severe and moderate patients, which accounted for 28.24% in the 85 patients treated with FVIII. The statistical significance(P< 0.05) associated with the FVIII inhibitor development was achieved regarding the following elements: the first enduring adminstration of FVIII, the condition of the patients, the gigantic dose of FVIII patients used when they undertook severe bleeding or the major operation.The occurrence of FVIII inhibitor by the Bethesda assay in Chinese hemophilia A patients is not rare, especially with low-titer inhibitor level,most of them are severe and moderate patients. And the FVIII inhibitor development was associated with the following elements: the first adminstration of FVIII more than 5 days, the severe or moderate conditions of patients, the gigantic dose of FVIII patients used when they undertook severe bleeding or the major operation.Relationship between polymorphisms in TNFA,CTLA-4 gene and inhibitor development in Chinese Han patients with hemophilia AThe single base change polymorphism in TNF-αand CTLA-4 gene were analyzed among 140 patients with hemophilia A and 108 normal controls by using PCR-restrictive fragment length polymorphism(RFLP). All of the HA patients, plasma samples were measured by Nijmegen-modified Bethesda assay simultaneously. In HA patients,G/G genotype,G/A genotype and A/A genotype were detected in 118 cases(84.3%),18 cases(12.8%) and 4 cases(2.9%) respectively. In normal controls, G/G genotype,G/A genotype and A/A genotype were detected in 92 cases(85.2%),11cases(10.2%)and 5cases(4.6%)respectively. In HA patients,C/Cgenotype,C/T genotype and T/T genotype were detected in 108cases(77.2%),30 cases(21.4%) and 2 cases(1.4%)respectively. In normal controls, C/Cgenotype,C/T genotype and T/T genotype were detected in 81cases(75%),25cases(23.1%)and 2 cases(1.9%)respectively.The difference in the genotype frequencies between HA patients and controls was nonsignificant(P>0.05). The FⅧinhibitor activity was detected by Nijmegen -modified Bethesda assay in 34 patients (24.3%), all of them with low-titer inhibitor. Patients who were carrier of homozygotes for A allele had a higher risk of inhibitor development compared with patients who were not(OR=7.519,95% confidence interval=3.168～17.844). Patients with severe HA who were carrier of homozygotes for A allele had a higher risk of inhibitor development compared with patients who were not(OR=8.163,95%Confidence interval=2.521～26.434). TNF-α-308 gene polymorphism is significantly associated with inhibitor development in Chinese Han patients with severe hemophilia A . TNF-αgene may be a useful marker and potential modulator of the immune response to replacement therapy in patients with hemophilia A .Measure of alloantibodies against factorⅧwith Hemophilia A using a newly-developed ELISA and the interaction between factorⅧC domain and alloantibodies140 patients with hemophilia A and 80 normal controls were enrolled, among them, 84 patients with plasma FⅧlevel less than 1%, 34 patients between 1% to 5%, and 22 patients more than 5%. The titer wells were coated with MoAb against FⅧdeveloped in our lab, then human recombinant FⅧconcentrates were applied. After incubation in room temperature for 2 hours, diluted plasma samples and HRP-conjugated goat anti-human IgG were added successively, finally A490 were recorded. The threshold of alloantibody of patient plasma was set as more than 3 SD than control. All of the plasma samples were measured by Nijmegen-modified Bethesda assay simultaneously. The results showed that the alloantibodies against FⅧwere found in 56 patients (40%) by ELISA assay, while the FⅧinhibitor activity was detected in 34 patients (24.3%), all of them with low-titer inhibitor.There were 25 patients, which were positive for FⅧalloantibodies by the ELISA assay, without FⅧinhibitor activity by the Bethesda assay.In order to investigate the interaction between factorⅧC domain and alloantibodies, The titer wells were coated with antibody against His, then human recombinant FⅧ-C1C2 developed in our lab were applied. After incubation in room temperature for 2 hours, diluted plasma samples and HRP-conjugated goat anti-human IgG were added successively, finally A490 were recorded. The threshold was set as more than 3 SD than control.82.1%(46/56) of this kind of alloantibody can interact with the C domain of FVIII.These results suggest the occurrence of FⅧinhibitor in Chinese hemophilia A patients is not rare, especially with low-titer inhibitor level. The ELISA-based assay for alloantibodies against FⅧcould provide a rapid and sensitive screening tool for estimating the state of patients and guiding the treatment for patients with hemophilia A., most of this kind of alloantibody can interact with the C domain of FVIII.