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【Objective】 To evaluate the clinical efficacy and safety of one kind of human prothrombin complex concentrate in treatment of patients with hemophilia B. 【Methods】 The clinical data of 36 patients with hemophilia B treated with human prothrombin complex concentrate produced by Shenzhen Weiguang Biological Products Co. Ltd. from May 2018 to April 2019 were retrospectively analyzed, and its clinical efficacy and safety were analyzed. 【Results】 A total of 35 subjects entered the full analysis set (FAS)and safety set (SS), 33 subjects entered the per protocol Set (PPS). Thirty minutes after the first infusion of FAS subjects, the activity of coagulation factor Ⅸ increased from (3.93±0.975) IU/dL to (25.61±9.337) IU/dL, and the infusion efficiency was (96.43±22.007)%. The increased value of coagulation factor Ⅱ activity was (73.25±14.874) IU/dL. The activity of coagulation factor Ⅶ was (42.79±16.847) IU/dL. The increased value of coagulation factor Ⅹ activity was (65.29±17.042) IU/dL. The increased value of coagulation factor Ⅸ activity was (21.68±9.434%) IU/dL. Twenty-four hours after the first infusion of FAS subjects, the improvement of bleeding symptoms and signs was excellent in 21 cases (60%), improved in 14 cases (40.0%), and the effective rate was 100%. The incidence of adverse reactions was 2.9%(1/35), and there was no antibody to human coagulation factor Ⅸ and new virus infection. 【Conclusion】 Infusion of human prothrombin complex concentrate produced by Shenzhen Weiguang Biological Products Co. Ltd. in the treatment of hemophilia B has significant clinical efficacy and good safety.
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【Objective】 To optimize the purification conditions of heparin affinity chromatography in the purification of human coagulation factor Ⅸ by response surface method and establish the optimal chromatography process parameters. 【Methods】 The effect of sample loading temperature on purification efficiency was analyzed through single factor test. Three-factor three-level response surface method was used to optimize the chromatographic elution conditions. The Folin phenol method and the automatic hemagglutination analyzer were used to determine the total protein content and human coagulation factor Ⅸ titer, respectively. The purification effect was evaluated by activity index and process recovery rate. 【Results】 The optimized optimal chromatographic conditions were loading at 5 ℃, washing 4 CV, eluent formulation of sodium citrate 20 mmol/L, arginine hydrochloride 18.7 mmol/L, NaCl 611.6 mmol/L and pH 7.5; under this optimal setting, the recovery rate of the chromatographic process was (46.6±2.9) %, titer of factor Ⅸ rated to (68.4±4.7) IU/mL and specific activity was (62.8±3.3) IU/mg. 【Conclusion】 The optimized parameters of heparin affinity chromatography process by response surface method can produce better purification effect on human coagulation factor Ⅸ.
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Objective To examine the short-term prognostic value of procalcitonin ( PCT ) combined with coagulation factors for cirrhotic patients complicated with spontaneous bacterial peritonitis (SBP).Methods Clinical data of 128 cirrhotic patients complicated with SBP admitted in Jinhua Central Hospital from June 2014 to October 2017 were retrospectively analyzed .In 3 months after admission , 83 patients survived ( survival group ) and 45 patients died ( fatal group ) .The factors related to prognosis were analyzed with Logistic regression and the prediction model was constructed with the weights derived from regression coefficients.The ROC curve and the area under the curve (AUC) of combination of PCT with coagulation factors were used to predict the survival of patients .Results Univariate analysis indicated that the level of PCT , total bilirubin ( TBil ) , serum creatinine ( Scr ) , prothrombin time ( PT ) , prothrombin activity ( PTA ) , blood coagulation factor Ⅱ, Ⅴ, Ⅶ, Ⅸ, Ⅹ, Ⅺ and Ⅻ were factors affecting the prognosis of cirrhotic patients complicated with SBP (P<0.01).Multivariate analysis showed that PCT , blood coagulation factors Ⅴ and Ⅸ were independent factors of short-term prognosis of cirrhotic patients complicated with SBP.The constructed predictive model was Logit (P) =1.200+0.099 ×PCT-0.026 × clotting factor Ⅴ-0.038 ×clotting factor Ⅸ.The sensitivity and specificity of the model were 0.822 and 0.675, respectively, and the AUC was 0.829.Compared with the classic MELD score , the difference was not statistically significant (P>0.05).Conclusions The predictive model based on PCT and coagulation factors Ⅴand Ⅸcan effectively predict the short-term survival of cirrhotic patients complicated with SBP . The overall prognostic ability is not different from MELD score , but the model is more simple and easier to apply.
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Objective@#To evaluate the efficacy and safety of a domestic human plasma derived coagulation Factor Ⅸ concentrate (pd-FⅨ) in patients with hemophilia B.@*Methods@#The study was a multicenter, open-label and single-arm study. The efficacy of pd-F Ⅸ was evaluated by objective performance criteria. The doses of pd-FⅨ were calculated according to the bleeding symptom and disease severity. The infusion efficiency of pd-FⅨ and improvement of bleeding symptoms were measured at 30 minutes and (24±4) h after the first infusion, respectively. Adverse events were recorded. Viral infection and FⅨ inhibitor were detected 90 d after the first infusion.@*Results@#All 36 subjects with hemophilia B were enrolled in the study. The median age of these patients was 31 years old and the median injection doses were 4 (1-17) times. The hemostatic effect of 27/36 (75.00%) and 9/36 (25.00%) acute bleeding events were rated as "excellent" and "better" , respectively. The recovery rate was 111.92% (65.55%-194.28%) at 30 minutes after infusion of FⅨ. There was no adverse event related to FⅨ. No reactivation of HBV, HCV or HIV and FⅨ inhibitor was detected at 90-104 d after the first FⅨ infusion.@*Conclusion@#This domestically made human plasma derived FⅨ concentrate is safe and effective in the treatment of acute bleeding in patients with hemophilia B.@*Clinical trial registration@#China food and Durg Administration, 2016L08027.
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Objective To study the influence factors on detection coagulation factors Ⅸ activity in human prothrombin complex concentrates(PCC).MethodsUsing Chinese Pharmacopoeia(2010)as reference,factor Ⅸ deficient plasma from different manufacturers,different types of instruments,different methods of heparin neutralization,different sample pretreatment methods were used to determine the activity of coagulation factor Ⅸ in PCC.The influence on the results of coagulation factor Ⅸ was analyzed.ResultsThere was a significant difference between factor Ⅸ deficient plasma from different manufacturers for the activity of coagulation factors Ⅸ(P<0.05).There was no significant difference between the results of coagulation factor Ⅸ measured by different types of Automatic Coagulation Analyzer.When the dilution ratio was more than 60 times,the neutralization of heparin had little effect on the coagulation factor Ⅸ activity.When the dilution ratio is less than 60 times,the detection results of coagulation factor Ⅸ by neutralization of heparin are higher than no neutralization of heparin,and the difference is significant(P<0.05).Samples for pre-temperature or not and whether dissolution for 15minutes or not,the coagulation factor Ⅸ activity showed no significant difference.Conclusion The coagulation factor Ⅸ activity in PCC was affected by factor Ⅸ deficient plasma from different manufacturers,different dilution times of heparin neutralization method and sample pre-treatment methods.It must be paid attention to in the detection process.Strengthen the quality control of the factor deficient plasma and the standardization of operation process are necessary.The External Quality Assessment for the detection of coagulation factors in PCC products should be edtablished.
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Objective To investigate current status and problems of coagulation factor Ⅷ and Ⅸ assay in domestic laboratories so as to provide the reference for implementing the standardization and quality improvement.Methods A questionnaire survey was carried out in 76 laboratories,and quality control materials were distributed to 54 laboratories for activity assay.The questionnaire information was analyzed statistically.Test results of quality control materials were classified into three groups according to the reagents and the ranked grading analysis were used to evaluate the performance.Results This research was investigative study.The amount of sample was less than 30 per month in 72％ (52/72)of laboratories.The frequencies of calibration were different,and 33％ (24/72) of laboratories did not perform calibration in a different assay batch.39％ (28/72)of laboratories did not run internal quality control,and about 21％ (15/ 72) of laboratories just performed the normal level quality control.Individual laboratories showed a high cumulative CV (＞ 30％) of intemal quality control.For normal FⅧ and FⅨ control materials,the CV of results were 11.3％-18.2％ and 11.3％-17.9％ respectively as well as 15.3％-20.3％ and 19.5％-21％ for abnormal.Of the three groups,the proportions of laboratories which the FⅧ test results out with consensus were18％,24％ and 22％ as well as 20％,24％ and 28％ for FⅨ.Conclusions The key requirements for quality control of coagulation factors active assay remain to be addressed and implemented.The repeatability and comparability in some laboratories are not satisfactory to meet the clinical needs.With the purpose of promoting quality improvement,we need to develop guidelines,organize related training and establish a national external quality assessment scheme.
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ObjectiveTo establish an effective method for F9 gene mutation detection by using high resolution melting ( HRM ) curve analysis.Methods Peripheral blood samples of 55 hemophilia B (HB) patients were collected from Shanghai Ruijin Hospital during January 2005 to June 2010.Genomic DNA was extracted from the peripheral blood.PCR amplification combined with sequencing was used to identify the F9 gene mutations in 40 patients.HRM assay was established on the 21 DNA samples with known mutations in exonl to exon7 of F9 gene.Mutation scanning of exonl to exon7 by HRM combined with direct sequencing of exon8 was used in the molecular diagnosis of 15 HB patients with unknown F9 gene mutations.ResultsF9 gene mutation was detected in each of the 40 HB patients by direct sequencing.By HRM,the different melting curve patterns were identified in 19 out of 21 cases.The detection rate was about 90％.Through mutation scanning of exonl to exon7 by HRM combined with direct sequencing of exon8,F9 gene mutations were detected in all the 15 HB patients with unknown F9 gene mutations.Thirty-four F9 gene mutations had been identified in the 55 HB patients.ConclusionsA new strategy of HB genetic diagnosis,scanning mutations of exonl to exon7 combined with DNA sequencing of exon8 of F9 gene,is established in this study.The new strategy is efficient and reliable.
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Objective To investigate function of Arg327Ile (R327I) and Arg327Ala(R327A) FⅨ mutants and to study the molecular pathogenesis of haemophilia B(HB) caused by R3271 mutation.Methods Hygromycin-resistant cell line was screened and the secretion of FⅨ antigen into the medium was measured by ELISA.The cell line with appropriate expression levels of F Ⅸ antigen was selected for culture.Recombinant F Ⅸ (rF Ⅸ ) was purified from concentrated medium by two step methods of Q-Sepharose Fast Flow and anion exchange chromatography.The concentration and purity of rF Ⅸ were determined by ELISA and SDS-PAGE,respectively.The activation of wild-type ( WT),R327I and R327A of rFⅨ by FⅦa/TF/Ca2+ or FⅪa/Ca2+ was identified by Western blot in different time periods.The FⅨa and FⅧa complex formed by interaction with different concentrations of FⅧa was used to activate F X,the apparent dissociation constant (Kd) for FⅧa binding was calculated by the kinetic results.The kinetic data of the activation of FX by WT,R327I and R327A FⅨa with or without FⅧa were calculated.Results The amount of WT,R327I and R327A rFⅨ were 450,210,64 μg,and the purity of rFⅨ was confirmed by SDSPAGE.Both R3271 and R327A could be normally activated by FⅧa/TF/Ca2+ or FⅪa/Ca2+.Kd for FⅧa binding showed that the binding capacities of R327I and R327A were 4 and 5 times lower than WT,respectively.The catalytic efficiencies of R327I and R327A F Ⅸ a for F X were 6 and 8 times lower with FⅧa,and 3 and 7.4 times lower without F Ⅷ a,respectively.Conclusions R327I and R327A rF Ⅸ mutants impair their binding to the FⅧa.The site on R327 contributes to FⅧa binding.It is partly related to the activation of FX.The low FⅧa binding to R327I FⅨa may cause HB.
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Objective To study two new factor Ⅸ mutations Cys82Ser and Ile288Ser in vitro and research the molecular mechanism of haemophilia B. Methods PcDNA3. 1 ( - ) FⅨwt expression plasmid was prepared. The mutated FⅨcDNA expression plasmids, PcDNA3.1 ( - ) FⅨM1 (Cys82Ser) and PcDNA3. 1 ( - ) F Ⅸ M2 (Ile288Ser) were constructed by megaprimer method respectively. Transient expression experiments were performed using HEK293 cells transfected with the expression vectors containing the wild-type or the mutation recombinant cDNA. PcDNA3. 1 ( - ) was used as a blank control. The expression proteins were detected by ELISA, factor activity assay and flourescence stain. Results The results suggested that the two FⅨ gene mutations did not induce the reduction of the mutant FⅨ mRNA compared with the wild-type FⅨ mRNA. The FⅨ:Ag in culture media and cell lysate of wild type conduct were assigned as 100. 0%. The results of PcDNA3.1 ( - ) FⅨ M1 mutation protein were (27. 1 ± 5. 2)% and (99.4 ±4. 1)% respectively. For PcDNA3. 1( - )FⅨM2, the results were (5.3 ± 1.8)% and (31.7 ±2. 5)% respectively. The FⅨ: C in culture media of wild type conduct was also assigned as 100. 0%. Then the two types of mutant protein were ( 8. 5 ± 3.2 ) % and ＜ 1%, respectively. Immunofluorescence microscopy result suggested that the intensity of perinuclear spot was reduced in cells transfected with PcDNA3.1 ( - ) FⅨM2 while staining for PcDNA3. 1 ( - ) FⅨM1 was predominantely diffuse without perinnclear enhancement. Conclusions These results strongly suggest that the FⅨ Cys82Ser mutation protein is not been correctly folded, by any possibility. The mutation protein has secretion defect. The secretion dysfunction and the protein degradation intracellular are possiblely the molecular pathology of Ile288Ser mutant protein.
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Objective:To investigate the effects of human factor Ⅸ (FⅨ) intron 1 inserted in forward orientation into retroviral vector on the expression of FⅨ in muscle cells. Methods: Two FⅨ mini-genes, FⅨm1 and FⅨm2, were inserted in forward orientation into retroviral vector backbone, LⅨSN, constructing 2 vectors named LⅨm1SN and LⅨm2SN, containing shortened intron of 1.4 and 0.3 kb, respectively. Retrovirus was made by transfecting PA317 packaging cells. Transient and stable expression of FⅨ was tested in SCID mouse muscle cells (SCID-MB). Results: The vector titers and expression levels of LⅨm1SN and LⅨm2SN in SCID-MB were 1 to 2 folds higher than that of LⅨSN. Some SCID-MB colonies transfected with LⅨm2SN contained intact FⅨm2 inton elements, suggesting that splicing of virus mRNA may not be complete in packaging cells. Conclusion: FⅨ intron 1 inserted in forward orientation in retroviral vectors can increase retroviral titer and the expression level of FⅨ in muscle cells.