Validation of the detection method for residual human coagulation factor Ⅺ in human prothrombin complex / 中国输血杂志

【Objective】 To establish an enzyme-linked immunosorbent assay (ELISA) method for the determination of residual human coagulation factor Ⅺ in human prothrombin complex and validate the method. 【Methods】 Human factor Ⅺ was reacted with the capture antibody coated on the microtiter plate. After appropriate washing steps, biotinylated primary antibody was bound to the captured protein. Excess primary antibody was washed away and bound antibody was reacted with horseradish peroxidase conjugated streptavidin. TMB substrate was used for color development at 450 nm. The dilution reliability, accuracy, specificity, repeatability, intermediate precision, linearity, range and durability were verified. 【Results】 The verification results showed that the accuracy and specificity of this method met the experimental requirements, with an average recovery rate of 109.2% and RSD of 6.93%. The repeatability RSD was 6.78%, and the intermediate precision RSD was 6.75%, indicating good precision. The linear regression correlation coefficient of standard curve was 0.999 9, showing good accuracy and precision within the linear range. The durability was verified by the incubation time and the validity period of reagent kit opening. The results showed that the RSD of the incubation time change was 6.62%, indicating that the incubation time of this detection method was controlled between 28 to 32 minutes, and there was no significant impact on the results. The RSD of the detection results before and after the reagent kit was opened and stored under conditions for 7 days was 3.84%, indicating that the preservation of the reagent kit according to the conditions for 7 days after opening has no effect on the FⅪ detection results. Both indicated that the method had good durability. The dilution reliability results showed that there was a "hook" effect in the detection of FⅪ residue in human prothrombin complex, which could be solved by diluting 100 to 200 times. 【Conclusion】 This method can be used for the determination of FⅪ residues of human prothrombin complex in laboratory.

Hemophilia A with reduced coagulation factor Ⅺ: a case report and literature review / 中国输血杂志

【Objective】 To investigate the possible molecular pathogenesis of a child with hemophilia A accompanied by coagulation factor Ⅺ reduction by testing coagulation-related indicators and genotyping in the child and his family. 【Methods】 Peripheral blood from the patient and his parents for detection of coagulation factors Ⅷ, Ⅸ, Ⅺ, Ⅻ, VWF∶Ag, lupus anticoagulants and F VIII, F XI inhibitors were collected. All exons and flanking sequences of the genes encoding FⅧ and FⅪ were sequenced and bioinformatically analyzed. 【Results】 The child had low FⅧ and FⅪ activity and no parental abnormalities were observed. The sequencing results showed that there was a c. 1834(exon12) C>T heterozygous mutation in the FⅧ gene and a c. 1817 (exon15) G>A heterozygous mutation in the FⅪ gene, which was de novo. Bioinformatics analysis shows that the FⅪ mutation changes the original protein structure and increases the number of carboxyl groups. 【Conclusion】 For patients with prolonged APTT, in addition to excluding factors that interfere with APTT testing, all coagulation factors related to APTT should be tested to clarify the diagnosis.

A study on gene mutation of coagulation factor Ⅺ protein secretion disorder and its mechanism / 中华检验医学杂志

Objective:To investigate the molecular pathogenesis of a newly discovered gene mutation in a family with hereditary coagulation factor Ⅺ(FⅪ) deficiency.Methods:The proband was admitted to the First Affiliated Hospital of Wenzhou Medical University in September 2021 due to "calculus of intrahepatic duct". The patient had no symptoms of spontaneous bleeding.The clinical data and blood samples of the proband and her family members (10 persons in 3 generations) were collected.The activated partial thromboplastin time (APTT) and FⅪ activity (FⅪ:C) were performed by the one-stage clotting assay. FⅪ antigen (FⅪ:Ag) were detected by enzyme linked immunosorbent assay (ELISA). Genomic DNA extracted from peripheral blood cells of subjects was used as template to analyze F11 gene mutation by DNA direct sequencing. Bioinformatics software was used to analyze the effects of mutations on protein structure and function. Wild-type and mutant FⅪ protein expression vectors were constructed and transient transfected into HEK293T cells. The total RNA was extracted from positive transfected cells and then reversely transcribed into cDNA. The mRNA expression level of F11 gene in transfected cells was detected by real-time fluorescence quantitative PCR (qRT-PCR). The content of FⅪ:Ag and the expression of FⅪ protein in transfected cell lysates and culture supernatant were detected by ELISA and western blot.Results:The APTT of the proband was significantly prolonged to 107.9s (reference range 29.0-43.0s), while FⅪ:C and FⅪ:Ag were significantly decreased to 2% (reference range 84%-122%) and 5% (reference range 76%-127%), respectively. Gene sequencing analysis indicated that the proband had c.536C>T (p.Thr161Met) heterozygous missense mutation and c.1556G>A (p.Trp501Ter) heterozygous nonsense mutation in exon 6 and 13 of the F11 gene, respectively. Bioinformatics analysis showed that the amino acids at site 161 of FⅪ protein were threonine (Thr) in the matrix composed of five different species, indicating that Thr161 site was highly conserved among homologous genes in different species. p.Thr161Met heterozygous mutation affected the stability of local intermolecular structure of FⅪ protein. In vitro expression experiments of p.Thr161Met mutation showed that FⅪ protein had a normal synthesis in the cells but secretion dysfunction.Conclusions:c.536C>T (p.Thr161Met) heterozygous missense mutation and c.1556G>A (p.Trp501Ter) heterozygous nonsense mutation were mainly responsible for the decrease of FⅪ in this family. p.Thr161Met mutation was first reported in the world and did not affect the normal synthesis of FⅪ protein, but caused secretion dysfunction.

Activated coagulation factor Ⅺ(FⅪa)activity in intravenous immunoglobulin preparations from China: An analysis test / 中国输血杂志

【Objective】 To investigate the activity level of coagulation factor Ⅺ(FⅪ) in IVIG products, which would provide support for improving the safety and quality control of IVIG products in China. 【Methods】 A total of 71 batches of IVIG products(half of which were closed to expiry date) from 23 domestic manufacturers (brands) were submitted for inspection and assayed for procoagulant activity including FⅪa, FⅪ and NAPTT activity with a chromogenic assay, coagulation proenzymes levels using one stage clotting assays and non-activated partial thromboplastin time (NAPTT). 【Results】 FⅪa(mIU/mL): 32 lots of IVIGs from 15 manufacturers(brands) possessed activities below 0.1 or the detection limit of the assays, 24 IVIG lots from 12 manufacturers were between 0.1~1(0.39±0.20), the remaining 15 IVIG lots from 6 manufacturers had FⅪa activity greater than 1 up to 47(13.27±15.61). FⅪ(mIU/mL): there were no detectable activities of FⅪ in 58 lots from 21 manufacturers, the other 13 lots from 6 manufacturers had IVIGs between 13~309(69.0±98.1), which included in 15 lots of IVIG whose FⅪa activity were greater than 1mIU/mL. NAPTT(s): The coagulation times for aforementioned 15 IVIG lots were all above 150s in the NAPTT test. The NAPTT ratios varied between 0.67 and 0.94(0.83±0.07), and 7 of 15 IVIGs had NAPTT ratios below or equal to 0.8. 【Conclusion】 There are substantial differences in the FⅪ and FⅪa activities in the IVIG preparations from 23 different manufacturers. Most of them had a lower activities; certain lots from specific manufacturers had relatively higher FⅪa activities. Manufactures should evaluate the manufacturing processes and monitor IVIG preparations for the presence of factor Ⅺ-like activity in case of thrombotic risks.

Investigation of the change in redox state of plasma factor Ⅺ in elderly type 2 diabetic patients with macroangiopathy / 中华老年医学杂志

Objective To investigate the potential effect of the redox state of plasma factor Ⅺ (FXI) on the pathogenesis of elderly diabetic hypercoagulability and macroangiopathy.Methods The plasma levels of reduced FXI were detected in elderly type 2 diabetic(T2DM)patients with/without macroangiopathy (T2DM group/DMAP group) and healthy subjects (control group),and variables associated with reduced FXI were analyzed.Results Elderly patients with T2DM had higher plasma levels of reduced FXI as compared with healthy controls.The level of reduced FXI was significantly higher in patients with macroangiopathy than without macroangiopathy [control group:(80.6± 15.6) ％,T2DM group:(94.7 ± 16.0) ％,DMAP group (142.6 ± 36.5) ％,all P＜ 0.05].The multiple stepwise regression analysis showed that plasma levels of triglyceride,cholesterol and HDL-cholesterol were the independent predictors for reduced FXI.Conclusions The plasma level of reduced FXI is increased in elderly T2DM patients with macroangiopathy.The abnormality of lipid profiles may associate with the increment of reduced FXI.These findings maybe provide the novel mechanisms for diabetic hypercoagulability and macroangiopathy.

Investigation on gene defects in a pedigree with inherited coagulation factor Ⅺ deficiency / 中华检验医学杂志

Objective To investigate the gene defects of a pedigree with inherited coagulation factor Ⅺ (FⅪ) deficiency by analyzing its phenotype and molecular genetic characteristics. Methods A pedigree with inherited FⅪ deficiency was enrolled in this study. The activated partial thromboplastin time (APTF), prothrombin time (PT), FⅪ activity (FⅪ: C) and FⅪ antigen (FⅪ: Ag) were determined for phenotype diagnosis. Fifteen exons and their flanks of F11 gene from the proband's genomic DNA were amplified by polymerase chain reaction (PCR), and the PCR products were directly sequenced to analyze the F11 gene mutation. The PCR products amplified from genomic DNA from the proband, her parents and 100 healthy donors were digested with restriction enzyme BssSI to exclude gene polymorphism and confirm the mutation site. The cleavage site in the signal peptide was predicted by the SignalP software. Results The values of APTT, PT, FⅪ: C and FⅪ: Ag of the proband were 69.5 s, 12.3 s, 2.6% and 2.5%, respectively, indicating that this case was cross-reacting material (CRM) negative. The same values of healthy controls were 35 s, 13 s, 100% and 100%, respectively. As compared with Genbank AY191837 sequence, four variants in F11 exons were found. G3733C heterozygous mutation in exon 2 causod Gly to Arg substitution at-1 amino acid position in signal peptide (G-1R). The G3733C mutation in exon 2 introduced a new BssSI enzyme digestion site. Further analysis of the 100 randomly collected DNA samples from the normal population excluded the possibility of G3733C as a polymorphism. CI6642T heterozygous mutation in exon 8 introduced a premature stop codon at 263 amino acid position (Q263Term). Conclusions G-1R mutation and Q263Term compound heterozygous mutation in F11 gene are the mechanism of FⅪ deficiency for the proband. G-1R mutation is a novel F11 gene mutation causing inherited FⅪ deficiency.

Mutation analysis of coagulation factor Ⅺ gene of inherited factor Ⅺ deficiency / 中华检验医学杂志

Ⅺ deficiency in Chinese Han population. Conclusion The 13 mutations of the F Ⅺ gene which were found in this study may unravel the molecular pathogenesis of the F Ⅺ deficiency in Chinese Han population.

Laboratory diagnosis of lupus anticoagulant associated by coagulation factors(F Ⅷ,F Ⅸ and F Ⅺ) deficiency:report of 13 cases / 中华检验医学杂志

Objective To explore laboratory diagnosis tests for lupus anticoagulant associated by coagulation factors(F Ⅷ,F Ⅸ and F Ⅺ)deficiency.Methods We investigated the coagulation factors'activity,coagulation factors'antibodies,anticardiolipin antibodies,lupus anticoagulant and thrombin generation in thirteen patients of lupus anticoagulant associated by coagulation factors(F Ⅷ,F Ⅸ and F Ⅺ)deficiency,ten hemophilia A patients, forty-eight healthy people. In these thirteen patients,there were five patients with schizophrenia and had been taking chlorderazin for a long time,two patients with antiphospholipid syndrome, one patient with pemphigus, five patients in which causes of disease were unknown.Among these patients,two patients has haemorrhage,eleven patients don't have reduced.The antibodies of these coagulation factors and LA were positive.Six patients in this group were ACA poslhve,and seven patients were ACA negative.The lag time of eleven non-haemorrage patients with LA positive associated by F Ⅷ,F Ⅸ and F Ⅺ deficiency was(5.60±1.18)min,higher than that of control group which was(1.88±0.25)min.This differenee had statistically significant(t=10.05,P＜0.01).The time to peak was(8.11±1.91)min,significantly higher than that of control group which was (4.10±0.36)min(t=8.83,P＜0.01).The endogenous thrombin potential(ETP)was(1 640.87±higher than that of hemophilia A patients which was(38.50±6.20)%(t=9.77,P＜0.01).The time to peak of hemophilia A patients was(8.01±1.81)min,significantly higher than that of control group which Was(4.10±0.36)min(t=8.20,P＜0.01).The lag time of control group and hemophilia A patients was (1.88±0.25)min and(2.01±0.84)min,respectively.There was no significant difierence of the lag time between these two groups(t=1.26,P＞0.05).Conclusions Diseases like lupus anticoagulant associated bv coagulation factors(F Ⅷ,F Ⅸ and F Ⅺ)deficiency and inherited hemophilia should use APTT,PT,should be used as confirmation tests. LA and ACA may serve as indication test for identifying the causes of these diseases. ETP and ETP percentage are quite good parametem for predicting the bleeding risk of patients with LA positive associated by coagulation factors(F Ⅷ,F Ⅸ and F Ⅺ)deficiency.