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OBJECTIVE@#To analyze the results of activated partial thromboplastin time (APTT) mixing test in coagulation factor Ⅷ inhibitor-positive hemophilia patients, so as to increase the value of APTT mixing test in the screen of factor Ⅷ inhibitor.@*METHODS@#Eighty plasmas samples with different titers of coagulation factor Ⅷ inhibitors had been collected and diluted for routine immediate APTT mixing test and at 37 ℃ 2 hours incubation APTT mixing test. Fifteen samples were selected for immediate and normal temperature incubation for 15 min, 30min, 1 hour, 2 hours and 37 ℃ for 30 min, 1 hour, 2 hours APTT mixing test.@*RESULTS@#The results of APTT mixing test were significantly correlated with the titers of coagulation factor Ⅷ inhibitors. The ROC curve result showed that the best diagnostic cut-off value for 2 hours incubation APTT mixing test at 37 ℃ to determine the presence or absence of coagulation factor Ⅷ inhibitors was 43.8 s (sensitivity and specificity was 85.90% and 100%, respectively), while the best diagnostic cut-off value for distinguishing high-titer and low-titer Ⅷ inhibitors was 52.4 s (sensitivity and specificity was 98.18% and 95.65%, respectively). The critical coagulation factor Ⅷ inhibitor titer that could not be corrected by immediate APTT was 5.14 BU/ml, while that could not be corrected by 37 ℃ 2 hours incubation APTT was 1.31 BU/ml. Paired samples t -test was performed on the APTT mixing test results at different times and temperatures, and the differences were statistically significant (P < 0.05).@*CONCLUSIONS@#The APTT mixing test can be used as a screening index for coagulation factor Ⅷ inhibitors. APTT mixing test result shows a significant time-temperature dependence with lower titers of coagulation factor Ⅷ inhibitor. Patients with hemophilia who cannot be corrected by immediate APTT mixing test should be alert to the possibility of high titer of coagulation factor Ⅷ.
Subject(s)
Humans , Factor VIII , Hemophilia A/diagnosis , Blood Coagulation Tests/methods , Partial Thromboplastin Time , Blood Coagulation FactorsABSTRACT
【Objective】 To screen the sterilizing-grade filters applicable for production of human coagulation factor Ⅷ/von Willebrand factor complex(FⅧ/VWF)and study the sterilization filtration process. 【Methods】 Four sterilizing-grade filters for FⅧ/VWF were evaluated through indicators such as filtration capacity, filtration flux, recovery rate of FⅧ activity, recovery rate of VWF activity, recovery rate of VWF antigen, recovery rate of protein and VWF molecular distribution. The sterilizing-grade filter with the best filtration performance was selected for further study. The study was designed by general full-factor design to determine the appropriate filitered protein concentration and filitered speed range through evaluating the total filtered protein amount, recovery rate of protein and filtration efficiency, and then the process operation parameters was determined. 【Results】 The filtration flux of Sartobran P, Sartopore 2 XLG, Sartopore Platinum and Sartopore 2 XLI were 1.71±0.01, 1.80±0.01, 1.34±0.01, and 1.81±0.04 L·(m2)-1·min-1, respectively; the recovery rates (%) of FⅧ activity were 97.09±2.82, 99.22±0.99, 96.87±1.85 and 93.76±1.21, respectively; the recovery rates (%) of VWF activity were 98.12±1.42, 99.95±1.85, 94.80±1.62 and 92.09±1.67, respectively. Between Sartopore 2 XLG and Sartobran P, the difference of filtration flux (P<0.001) was statistically significant; between Sartopore 2 XLG and Sartopore Platinum, the differences of the filtration flux (P<0.001) and VWF potency recovery rate (P<0.05) were statistically significant; between Sartopore 2 XLG and Sartopore 2 XLI, the differences of FⅧ potency recovery rate (P<0.01) and VWF potency recovery rate (P<0.01) were statistically significant. The optimal process operating space of Sartopore 2 XLG was protein concentration of 0.45-0.58 mg/mL, and filtration rate of 1.48-2.95 L·(m2)-1·min-1. 【Conclusion】 Sartopore 2 XLG is the most suitable filter for the production of FⅧ/VWF and the DoE test proves that it has good process operation space.
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【Objective】 To evaluate the efficacy and safety of plasma-derived human coagulation factor Ⅷ (FⅧ) in the treatment of patients with hemophilia A. 【Methods】 A multi-center and open, SAT(single-arm trials) clinical study was conducted. A total of 54 subjects with hemophilia A were enrolled in 5 research centers. FⅧ was injected according to the subjects' weight, severity of disease and other factors, and the transfusion efficiency of FⅧ activity at 10 min after the first infusion of the first bleeding event was taken as the main efficacy indexes. The improvement scores of bleeding symptoms and signs within 24 h after the first infusion of the first bleeding event were the secondary efficacy indexes. The pathogenic microbial indexes and FⅧ inhibitors were detected on 90(th) and 180(th) day after treatment. 【Results】 The transfusion efficiency of FⅧ activity of 54 subjects at 10 min after the first infusion was 171.9% on average, with median of 169.5%, both higher than the target value of 100%. Within 24 h after the first infusion, the improvement of bleeding symptoms and signs of the subjects were scored, among which 19 cases (35.2%) were "obvious", 35 cases (64.8%) were "good", and the total clinical effective rate reached 100%. Five subjects (9.3%) had six drug-related adverse events. On 90(th) and 180(th) day after treatment, hepatitis B surface antigen, hepatitis C antibody, HIV antibody, treponema pallidum antibody and FⅧ inhibitors were detected, and no negative to positive cases were found. 【Conclusion】 After infusion, the FⅧ preparation can significantly improve the FⅧ activity level in hemophilia A patients in a short period of time, which has high infusion efficiency and can achieve better treatment efficacy, and can also effectively control and relieve bleeding symptoms and signs, with good overall safety.
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Objective: To evaluate the clinical effects of low- and intermediate-dose factor Ⅷ (F Ⅷ) prophylaxis in Chinese adult patients with severe hemophilia A. Methods: Thirty adult patients with severe hemophilia A who received low- (n=20) /intermediate-dose (n=10) F Ⅷ prophylaxis at Nanjing Drum Tower Hospital affiliated with Nanjing University Medical College were included in the study. The annual bleeding rate (ABR), annual joint bleeding rate (AJBR), number of target joints, functional independence score of hemophilia (FISH), quality of life score, and health status score (SF-36) before and after preventive treatment were retrospectively analyzed and compared. Results: The median follow-up was 48 months. Compared with on-demand treatment, low- and intermediate-dose prophylaxis significantly reduced ABR, AJBR, and the number of target joints (P<0.05) ; the improvement in the intermediate-dose prophylaxis group was better than that in the low-dose prophylaxis group (P<0.05). Compared with on-demand treatment, the FISH score, quality of life score, and SF-36 score significantly improved in both groups (P<0.05), but there was no significant difference between the two groups (P>0.05) . Conclusion: In Chinese adults with severe hemophilia A, low- and intermediate-dose prophylaxis can significantly reduce bleeding frequency, delay the progression of joint lesions, and improve the quality of life of patients as compared with on-demand treatment. The improvement in clinical bleeding was better with intermediate-dose prophylaxis than low-dose prophylaxis.
Subject(s)
Humans , Hemophilia A/drug therapy , Factor VIII/therapeutic use , Quality of Life , Retrospective Studies , Hemarthrosis/prevention & control , Hemorrhage/drug therapyABSTRACT
Objective To evaluate the performance of myPKFiT,a tool guiding the dosing of antihemophilic factor (recombinant) plasma/albumin-free method (rAHF-PFM),in maintaining the coagulation factor Ⅷ (FⅧ) level above a target threshold at the steady state and estimating the pharmacokinetics (PK) parameters in hemophilia A patients in China. Methods The data of 9 patients with severe hemophilia A in a trial (CTR20140434) assessing the safety and efficacy of rAHF-PFM in the Chinese patients with hemophilia A were analyzed.The myPKFiT was used to predict the adequate dose to maintain a patient's FⅧ level above target threshold at the steady state.Furthermore,the performance of myPKFiT in estimating the pharmacokinetics parameters of individuals was evaluated. Results Twelve combinations of two dosing intervals and six sparse sampling schedules were investigated,and 57%-88% of the patients remained the FⅧ level above the target threshold of 1 U/dl (1%) for at least 80% of the dosing interval.The clearance and time to FⅧ level of 1% obtained from sparse sampling by myPKFiT were similar to those obtained from extensive sampling. Conclusions The myPKFiT can provide adequate dose estimates to maintain the FⅧ level above the target threshold at the steady state in Chinese patients with severe hemophilia A.Moreover,it demonstrates good performance for estimating key pharmacokinetics parameters,including clearance and time to FⅧ level of 1%.
Subject(s)
Humans , China , East Asian People , Factor VIII/pharmacokinetics , Hemophilia A/drug therapyABSTRACT
The detection of coagulation factor Ⅷ activity plays an important role in the diagnosis, typing, efficacy monitoring and detection of inhibitor titer in hemophilia A, acquired hemophilia A and von Willebrand disease. However, due to the diversity of detecting systems, the difference of reagent composition, the existence of interfering substances and other influence factors, the detection of coagulation factor Ⅷ activity in the laboratories in China still needs to be improved.
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@#Hemophilia A is an X-chromosome-linked recessive genetic disease that lacks coagulation factor Ⅷ (Factor Ⅷ, FⅧ) and is clinically manifested as spontaneous or excessive bleeding after injury.The current main treatment for hemophilia A is alternative infusion of FⅧ, but the fixed infusion mode is still used for the dosage and frequency of infusion, which cannot achieve the optimal curative effect under the principle of individualized treatment.Among the factors that affect the efficacy of FⅧ replacement therapy, the difference in the pharmacokinetics (PK) of FⅧ products by individuals is an important factor.The clinical understanding of individualized FⅧ replacement therapy under the guidance of PK is not sufficient.Therefore, this article reviews the PK characteristics, analysis models, clinical application scenarios and specific treatment plan formulation of FⅧ.
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【Objective】 To investigate the matrix effect on the determination of potency in Recombinant Human Coagulation Factor Ⅷ for Injection (rFⅧ). 【Methods】 Two different detection matrices were used to establish two methods for detecting the potency in Recombinant Human Coagulation Factor Ⅷ for Injection. And the matrix effect on the determination of potency was determined, including specificity, linearity, repeatability, accuracy and intermediate precision. 【Results】 As to the specificity, the recoveries of the two substrates at high vs low concentration level were 112% and 110% vs 104% and 109%, respectively. As to the linearity, in the range of (0.125-1.000) IU/mL, the correlation coefficient between concentration and coagulation time of standard/ sample was higher than 0.99. As to the accuracy/repeatability, the recoveries of two matrices was 104% and 102%, and RSD was 2.4% and 1.9%. As to the intermediate precision, personnel factor of two matrices was 0.72 and 0.23, date factor was 0.79 and 0.85, and RSD(for 12 times) was 4.2% and 3.0%. Comparison of two matrices was as follows: Deviation in test results of 6 batches of rFⅧ was all lower than 5%. There was no significant difference between two matrices. 【Conclusion】 The two matrices for potency detection show good performance including specificity, linearity, repeatability, accuracy, and intermediate precision. They are suitable for the determination of potency in rFⅧ products.
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【Objective】 To evaluate the efficacy and safety of human coagulation factor Ⅷ developed by Shenzhen Weiguang Biological products Co, Ltd in the treatment of patients with hemophilia A. 【Methods】 A prospective, multi-center, open, single-group clinical study was conducted. A total of 65 subjects with hemophilia A were enrolled, and human coagulation factor Ⅷ(FⅧ) was injected according to the patients’ bleeding severity. The improvement score of bleeding symptoms and signs after the first infusion of the first bleeding event and the transfusion efficiency of FⅧ activity at 10 min and 1 hour after infusion were taken as the main efficacy indexes. The improvement scores of bleeding symptoms and signs after the first infusion and the increase of FⅧ activity at 10 min and 1 hour after infusion were the secondary efficacy indexes. 【Results】 The 65 subjects were enrolled in safety analysis set (SS) and full analysis set (FAS), and 58 of them were enrolled in protocol analysis set (PPS). Ten minutes and one hour after the first infusion, the level of factor Ⅷ activity in the subjects increased significantly, and the FⅧ activity increased by 100% or more in more than 79% of the subjects. The average infusion efficiency of FⅧ activity in all subjects was more than 100%. In 70% of the subjects, the pain was relieved rapidly and /or the bleeding symptoms were significantly improved 8 hours after each bleeding infusion, and the improvement rate of bleeding symptoms and signs reached 100% 72 hours after infusion. 【Conclusion】 After infusion of human coagulation factor Ⅷ, the activity level of factor Ⅷ in patients with hemophilia A significantly increased. The infusion efficiency can reach a optimal level, and the bleeding symptoms can be significantly improved.
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【Objective】 Tostudy the effect of ABO blood group on the FⅧ∶C and Fib content in human plasma, so as to provide the oretical guidance for the quality control of fresh plasma products and the establishment of relevant quality standards. 【Methods】 Samples determined included fresh plasma collected and fresh plasma separated manually. The FⅧ∶C and Fib content were determined by coagulation method. The exon6 of ABO gene was amplified and sequenced to determine the genotype. 【Results】 The FⅧ∶C in fresh plasma collected was (147.421±45.773)%, and that in fresh plasma separated manually was (119.083±35.130)%, showing significant differences(P0.05). The FⅧ∶C in non-O type (A, B, AB type) fresh plasma collected and fresh plasma separated manually were (167.048±40.862)% and (129.251±33.503)%, respectively, significantly higher than that in O type fresh plasma collected and fresh plasma separated manually as(121.386±38.632)% and (91.589±22.328)%, respectively. The Fib contents in non-O type fresh plasma collected and fresh plasma separated manually were (2.242±0.385)g/L and (2.329±0.472)g/L, respectively. The Fib contents in O type fresh plasma collected and fresh plasma separated manually were (2.287±0.370)g/L and (2.307±0.462)g/L, respectively, and no significant difference was noticed (P>0.05). 【Conclusion】 There was no significant correlation between Fib content and ABO blood group, while FⅧ∶C was significantly correlated with ABO blood group. In the preparation and quality control of FⅧ related blood products, the effect of ABO blood group on the FⅧ∶C should be considered, and the quality standard of FⅧ in plasma products should be established based on the ABO blood group.
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【Objective】 To explore the difference of total protein (TP) content, coagulation factor VIII (FⅧ) activity and Fib content in different common plasma products, and to further provide basis for the establishment and refinement of relevant quality standards of common plasma products. 【Methods】 Samples involved in the experiment included frozen plasma and cryoprecipitated frozen plasma derived from whole blood and eukocyte-depleted whole blood. The TP content determination was carried out by biuret method. The FⅧ activity (FⅧ: C) and Fib content were determined by coagulation method. 【Results】 The TP content( g/L) in frozen plasma and cryoprecipitated frozen plasma derived from whole blood and eukocyte-depleted whole blood were 59.64±4.78 vs 58.09±4.1 vs 52.20±3.57 vs 51.89±1.50, respectively, and the FⅧ: C( %) were 109.63±43.38 vs 27.06±7.09 vs 71.83±21.64 vs 21.66±3.86,, and the Fib content (g/L) were 2.19±0.39 vs 1.30±0.24 vs 2.04±0.37 vs1.22±0.15, respectively. There was a significant difference in TP content between other common plasma products (P0.05). There was significant difference in FⅧ: C among four common plasma products (P0.05). 【Conclusion】 The TP content and FⅧ: C of common plasma products are closely related to the initial blood and preparation process. It is suggested that the quality standard of common plasma products should be further refined, and the establishment of cryoprecipitated frozen plasma relevant quality standard and clinical indications should be considered.
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【Objective】 To establish a novel preparation method of cryoprecipitate coagulation factor from overcooled liquid-state plasma. 【Methods】 The fresh liquid plasma was kept at -11℃ to -13℃ for a period of time. It can remain in the liquid state with some coagulation factors generated due to supercooling. Then cryoprecipitate can be obtained from the liquid plasma by siphon method. 【Results】 The average fibrinogen content yielded in cryoprecipitate, prepared from 50 samples of 16-hour-stored fresh liquid plasma, was (186.02±22.72) mg, with the average recovery rate of (37.51±7.42) %, and the average content of coagulation FⅧ was (104.66±22.88) IU, with the average recovery rate of (46.62±5.58) %. 【Conclusion】 The cryoprecipitate coagulation factors could be obtained not only from fresh frozen-thawed plasma, but also from overcooled liquid plasma which is simple and stable, also meets the requirements of relative standards.
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Objective To investigate the changes of plasma protein Z and coagulation factor Ⅷ activity in children with primary nephrotic syndrome.Methods 94 children with primary nephrotic syndrome were se-lected as the observation group,and 63 healthy children were selected as the control group.The blood samples of peripheral blood were collected from the study group,and plasma protein Z and coagulation factor Ⅷ were measured by enzyme linked immunosorbent assay.Enzyme linked immunosorbent assay was used to measure plasma protein Z and coagulation factor Ⅷ.The changes of plasma protein Z and coagulation factor Ⅷ in the two groups were compared,and the changes of plasma protein Z and coagulation factor Ⅷ in the acute and re-covery phase,and the correlation between plasma protein Z and coagulation factor Ⅷ were observed.Results The observation group of plasma protein Z level is lower than the control group,blood coagulation factor Ⅷlevels higher than the control group,and the difference was statistically significant(P<0.05);plasma protein Z level in acute stage is lower than the recovery period,and coagulation factor Ⅷ level is higher than the recov-ery period,the differences were statistically significant(P<0.05);plasma protein Z and coagulation factor Ⅷwas negatively correlated.Conclusion The plasma protein Z level in children with primary nephrotic syn-drome is significantly reduced,and the activity of coagulation factor Ⅷ is significantly increased.Detection of plasma protein Z and coagulation factor Ⅷ level can predict primary nephrotic syndrome in children.
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Objective To establish the method for determination content of glycine in the pharmaceutical of human coag‐ulation factor Ⅷ by HPLC .Methods The analysis was carried on a Shim‐Pack CLC‐ODS column with a mobile phase of 50%acetonitrile‐0 .05 mol/L sodium acetate buffer (35∶65) at the detection wavelength of 360 nm ,using alanine and 2 ,4‐dini‐trofluorobenzene as the internal standard and derivation agent ,respectively .Results The method showed a good linearity in the range of 0 .006‐0 .030 mg/ml (r=0 .999 3) for glycine .The average recovery was 101 .4% ,and the RSD was 0 .14% (n=9) .Conclusion This method was simple ,sensitive ,accurate ,reliable ,and suitable for determination of glycine in the pharma‐ceutical of human coagulation factor Ⅷ .
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Objective To identify the expression of sphingosine-1-phosphate receptor (S1PR) subtypes, C-myc and His tag proteins of human umbilical vein endothelial fusion cell line, EA.hy926 and human umbilical vein endothelial cells (HUVEC), CRL-1730 for studying the function of apolipoprotein M (ApoM)-S1P axis. Methods Two kinds of cells (EA. hy926 and CRL-1730) were cultured to reach the density of 60%-70% in vitro. Immunofluorescence technique was em?ployed to investigate the expressions of coagulation factorⅧ(FⅧ), ApoM, S1PR1-S1PR5, C-myc and His tag proteins. Re?sults (1) Two kinds of cells both expressed FⅧand ApoM. FⅧpresented scattered particle distribution in CRL-1730, while uniform distribution in EA.hy926. However, ApoM was strongly expressed and widely distributed in cytoplasm of two kinds of cells. (2) S1PR1-3 can be detected on their membrane other than S1PR4 and S1PR5. S1PR1 was highly expressed but S1PR2 and S1PR3 were in a low level expression. (3) Two kinds of cells both expressed C-myc and His tag proteins in cytoplasm. Conclusion Two kinds of cells have the properties of endothelial cells and can express FⅧ, ApoM, C-myc and His tag proteins. It is not suitable for choosing C-myc and/or His tag–conjugated recombinant ApoM to study the fuction of ApoM-S1P axis with these two kinds of cells.
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Objective To compare four pretreatment methods of human plasma coagulation factor Ⅷ( FⅧ) before column purification.Methods Cryoprecipitate was dissolved in Tris , before the supernatant was treated with glycine precipitation, PEG precipitation, acid precipitation and aluminum hydroxide gel adsorption , respectively.Activated partial thromboplastin (APTT) was used to measure the activity of the supernatant clotting FⅧ after treatment.The total activity recovery and specific activity of the final samples were used to weigh the efficacy of those methods .The purity of the intend-ed protein was estimated by non-reducing SDS-PAGE electrophoresis .Results Total activity recovery of glycine precipitati-on was the highest (94.00%±7.60%), followed by that of acidic precipitation (89.47%±2.60%) and PEG precipita-tion (80.92%±9.67%) methods.The lowest was aluminum hydroxide gel adsorption (78.65%±7.52%).Glycine precipitation and PEG precipitation could more effectively remove contaminating protein than acid precipitation and aluminum hydroxide gel adsorption .Treated by four different methods , the specific activity of FⅧ of glycine precipitation sample was the highest (0.6856 ±0.1258 IU/mg), followed by PEG precipitation (0.5773 ±0.0787 IU/mg) and acidic precipitation (0.3885 ±0.0301 IU/mg).The specific activity of aluminum hydroxide gel adsorption was the lowest (0.2879 ±0.0472 IU/mg).Conclusion PEG precipitation is more effective for the actual production process than the other three methods .
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Objective To study effect of virus inactivation/removal treated by solvent/detergent method and dry heating at 80℃, 72 h for inactivation in human coagulation factor Ⅷ.Methods Human coagulation factor Ⅷextracted from healthy human plas-ma were treated by solvent/detergent method and dry heating at 80℃, 72 h for inactivation .The virus inactivation effect was validated by adding the indicator virus ( PRV, Sindbis, HIV, EMCV, PPV).Results The methods could effectively inactivate lipid-enveloped and non lipid-enveloped viruses which could be used for virus inactivation /removal during human coagulation factor Ⅷexperiments , the residual amount of TNBP in production was less than one percent ten thousand (10 ppm), the residual Tween-80 concentration was less than one percent hundred thousand (100 ppm),which all met the safety standards .Conclusion and no significant change was ob-served in the activation and other indicators of human coagulation factor Ⅷ.
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Objective To set up the molecular cytobiological model of endogenous coagulation factor Ⅷ (FⅧ) re-expressing in human liver cells L02,and to study the regulation pathway and molecular basis of the re-expression of FⅧ in L02 cells activated by NO signal.Methods The L02 cells at logarithm growth phase were selected and randomly divided into blank control group and experimental group, inhibitor group and inhibitor control group;they were cultured for 0,12,24,36,48,and 60 h.Flow cytometry was used to detect the expression of human FⅧ protein in L02 cells after treated for 48 h.Griess experiment was performed to detect the levels of NO in L02 cells at different time points;the transcription levels of human FⅧ gene,iNOS gene,NF-κB1 gene and I-κB alpha gene were detected by RT-PCR method.Western blotting method was used to detect the expression levels of human phosphorylated I-kappaB (phosphorylated I-κB)in L02 cells.Results The results of flow cytometry showed that the expression of human L02 FⅧ protein was found after treated with L-arginine for 48 h. The Griess results showed that the levels of NO in L02 cells in experimental group were significantly increased at 3,6,12,and 24 h (P<0.05)and the levels of NO in blank control group,inhibitor group and inhibitor control group had no changes. The RT-PCR results showed that the transcription of human FⅧ mRNA in L02 cells was found in experimental group,but there was no transcription of human FⅧ mRNA in blank control group,inhibitor group and inhibitor control group;the transcription levels of iNOS,NF-κB1 and I-κB alphain experiment group were increased(P<0.05)and the transcription levels of these genes in blank control group,inhibitor group and inhibitor control group had no changes. The Western blotting results showed that after adding L-arginine the expression level of phosphorylated I-κB was significantly increased (P < 0.05 ), other groups had no such change. Conclusion L-arginine can activate the phosphorylation of I-κB by NO signal pathway to lead to the changes in the expression of human FⅧ gene promoter upstream regulatory-related transcription factors NF-κB to activate the expression of human FⅧ in human liver cells L02.