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

BACKGROUND/AIMS: This study aimed to investigate the risk factors associated with provoked pulmonary embolism (PE). METHODS: This retrospective cohort study included 237 patients with PE. Patients that had transient risk factors at diagnosis were classified as having provoked PE, with the remaining patients being classified as having unprovoked PE. The baseline clinical characteristics and factors associated with coagulation were compared. We evaluated the risk factors associated with provoked PE. RESULTS: Of the 237 PE patients, 73 (30.8%) had provoked PE. The rate of respiratory failure and infection, as well as the disseminated intravascular coagulation score and ratio of right ventricular diameter to left ventricular diameter were significantly higher in patients with provoked PE than in those with unprovoked PE. The protein and activity levels associated with coagulation, including protein C antigen, protein S antigen, protein S activity, anti-thrombin III antigen, and factor VIII, were significantly lower in patients with provoked PE than in those with unprovoked PE. Multivariate analysis showed that infection (odds ratio [OR], 3.2; 95% confidence interval [CI], 1.4 to 7.4) and protein S activity (OR, 0.97; 95% CI, 0.95 to 0.99) were significantly associated with provoked PE. CONCLUSIONS: Protein S activity and presence of infection were important factors associated with provoked PE. We should pay attention to the presence of infection in patients with provoked PE.
Cohort Studies ; Diagnosis ; Disseminated Intravascular Coagulation ; Factor VIII ; Humans ; Multivariate Analysis ; Protein C ; Protein S ; Pulmonary Embolism* ; Respiratory Insufficiency ; Retrospective Studies ; Risk Factors*

OBJECTIVETo assess the value of thrombotic biomarkers in estimation of venous thromboembolism (VTE) risk in cancer patients.

METHODSA total of 1473 cancer patients treated in the Tianjin Medical University General Hospital from 2009 to 201 were selected, including 845 males and 628 females in the age of 56 ± 17 years. The activities of von Willebrand factor antigen (vWF:Ag), factor VII (F VII:A), factor VIII (F VIII:A), antithrombin (AT:A), protein C (PC:A) and protein S (PS:A) were assayed using an ACL TOP 700 blood coagulation analyzer. The level of D-dimer (D-D) was assayed using the Biomerieux Mini Vidas Automated Immunoassay Analyzer. Receiver operating characteristic curve (ROC) was used to analyze the diagnostic performance of the parameters. Cox regression analysis model was applied to evaluate the effect on prognosis, and Kaplan-Meier curve was used to implement the survival analysis.

RESULTSThe levels of vWF:Ag, D-D, and F VIII:A were significantly higher in all the specified tumor groups ( except the other tumor group ) than that of the control groups (P < 0.05). F VIII:A was significantly higher than that in the control group in all tumor groups except the renal carcinoma, prostatic cancer, lymphoma groups and the other tumor group (P < 0.05). The PC:A level was significantly lower in all tumor patients groups than in the control group, except glioma, breast cancer, gastric carcinoma, renal carcinoma and the other tumors groups (P < 0.05). The PS: A level was significantly lower in all tumor groups than in the control group, except the glioma, breast cancer, prostatic cancer, lymphoma and the other tumors groups (P<0.05). The AT: A level was significantly lower in all tumor groups than in the control group (P<0.05). When the optimum cut-off point of vWF:Ag for VTE diagnosis was 192% in the cancer group, the area under ROC curve = 0.828 (95% CI: 0.716 to 0.939). When the optimum cut-off point of D-dimer for VTE diagnosis was 1484 ng/ml in the cancer group, the area under ROC curve = 0.915 (95% confidence interval: 0. 840 to 0.988). When the optimum cut-off point of PC: A for VTE diagnosis was 75.2% in the cancer group, the area under ROC curve = 0.764 (95% confidence interval: 0.630 to 0.898). The Cox analysis showed that age, surgery, chemotherapy and D-dimer were independent risk factors for VTE event within three months in cancer patients. The cumulative probability of VTE was increased significantly in the cancer patients if whose plasma D-dimer level was over the cut-off value.

CONCLUSIONSThe plasma D-dimer level is obviously increased in cancer patients, and there is a relevance to thrombosis risk stratification and VTE cumulative probability. It is with good diagnostic performance, and may be used as an effective marker in estimation of VTE risk within 3 months in cancer patients.

Aged ; Antithrombins ; blood ; Biomarkers ; blood ; Factor VII ; analysis ; Factor VIII ; analysis ; Female ; Fibrin Fibrinogen Degradation Products ; Humans ; Male ; Middle Aged ; Neoplasms ; blood ; Prognosis ; Protein C ; analysis ; Protein S ; analysis ; ROC Curve ; Regression Analysis ; Risk Assessment ; Risk Factors ; Venous Thromboembolism ; etiology ; von Willebrand Factor ; analysis

OBJECTIVETo study the current situation of coagulation factor VIII (FVIII) inhibitor development in children with hemophilia A (HA) through a cross-sectional survey, and to explore the risk factors of inhibitor development in order to provide evidence for further prevention and management strategies.

METHODThe clinical data of outpatients with hemophilia A in Beijing Children's Hospital seen from November 2012 to May 2013 were collected, FVIII inhibitor was screened and analyzed its risk factors.

RESULTA total of 102 HA children were enrolled, 5 were mild cases, 32 were moderate, and 65 were severe cases; the median age on enrollment was 55.5 (3.0-200.0) months:19(18.6%) of patients had inhibitors and 9 (8.8%) had low-titer inhibitors, 10 (9.8%) had high-titer inhibitors. Receiving FVIII treatment for life-threatening bleeding (P = 0.03) ,OR 4.10 (95%CI:1.17-14.32) was a risk factor for inhibitor generation and patients within 20 exposure days have more chances of inhibitor development (P = 0.04) ,OR 3.32 (95%CI:1.02-10.86) . High and intense FVIII exposure within short term was the risk factor for high titer inhibitor development (P = 0.01) ,OR 5.25 (95%CI:1.45-21.92) .

CONCLUSIONIntense FVIII exposure for severe hemorrhage was the risk factor of inhibitors development especially of high titer inhibitors.

Adolescent ; Blood Coagulation Factor Inhibitors ; blood ; Child ; Child, Preschool ; Cross-Sectional Studies ; Dose-Response Relationship, Drug ; Factor VIII ; administration & dosage ; antagonists & inhibitors ; Female ; Hemophilia A ; blood ; therapy ; Humans ; Infant ; Male ; Multivariate Analysis ; Risk Factors ; Time Factors

OBJECTIVETo study the effects of desmopressin (DDAVP) on coagulation factor Ⅷ (FⅧ) and activated partial thromboplastin time (APTT) in children with mild hemophilia A.

METHODSEighteen children with mild hemophilia A were enrolled. DDAVP (0.3 μg/kg•d) was injected intravenously for 5 days. Plasma FⅧ levels and APTT were measured before and after DDAVP treatment.

RESULTSIn 16 of 18 children with mild hemophilia A, the bleeding symptoms, including the articular or musclar hematoma, were significantly alleviated as a result of DDAVP treatment. The plasma FⅧ levels increased significantly to (27±4)% and APTT was shortened to (66±10)s 60 minutes after the first dose of DDAVP treatment. The plasma FⅧ remained at the levels of 25%-30% during 3-4 days of DDAVP treatment. Five days after DDAVP treatment, the plasma FⅧ levels decreased [(21±3)%], and APTT was prolonged when compared with 1-4 days of DDAVP treatment.

CONCLUSIONSDDAVP treatment can increase plasma FⅧ levels and shorten APTT in children with mild hemophilia A. DDAVP is effective in the treatment of mild hemophilia A. The duration of DDAVP therapy for mild hemophilia A is recommended as 3 to 4 days.

Child ; Child, Preschool ; Deamino Arginine Vasopressin ; therapeutic use ; Factor VIII ; analysis ; Hemophilia A ; blood ; drug therapy ; Humans ; Infant ; Male ; Partial Thromboplastin Time

OBJECTIVETo identify the F VIII gene mutations of patients and suspected female carriers in 10 Hemophilia A (HA) families, and to guide the prenatal diagnosis.

METHODSPCR, denaturinghigh performance liquid chromatogramphy (DHPLC) and DNA sequencing technologies were applied to screen the F VIII gene of 8 HA patients and 12 suspected female carriers in the 10 families. Linkage analysis was performed by using St 14(DXS 52), intron 13 (CA)n and EX18/Bcl I of the F VIII gene in the HA families. In prenatal diagnosis, we screened the same mutation found in the patients. PCR-restriction fragment length polymorphism was applied to detect the new missense mutations of F VIII gene in 100 unrelated healthy individuals to exclude the possibility of polymorphism.

RESULTSFive missense mutations, 3 frameshift mutations, 2 nonsense mutations and 2 single nucleotide polymorphism (SNP) were identified in 10 the HA families. Among them, c.878A to G, c.1015A to G, c.6870G to T, c.1282delA, c.3072_3073insT, c.4880_4881insA and c.5000G to A were novel mutations or polymorphism. No missense mutations c.878A G, c.1015A to G and c.6870G to T, were found in the 100 healthy unrelated controls. (2) Nine suspected female carriers were confirmed at the gene level. (3) X risk chromosome could be determined to in 4 HA families by genetic linkage analysis. (4) Among the four fetuses for prenatal diagnosis, 2 were normal, 1 was carrier and the remaining 1 was a patient.

CONCLUSIONSix novel mutations, i.e., c.878A to G, c.1015A to G, c.6870G to T, c.1282delA, c.3072_3073insT and c.4880_4881insA, were identified in this study. PCR, DHPLC and DNA sequencing could be used to screen the gene mutations of HA patients, to carry out carrier detection and prenatal diagnosis of HA families efficiently, by combining with restriction endonuclease analysis and genetic linkage analysis.

Chromosomes, Human, X ; DNA Mutational Analysis ; methods ; DNA Restriction Enzymes ; genetics ; Factor VIII ; genetics ; Female ; Genetic Testing ; methods ; Hemophilia A ; diagnosis ; genetics ; Heterozygote ; Humans ; Male ; Mutation ; Pedigree ; Polymorphism, Single Nucleotide ; Prenatal Diagnosis ; methods ; Sequence Analysis, DNA ; methods

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

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

OBJECTIVEScreening the intron 1 inversion of factor VIII (FVIII) in the population of severe haemophilia A(HA) in China and performing carrier detection and prenatal diagnosis.

METHODSUsing LD-PCR to detect intron 22 inversions and multiple-PCR within two tubes to intron 1 inversions in severe HA patients. Carrier detection and prenatal diagnosis were performed in affected families. Linkage analysis and DNA sequencing were used to verify these tests.

RESULTSOne hundred and eighteen patients were seven diagnosed as intron 22 inversions and 7 were intron 1 inversions out of 247 severe HA patients. The prevalence of the intron 1 inversion in Chinese severe haemophilia A patients was 2.8% (7/247). Six women from family A and 2 from family B were diagnosed as carriers. One fetus from family A was affected fetus.

CONCLUSIONIntron 1 inversion could be detected directly by multiple-PCR within two tubes. This method made the strategy more perfective in carrier and prenatal diagnosis of haemophilia A.

Adult ; Chromosome Inversion ; genetics ; DNA Mutational Analysis ; Factor VIII ; genetics ; Female ; Hemophilia A ; diagnosis ; genetics ; Humans ; Introns ; genetics ; Male ; Polymerase Chain Reaction ; Pregnancy ; Prenatal Diagnosis ; methods