This study was purposed to detect the alloantibodies against Factor VIII (FVIII) by ELISA for estimating the incidence of the alloantibodies against Factor VIII (FVIII) in patients with hemophilia A, and to investigate the relationship between factor VIIIC domain and alloantibodies. Total of 140 patients with hemophilia A and 80 normal controls were enrolled in this study, among them plasma FVIII level of 84 patients was less than 1%, plasma FVIII level of 34 patients was between 1% and 5%, and plasma FVIII level of 22 patients was more than 5%. All patients were treated with plasma-derived FVIII concentrate or plasma before. The ELISA plate was coated with McAb (SZ-132) against FVIII prepared in our laboratory, then human recombinant FVIII concentrates 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 was recorded. The threshold of alloantibody of patient plasma was set as mean value>3 SD more than control. The plate was coated with antibody against His, then human recombinant FVIII-C1C2 prepared in our laboratory was added. 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 the mean value>3 SD more than control. The results showed that the alloantibodies against FVIII were found in 56 patients (40%) by ELISA, and 82.1% (46/56) of this kind of alloantibody could interact with the C domain of FVIII. It is concluded that C domain of FVIII is one of the primary binding sites for the alloantibodies against FVIII in Chinese patients with hemophilia A.
Adolescent ; Adult ; Binding Sites, Antibody ; Case-Control Studies ; Child ; Child, Preschool ; Factor VIII ; immunology ; Hemophilia A ; blood ; immunology ; Humans ; Infant ; Isoantibodies ; blood ; Male ; Middle Aged ; Protein Interaction Domains and Motifs ; Young Adult

PURPOSE: Hemophilia A (HA) is the most common X-linked inherited bleeding disorder. In some patients with HA, particularly those with severe HA, replacement therapy results in the production of high-responding clotting factor VIII inhibitors. The economic burden of this complication is the highest reported for a chronic disease. Our aim was to investigate the direct medical expenditure burden of high-responding inhibitors in patients with HA. MATERIALS AND METHODS: A retrospective study was conducted using the National Health Insurance Research Database, utilizing data covering the period of 2004-2007. RESULTS: In total, 638 males with HA, including 37 patients with high-responding inhibitors were evaluated. Over 99% of the annual median medical expenditure was attributable to the cost of clotting factor concentrates (CFCs) in patients with high-responding inhibitors. The annual median expenditure related to CFCs of the total medical care and outpatient care were US$170611 and US$141982, respectively, and were 4.6- and 4.3-fold higher in these patients during the study period, respectively. In patients with high-responding inhibitors, the median hospitalization expenditure and daily hospitalization cost with or without surgical procedures were 3.0- and 2.4-fold higher, respectively, and 4.3 and 5.6-fold higher, respectively. CONCLUSION: Our data reveal higher medical expenditures burden for patients with HA and high-responding inhibitors in Taiwan. Future research is encouraged to evaluate the impact of this burden on patient quality of life.
*Cost of Illness ; *Drug Resistance ; Factor VIII/immunology/therapeutic use ; Hemophilia A/*complications/drug therapy/economics ; Hospitalization/economics ; Humans ; Male ; *Quality of Life ; Retrospective Studies ; Taiwan

Blood Coagulation Factor Inhibitors ; antagonists & inhibitors ; blood ; Factor VIII ; administration & dosage ; antagonists & inhibitors ; immunology ; Hemophilia A ; drug therapy ; genetics ; immunology ; Humans ; Immune Tolerance ; Isoantibodies ; blood ; immunology ; Recombinant Proteins ; adverse effects ; immunology ; therapeutic use ; Risk Factors ; Time Factors

Acquired hemophilia A (AHA) is a rare coagulopathy caused by autoantibodies to coagulation factor VIII (FVIII). Most patients with AHA have been previously healthy; however, a variety of morbidities have been associated with the condition including pregnancy. A 40-yr-old woman visited our institution with extensive hematoma on the right hip area. Her medical history revealed no personal or familial history of bleeding diathesis. Her coagulation tests showed markedly prolonged aPTT (117 sec), markedly decreased level of FVIII activity (0.4%) and high-titer FVIII inhibitor (77 BU). Collectively, she was diagnosed as having postpartum AHA and was treated with bypassing agents and corticosteroids. Her aPTT was normalized on the 174th postpartum day and FVIII inhibitor showed negative conversion on the 224th postpartum day. This is the first case of postpartum AHA with high-titer FVIII inhibitor in Korea. Timely diagnosis and management can reduce morbidity and mortality of this potentially life-threatening condition.
Adrenal Cortex Hormones/therapeutic use ; Adult ; Autoantibodies/blood ; Blood Coagulation Factors/therapeutic use ; Factor VIII/immunology ; Factor VIIa/therapeutic use ; Female ; Hematoma/diagnosis ; Hemophilia A/*diagnosis/therapy ; Humans ; Partial Thromboplastin Time ; Postpartum Period ; Pregnancy ; Recombinant Proteins/therapeutic use ; Republic of Korea

This study was purposed to investigate the diagnosis, typing and influencing factors of the antibody (inhibitor) to coagulation factors in hemophilia. 500 hemophilia patients were enrolled in this study. The activities of coagulation factor FVIII and FIX were tested by one stage assay. The antibodies of FVIII and FIX were detected by Bethesda assay. All data were analyzed by statistical soft SPSS 10.0. The results indicated that there were 411 cases of hemophilia A, out of which 151 cases (30.2%) showed FVIII antibody positive, the titer was 3.50 ± 2.84 Bu/ml; there were 79 cases of hemophilia B, out of which 18 cases (3.6%) showed FIX antibody positive, the titer was 2.92 ± 2.19 Bu/ml. The other 10 cases were acquired autogeneic hemophilia (2.0%). The antibody was divided into three types: high-response (3 cases), intermediate-response (47 cases), and low-response (119 cases). Among the 169 cases with antibody positive, 157 cases (92.9%) were younger than 30 years old; among 151 (89.35%) cases of hemophilia A; 138 cases (81.66%) were moderate or severe hemophilia; 166 case (98.22%) showed intermediate or low-response antibody. There were 158 cases with allogeneic antibody positive, all of which received blood transfusion. It is concluded that the moderate and low responsive antibodies are the dominant in hemophilia patients, the age of patients and transfusion frequency of blood preparation are the influencing factors. The results of this study provide the basis for the hemophilia diagnosis, antibody typing and evaluation of factors influencing hemophilia, and also suggest that the repeated transfusion of blood preparation may influence the production of antibodies.
Adolescent ; Adult ; Aged ; Autoantibodies ; blood ; Child ; Child, Preschool ; Factor VIII ; immunology ; Female ; Hemophilia A ; blood ; diagnosis ; immunology ; Humans ; Infant ; Male ; Middle Aged ; Young Adult

PURPOSE: This study was designed to investigate whether transduction of lentiviral vectors (LV) carrying human coagulation factor VIII (hFVIII) cDNA into skeletal muscle could increase circulating hFVIII concentrations. MATERIALS AND METHODS: A LV containing bacterial LacZ gene as a control or human FVIII gene was intramuscularly administered into the thigh muscle of 5 weeks old Sparague-Dawley rats. The plasma human FVIII concentration and neutralizing anti-FVIII antibodies were measured for up to 12 weeks in these experimental animals. RESULTS: The plasma human FVIII levels in the rats injected with LV carrying FVIII cDNA peaked at post-injection 1st week (5.19 +/- 0.14 ng/mL vs. 0.21 +/- 0.05 ng/mL in control rats , p < 0.05). Elevated hFVIII concentrations were maintained for 4 weeks (2.52 +/- 0.83 ng/mL vs. 0.17 +/- 0.08 ng/mL in control rats, p < 0.05) after a single intramuscular injection. In the Bethesda assay, neutralizing antibodies for FVIII protein were detected only in FVIII-LV injected rats by the 10th week, but not in control rats. CONCLUSION: This study suggested that a single administration of an advanced generation LV carrying the human FVIII cDNA resulted in elevation of FVIII level in immune competent rats, and that this gene transfer approach to the skeletal muscle could be an effective tool in treatment of hemophilia A.
Animals ; Antibodies/blood/immunology ; Factor VIII/genetics/immunology/*metabolism ; Gene Therapy ; Genetic Vectors/genetics ; Hela Cells ; Hemophilia A/therapy ; Humans ; Lentivirus/genetics ; Male ; Muscle, Skeletal/*metabolism ; Rats ; Rats, Sprague-Dawley ; Transduction, Genetic ; beta-Galactosidase

OBJECTIVETo explore the immune tolerance induction (ITI) in a severe hemophilia A patient with inhibitor, and to improve the therapeutic efficacy for patient.

METHODSThe FVIII:C was assayed by one-stage method and FVIII antibody by Bethesda method. Mutation screening of FVIII gene intron 22 inversion was performed using LD-PCR.

RESULTSFVIII gene intron 22 inversion was detected in this patient. Clinical tolerance to FVIII was successfully induced after administration of the ITI regimen combined with immunosuppression. A fall of inhibitor titer from 8 BU to 0 BU after treatment for 3 months, and in vivo FVIII recovery (> 66%) was normalized. The patient had no bleeding episode in the following 6 months.

CONCLUSIONThis is the first case report on successful immune tolerance induction therapy in Chinese hemophilia A patient. ITI is the most effective therapy for hemophilia A with inhibitor.

Autoantibodies ; immunology ; Factor VIII ; genetics ; Hemophilia A ; genetics ; Humans ; Immune Tolerance ; drug effects ; Immunosuppression

OBJECTIVETo compare the sensitivity and practicability of modified Bethesda assay and Nijmegen assay in detecting factor VIII (FVIII) inhibitor.

METHODSModified Bethesda assay and Nijmegen assay were used to screen FVIII inhibitors in 237 patients with hemophilia A. The buffer plus universal coagulation reference plasma (UCRP) was used to establish a standard curve for FVIII: C assay in modified Bethesda method, instead of Nijmegen plasma plus FVIII deficiency plasma in Nijmegen method. The cutoff value for positive FVIII inhibitors is > or = 0.6 BU/ml.

RESULTSThe positive rate of FVIII inhibitors was 5.5% (n = 13) when using modified Bethesda assay and was 8.4% (n = 20) when using Nijmegen assay (P > 0.05).

CONCLUSIONModified standard Bethesda assay is a convenient and feasible method for detecting FVIII inhibitors.

Adolescent ; Adult ; Autoantibodies ; blood ; Blood Coagulation Tests ; methods ; Child ; Child, Preschool ; Factor VIII ; immunology ; Female ; Hemophilia A ; blood ; immunology ; Humans ; Male ; Middle Aged ; Sensitivity and Specificity ; Young Adult

OBJECTIVETo develop a monoclonal antibody (mAb) directed to FVIII C2 domain and investigate its effect on FVIII activity.

METHODSFVIII C2 protein was expressed in E. coli and purified. A murine antihuman FVIII C2 domain mAb SZ-132 was developed by standard hybridoma technology and characterized. In coagulation assays, different concentrations of SZ-132 were incubated with freshly collected pooled human plasma and the residual activity of FVIII and activated partial thromboplastin time (APTT) were determined. The effects of SZ-132 on rhFVIII binding to purified human vWF, phosphatidylserine (PS) and platelets were assessed by enzyme linked immunosorbent assays (ELISA).

RESULTSSZ-132 could inhibit FVIII procoagulant activity in a dose-dependent manner within the concentrations of 0-25 microg/ml and the FVIII activity was completely inhibited on above 25 microg/ml. It could also prevent rhFVIII from binding to vWF, PS and platelets.

CONCLUSIONSSZ-132 is a neutralizing mAb against FVIII C2 domain and can inhibit FVIII procoagulant activity by preventing FVIII from binding to vWF and PS.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Neutralizing ; biosynthesis ; immunology ; Factor VIII ; immunology ; metabolism ; Humans ; Male ; Mice ; Mice, Inbred BALB C

Acquired hemophilia is a rare disorder caused by autoantibodies to factor VIII (FVIII) (also referred to as factor VIII inhibitors or anti-FVIII) and may be associated with pregnancy, underlying malignancy, or autoimmune disorders. A 33-month-old girl who presented with hematochezia and ecchymotic skin lesions was diagnosed with Mycoplasma pneumoniae pneumonia by serology and polymerase chain reaction. Hematologic studies showed a prolonged activated partial thromboplastin time (aPTT), partially corrected mixing test for aPTT, reduced levels of FVIII, and the presence of antibodies against FVIII. She was treated conservatively with prednisone and intravenous immunoglobulin (IVIG) without FVIII transfusion and recovered without sequelae. This report provides the first description of acquired hemophilia due to anti-FVIII in association with M. pneumoniae in Korea. We discuss this case in the context of the current literature on acquired hemophilia in children.
Autoantibodies/blood ; Child, Preschool ; Factor VIII/immunology ; Female ; Hemophilia A/*etiology ; Humans ; Partial Thromboplastin Time ; Pneumonia, Mycoplasma/*complications/immunology ; Time Factors