Classic hemophilia, (hemophilia A), is an X-linked hereditary bleeding disorder affecting half of the male offspring of female carriers. Prenatal diagnosis offers an option, namely to restrict abortions to hemophilic fetuses only, and thus retain the chance of bearing normal sons. Recently, the authors have made a prenatal diagnosis of hemophilia A in an obligate carrier with a male fetus at 24 weeks of gestation by pure fetal sampling and accurate factor VIII coagulant assay, which was repeatedly less than 1% at 28 weeks of gestation.
Adult ; Factor VIII/analysis ; Female ; Fetal Diseases/*diagnosis ; Hemophilia A/*diagnosis ; Humans ; Male ; Pregnancy ; *Prenatal Diagnosis

In order to analyze the clinical features and laboratory findings in patients with acquired hemophilia A, one case of acquired hemophilia A was studied, the medical history, clinical features, ultrasonography and laboratory examination including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and FVIII:C, FIX:C, FXI:C, FXII:C ratio, as well as medical treatment were analyzed. The results showed 99.3 sec of APTT, 13 sec of PT, and 13.5 sec of TT, 2% of FVIII:C, 7% of FIX:C, 9% of FXI:C and 21% of FXII:C. The prolongation of APTT could not be completely corrected by mixing the patient plasma with an equal volume of normal fresh plasma; the APTT increased with prolongation of incubation time, when patient plasma was mixed with an equal volume of normal fresh plasma and incubated at 37 degrees C. The plasma FVIII:C, FIX:C, FXI:C and FXII:C levels in patient were 6%, 75%, 95% and 123% respectively, when patient's plasma was diluted by tenfold and mixed with an equal volume of non-diluted normal plasma. FVIII inhibitor in the patient's serum was at a level >32.0 Bethesda units/ml after acquired hemophilia was diagnosed, the patient was admitted to hospital and given orally prednisone and azathioprine therapy. One month later, clinical status of the patient were improved with 33.3 seconds of APTT, 128% of FVIII level and elimination of FVIII inhibitor. In conclusion, inquiring case history, analyzing imaging results, detecting level of APTT, performing dilution test and assaying titer of FVIII inhibitor can reduce misdiagnosis and wrong therapy for patients with acquired hemophilia A. The FVIII inhibitor can be eliminated and function of clotting can be recovered by using immunosuppressive therapy.
Factor VIII ; analysis ; antagonists & inhibitors ; Female ; Hemophilia A ; blood ; diagnosis ; Humans ; Middle Aged

OBJECTIVETo investigate the molecular mechanism of a Chinese hemophilia A patient in whom there was a discrepancy between the clinical bleeding symptoms and laboratory assay of FVIII activity (FVIII: C).

METHODSFVIII: C was detected by chromogenic and one-stage methods, and FVIII: Ag by ELISA. The APTT corrected test was used to screen the FVIII inhibitor and PCR amplification to analyze all the exons and flanking sequences of F8 gene of the proband, PCR products were purified and sequenced directly. The corresponding gene sites of family members were detected according to the gene mutation sites. Two B domain deleted human FVIII mutant expression plasmids His99Arg and His99Ala (pRC/RS V - BDhFVIIIcDNA) were constructed and transfected into HEK293T transiently. FVIII: Ag and FVIII: C of the expression products were assayed.

RESULTSThe proband APTT was prolonged, FVIII: Ag was 120% but FVIII: C <1% and no FVIII inhibitor in plasma. The results of anticoagulation and fibrinolytic functions were normal. The cross reacting material positive (CRM+) hemophilia A was diagnosed. Gene analysis revealed a A28828G substitution in exon 3 resulted in a H (His) to R (Arg) missense mutation and the same heterozygous was identified in his mother. In vitro expression of FVIII: Ag and FVIII: C of His99Arg were 180.0% and 5.8% , respectively, while FVIII: Ag and FVIII: C of His99Ala were 45.0% and 20.0% of that of wild type, respectively. His99Arg and His99Ala were diagnosed as CRM+ and CRM- mutations, respectively.

CONCLUSIONBoth the two F VIII mutations could express FVIII protein. However, CRM His99Arg mutant protein has little FVIII procoagulant activity and His99Ala has reduced FVIII function by routine methods.

Adult ; DNA Mutational Analysis ; Factor VIII ; genetics ; Genotype ; Hemophilia A ; etiology ; genetics ; Humans ; Male ; Mutation, Missense

OBJECTIVE: To investigate the relationship between the type of FⅧgene mutation and the development of FⅧ inhibitors in patients with severe haemophilia A (HA). METHODS: The medical records of 172 patients with severe hemophilia A from January 2009 to September 2020 were reviewed. The types of FⅧgene mutations and the production of factor Ⅷ inhibitors were collected and divided into high-risk mutation group ( intron 1 inversions, large deletions, nonsense mutations), low-risk mutation group (missense mutations, small deletions and insertions, splice site mutations) and intron 22 inversions group. The correlation of FⅧgenotype and the production of FⅧ inhibitors in patients with HA were analyzed. RESULTS: Among 172 patients with severe HA, 21 cases(12.21%) developed FⅧ inhibitors. The cumulative incidence of FⅧ inhibitor development was 32%(10/31) in high risk group (75% patients with large deletions, 43% patients with intron 1 inversions, 20% patients with nonsense mutations) and 5%(2/43) in low risk group(6% patients with missense mutations, 5% patients with small deletions or insertions and 0% patient with a splice site mutation) and 9%(9/98) in intron 22 inversions group. Compared with the risk of FⅧ inhibitor development in intron 22 inversions group, the risk of FⅧ inhibitor development in high risk group was higher (OR＝4.7, 95% CI： 1.7-13.0), the risk of FⅧ inhibitor development in low risk group was equal (OR＝0.5, 95% CI： 0.1-2.3). Compared with the risk of inhibitor development in low risk group, the risk of FⅧ inhibitor development in high risk group was higher (OR＝9.8, 95% CI： 2.0-48.7). CONCLUSION Gene mutations of patients with severe HA in high-risk group which include intron 1 inversions, large deletions, nonsense mutations are a risk factor for FⅧ inhibitor production.
Codon, Nonsense ; DNA Mutational Analysis ; Factor VIII/genetics* ; Hemophilia A/genetics* ; Humans ; Introns ; Mutation

PURPOSE: We report a case of branch retinal artery occlusion (BRAO) in a healthy pregnant woman. METHODS: A 29-year-old pregnant woman presented with decreased vision in her left eye. She had a pale retina with macular edema consistent with BRAO. An extensive workup was performed to determine an etiologic factor. All test results were within normal limits except for her factor VIII activity. Her visual acuity improved from finger counting to 20/30 over 2 months without any treatment. RESULTS: This case suggests that BRAO can occur in healthy patients without any systemic or ocular disorders. CONCLUSIONS: BRAO can occur in healthy patients without any systemic or ocular disorders, despite an extensile evaluation.
Adult ; Factor VIII/analysis ; Female ; Humans ; Macular Edema/etiology ; Pregnancy ; *Pregnancy Complications, Hematologic ; Retinal Artery Occlusion/*etiology ; Vision Disorders/etiology

PURPOSE: We report a case of branch retinal artery occlusion (BRAO) in a healthy pregnant woman. METHODS: A 29-year-old pregnant woman presented with decreased vision in her left eye. She had a pale retina with macular edema consistent with BRAO. An extensive workup was performed to determine an etiologic factor. All test results were within normal limits except for her factor VIII activity. Her visual acuity improved from finger counting to 20/30 over 2 months without any treatment. RESULTS: This case suggests that BRAO can occur in healthy patients without any systemic or ocular disorders. CONCLUSIONS: BRAO can occur in healthy patients without any systemic or ocular disorders, despite an extensile evaluation.
Adult ; Factor VIII/analysis ; Female ; Humans ; Macular Edema/etiology ; Pregnancy ; *Pregnancy Complications, Hematologic ; Retinal Artery Occlusion/*etiology ; Vision Disorders/etiology

The aim is to observe the expression of human factor VIII gene in mice tranduced in vivo and ex vivo. The vector pLNC-FVIII BD was generated by cloning a B-domain-deleted (760aa-1639aa) FVIII cDNA (FVIIIBD cDNA) into retroviral vector pLNCX. 2 x 10(6) of mouse bone marrow stroma cells transduced by LNC-FVIII BD were infused into 4-week-old BALB/c mice by tail-vein injection. pLNC-FVIII BD was conjugated with PAMAM dendrimer to form complex PAMAM-pLNC-FVIII BD, with which C57BL/6J were injected by tail vein (200 micro l contained 15 micro g/mouse) and sacrificed at days 1, 2, 7, 14, 21 and 28, respectively after injection. Tissue such as liver, spleen, lung and kindney were harvested, with which the transcription were detected by means of RT-PCR. In addition, blood was collected to be measured human FVIII Ag, human FVIIIc and anti-FVIII of human inhibitors. The results showed that the highest level of human FVIII in the recipient BALB/c mice was 8.6 +/- 1.44 ng/ml detected on the first day post-injection; anti-FVIII antibodies were detected from the first week post-injection, and then the level of FVIII Ag decreased and cannot be measured on the fourth week. In the C57BL/6J mice physiological level of human FVIII was expressed in plasma at 48 hours after injection and the average human FVIIIc was 0.62 U/ml and the average human FVIII Ag was 115.5 ng/ml, and gradually reduced later. Anti-FVIII of human inhibitors was not revealed all the time. Syngene image scanning demonstrated that the transcription of the human FVIII BD cDNA occurred mainly in spleen and lung, and secondarily in liver and kidney. No side effects of PAMAM-pLNC-FVIII BD were observed in mice tissue by pathological examination at 4 weeks. In conclusion, retrovirus-transduced bone marrow stroma cells effectively produced human FVIII after ex vivo transduction, but the development of anti-FVIII antibodies in recipient mice influenced the expression level. The human FVIII gene can successfully be transduced in vivo through injecting PAMAM-pLNC-FVIII BD cDNA into mice intravenously. There was physiological level expression of human FVIII in plasma at 48 hours after injection and the average human FVIIIc is 0.62 U/ml and the peak in the six mice was 0.89 U/ml, and gradually reduced later.
Animals ; DNA, Complementary ; analysis ; Factor VIII ; genetics ; Genetic Therapy ; Hemophilia A ; therapy ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Transfection

This study was aimed to investigate the suitability of FVIII gene BclI (intron 18)and HindIII (intron 19) site polymorphism for genetic diagnosis of patients with hemophilia A (HA) and their families, and for detection of carriers. The FVIII gene bclI (intron 18) and HindIII (intron 19) site polymorphism on the X chromosome of 8 patients with HA and 45 families members were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), the pedigree of patients was drawn by means of PediDraw software online provided by China Genetic Counseling Network. The results indicated that combination detection of BclI and HindIII sites could provide the diagnosis information for 5 out of 8 HA families with diagnostic ratio of 62.57%, especially 2 HA families were accompanied by mutation of 2 sites. Besides, the definite diagnosis for 6 out of 11 suspicious carriers in 8 families could be made with diagnostic ratio of 54.5%. It is concluded that the combination detection of BclI and HindIII sites for analysis of HA patient family can elevate the diagnostic rate of HA patients and carriers.
DNA Mutational Analysis ; Factor VIII ; genetics ; Female ; Hemophilia A ; diagnosis ; genetics ; Humans ; Introns ; Male ; Pedigree ; Polymerase Chain Reaction ; methods ; Polymorphism, Genetic

Factor VIII inhibitors are produced during or after coagulation factor VIII (FVIII) therapy in hemophilia A patients. These inhibitors are usually detected by a modified Bethesda assay or an enzymelinked immunosorbent assay (ELISA). In this study, we used the Bethesda assay to determine the incidence of FVIII inhibitors in 75 fresh plasma samples obtained from 50 hemophilia A patients, and then used ELISA and the Bethesda assay to determine the titres of these inhibitors after the samples had been frozen and thawed. The samples from the screening Bethesda assay were centrifuged and stored at -70degrees C in accordance with the assay guidelines. Subsequently, these samples were thawed and analyzed using ELISA and the Bethesda assay. The incidence of inhibitors in hemophilia A patients was 20.0%. Among the 35 inhibitor-positive samples identified in the screening Bethesda assay, 16 were positive in ELISA while only 4 were positive in the repeated Bethesda assay. In this study, the ELISA technique showed a higher sensitivity than the Bethesda assay in the detection of FVIII inhibitors in samples that were subjected to freezing and thawing procedures; this was because the Bethesda assay could not identify the FVIII inhibitors that were degraded after freezing and thawing.
Blood Coagulation Factor Inhibitors/*analysis ; *Enzyme-Linked Immunosorbent Assay ; Factor VIII/*antagonists &inhibitors/metabolism ; Hemophilia A/*blood/diagnosis ; Humans ; Immunologic Tests ; Male

The aim of current study was to detect intron 22 inversion of factor VIII gene in severe hemophilia A (HA) patients and screen the carriers of the gene inversion. Fifty-five cases of severe HA were involved and factor VIII gene inversion was detected and identified by long distance-PCR (LD-PCR) and 0.6% agarose gel electrophoresis. The 11 kb and 12 kb bands indicate the factor VIII gene inversion and non-inversion, respectively. Occurring of both 11 kb and 12 kb bands indicates a carrier of the inversion. The results showed that factor VIII gene inversion existed in 22 out of 55 cases, which accounted for about 40% of total detected patients. Five carriers of factor VIII gene inversion were diagnosed from the members in 15 families. In conclusion, LD-PCR assay is a simple, rapid and accurate method for detection of factor VIII gene inversion, and this approach is helpful in screening, carrier testing, and prenatal diagnosis of severe hemophilia A.
Adolescent ; Adult ; Antigens ; analysis ; Child ; Child, Preschool ; Chromosome Inversion ; Factor VIII ; genetics ; Hemophilia A ; blood ; genetics ; Humans ; Infant ; Male ; Polymerase Chain Reaction ; methods ; von Willebrand Factor ; immunology