亚甲蓝光化学法病毒灭活血浆对凝血因子的影响

目的:探讨亚甲蓝(methylene blue,MB)光化学法病毒灭活血浆对凝血因子的影响.方法:制备过程中随机抽取新鲜血浆120人份,根据新鲜血浆进行病毒灭活情况分为对照组和试验组,分别对上述两组标本进行PT、APTT、TT、Fg、FⅤ、FⅧ、TP测定.对照组,即新鲜冰冻血浆(fresh frozen plasma,FFP)组,不进行病毒灭活,直接进行检测;试验组,即病毒灭活血浆组,先对其进行MB法病毒灭活,后再检测.结果:试验组中APTT、FⅤ、FⅧ水平与对照组比较差异有显著性(P＜0.01),PT、TT、Fg、TP较对照组无显著变化(P＞0.05).结论:MB法病毒灭活对于新鲜血浆中内源性凝血因子,特别是不稳定因子FⅤ、FⅧ有显著影响,对外源性凝血因子及血浆蛋白成分影响不大.     

血液制品病毒灭活因子与动力学的评价

目的分析血液制品特定灭活因子的时效动力学曲线，科学性地评价与改进不合理的病毒灭活参数。方法系统性整理针对水疱性口炎病毒、黄热病毒、脊髓灰质炎病毒、伪狂犬病毒、脑心肌炎病毒、人类免疫缺陷病毒，人血白蛋白采用巴氏消毒法，狂犬病人免疫球蛋白采用低pH常温孵放法，静注人免疫球蛋白、静注人乙肝免疫球蛋白采用低pH／巴氏消毒法；人纤维蛋白原、人凝血因子Ⅷ采用有机溶剂／去污剂与干热法灭活病毒的验证资料，统计3批样品多个取样点的残余病毒滴度均值、标准差与变异系数，制作灭活病毒动力曲线图并进行分析。结果人血白蛋白巴氏灭活VSV与Sindbis病毒，HRIG低pH灭活VSV和Sindbis病毒，HRIG低pH灭活HIV病毒，IVIG低pH／巴氏灭活HIV病毒，WIG低pH灭活HIV病毒，HBIG低pH／巴氏灭活VSV、Sindbis与Polio病毒。结论建议改进病毒灭活验证标准与不合理的灭活参数；采用终产品样品通过合适细胞或方法直接培养目标病毒来有效监测血液制品的病毒安全性。     

亚甲兰/光化学法病毒灭活对血浆成分的影响

目的 探讨亚甲兰(methylene blue,MB)光化学法病毒灭活血浆对血液成分结构和功能的影响.方法 新鲜血浆经MB病毒灭活后.检测照射前后样品的血浆量、MB浓度、FⅧ:C含量的变化.结果 血浆病毒灭活后血浆容量、FⅧ:C、的回收率分别为:(95.11±1.36)%、(79.23±3.95)%、MB去除率(77.12±6.44)%.结论 利用MB法灭活血浆病毒对血浆成分有一定影响,但血浆质量符合国家标准,可以满足临床安全输血的需求.     

亚甲蓝光化学法灭活寨卡病毒的研究

目的探讨亚甲蓝光化学反应对血浆中寨卡病毒的灭活效果。方法血浆中加入寨卡病毒后,使用一次性病毒灭活输血过滤器材在病毒灭活照射仪内进行亚甲蓝光化学法病毒灭活(亚甲蓝浓度为1μM、照射强度为35 000 Lx、灭活时间为30 min)。分别在灭活处理前以及灭活处理的第5、15、30 min留样。所取样品用免疫荧光法检测病毒滴度,并用RT-qPCR检测病毒RNA载量。结果灭活前,血浆中寨卡病毒的滴度和核酸载量分别为(5.55±0.18)log TCID_(50)/0.1mL和(9.08±0.75)log copies/mL。灭活后以及经过Vero细胞连续3代培养后,均未检测到病毒感染性,滴度下降(5.05±0.19)log TCID_(50)/0.1mL。病毒RNA载量在灭活后仍有(8.07±0.68)log copies/mL,传代培养从第1代到3代,都没有检测到病毒RNA。结论亚甲蓝光化学法能有效灭活血浆中的寨卡病毒。     

病毒灭活新鲜冰冻血浆制备冷沉淀的可行性探讨

目的探讨亚甲蓝光化学法(MB-P)病毒灭活新鲜冰冻血浆制备冷沉淀凝血因子的可行性。方法选取2014年1~6月无偿献血者捐献的400 mL全血93袋,分离新鲜冰冻血浆200 mL后再均分为2袋,各100mL,分别作为对照组和试验组。对照组直接制备冷沉淀凝血因子,试验组经MB-P法病毒灭活后再制备冷沉淀凝血因子。检测每袋冷沉淀凝血因子中FⅧ和Fbg水平。结果对照组FⅧ和Fbg水平分别为(82.9±7.1)IU/100mL、(102.4±8.5)mg/100mL,试验组分别为(56.6±5.3)IU/100mL、(83.3±5.6)mg/100mL,试验组FⅧ和Fbg水平均低于对照组,组间比较差异均有统计学意义(P0.05),且均符合相关国家标准。结论加强血液采集、储存、运输和制备过程中的质量控制,使用经MB-P法病毒灭活新鲜冰冻血浆制备的冷沉淀凝血因子符合相关国家标准。     

S/D法用于人凝血因子Ⅷ病毒灭活效果验证

人凝血因子Ⅷ(FⅧ)浓制剂是用于甲型血友病治疗的血液制品,患者需终身输注.本所用低温乙醇法制备了纯度>3IU/mg蛋白的FⅧ浓制剂,为减少该剂品传播病毒的危害并保证制品的质量,在制作过程中采用了Solvent/detergent (S/D) 法[1]进行病毒灭活.S/D法是由美国纽约血液中心[2]于20世纪8 0年代中期开发的,采用有机溶剂和表面活性剂进行协同处理,用于灭活血浆制品中的脂包膜病毒而不影响蛋白质的性质,已获得美国FDA认可.为验证该方法的效果,笔者对制品在处理前后的活性和理化性质进行了比较,采用HIV、VSV、Sindbis 3个指示病毒对S/D处理人凝血因子Ⅷ进行病毒灭活验证. 1 材料与方法     

FFP经亚甲蓝光化学法灭活病毒并直接冻干于血袋的冻干血浆研制

目的探索制备冻干血浆的新工艺,研制适合在艰苦环境下储运和方便使用的新型冻干血浆。方法采用亚甲蓝光化学技术对新鲜冰冻血浆(FFP)做病毒灭活处理,再将FFP直接冻干于血袋中,观察不同条件下的保存效果。结果亚甲蓝光化学技术(终浓度为1μmol/L)能有效灭活血浆中的Sindbis病毒、PRV和VSV等指示病毒,处理30min后灭活效果>4.75LgTCID50,经细胞3代盲传证明灭活病毒效果可靠。新型冻干血浆外观成淡黄色疏松体,残水量为2.2%—2.5%,室温下<2min完全溶解;储存稳定性试验表明,新型冻干血浆中的FⅡ、FⅤ、FⅧ、FⅨ和FⅪ在25℃和4℃比较稳定,高温(40℃)保存对其活性有一定影响。结论采用亚甲蓝光化学灭活病毒技术和袋装血浆冻干技术制备新型冻干血浆是可行的。     

核黄素光化学法灭活Sindbis病毒的研究

目的探究核黄素光化学反应对Sindbis病毒的灭活效果。方法将10 mmol/L的核黄素0.050 mL加入到4.95 mL的Sindbis病毒(XJ-160株)悬液中,经440 nm波段的可见光(40 J/cm2)双侧照射后,接种至幼仓鼠肾细胞(BHK-21)中培养,观察细胞病变情况(CPE),检测病毒滴度,用PCR检测病毒核酸的变化,并在透射电镜下观察病毒形态。结果经终浓度为100μmol/L核黄素结合440 nm可见光作用,可将滴度为6.5 log TCID50Sindbis病毒灭活至≤0.5 logTCID50;PCR法未能扩增出病毒核酸片段;透射电镜显示病毒颗粒塌陷。结论核黄素光化学法能有效灭活Sindbis病毒,其主要作用靶点为核酸。     

病毒灭活新鲜冰冻血浆质量控制指标分析

目的探讨MB法病毒灭活新鲜冰冻血浆的质量控制指标。方法随机抽取200 ml新鲜血浆,每例平均分成两份,一组进行病毒灭活,一组不进行,分别检测两组中Fib、TP、FⅧ因子含量,用t检验进行统计学分析。结果两组血浆中Fib、TP含量均无统计学意义（P〉0.05）,FⅧ因子病毒灭活后回收率为79.5%,含量下降,与灭活前相比差异有统计学意义。结论MB法病毒灭活新鲜冰冻血浆可以沿用GB18469-2001国家标准。     

丙型肝炎病毒样颗粒作为亚甲蓝光化学法灭活HCV效果评价物的可行性研究

目的探讨丙型肝炎病毒样颗粒(HCVLPs)在亚甲蓝光化学灭活处理过程中的变化趋势及其作为亚甲蓝光化学法灭活HCV效果评价物的可行性。方法以HCV阳性血浆作为平行对照组(HCV RNA载量约6.53 logcopies/m L)(n=6),将HCVLPs用代血浆调整成合适浓度(HCV RNA定量约为6.74 logcopies/m L)的悬液作为实验组(n=5),将2组分装至PVC血袋中,MB+L 1μmol/L进行病毒灭活处理,分别于光照处理0、5、10、20、30 min时取样,用荧光定量PCR技术检测病毒核酸载量;同时以细胞病变法测定HCV模型病毒sindbis病毒的残余滴度,验证MB+L灭活HCV的效果。结果模型病毒sindbis的病毒滴度降至检测限以下(log TCID50/m L≤0.5);MB+L处理过程中,随着光照处理时间的增加,HCV及HCVLPs的核酸载量明显下降,分别从6.53与6.74下降至4.51和2.89log copies/m L(P0.05),且2组在不同取样点的HCV RNA载量之间具有相关性(R20.98,Significance F0.01)。结论 HCVLPs能够反映MB+L灭活处理中HCV RNA动态变化,或有望成为安全而有效的HCV灭活评价物来监控HCV灭活效果。     

West Nile virus in plasma is highly sensitive to methylene blue-light treatment

BACKGROUND: The epidemic of West Nile virus (WNV) in the US resulted in cases of transfusion-transmitted WNV. Effective pathogen reduction methods could have removed this infectious agent from the blood supply We have evaluated the efficacy of photodynamic treatment of fresh frozen plasma (FFP) with methylene blue (MB), a decontamination method applied in several European countries. STUDY DESIGN AND METHODS: FFP units (300 ml each) were spiked with WNV. MB was added, and the units were illuminated with white or monochromatic yellow light. WNV infectivity was determined by bioassay. WNV-RNA was quantitated by real-time PCR. The inactivation of WNV was investigated under standard and under suboptimal conditions, respectively. In addition, rechallenge experiments with multiple addition of WNV at maximal load (approx. 105 CFU/ml) and repeated illumination without replenishing MB were performed. RESULTS: Complete inactivation of WNV was achieved by MB (0.8-1 mmol/l) and illumination with white light (30,000-45,000 Lux) within 2 min. White yellow light 20-40 J/cm(2) (2.5-5 min) were sufficient for inactivation by 5.75 log10-steps. The rechallenge experiments revealed the substantial reserve capacity of the procedure to inactivate WNV. Quantitative PCR indicated that the viral RNA was rapidly destroyed. CONCLUSION: All experimental data demonstrate the enormous potency of phototreatment with MB to inactivate WNV in plasma.     

S/D法对人凝血因子Ⅷ产品中人免疫缺陷病毒灭活效果的研究

目的研究S/D法对人凝血因子Ⅷ中污染的人免疫缺陷病毒灭活效果。方法采用细胞培养法,对S/D处理人凝血因子Ⅷ灭活HIV的效果进行了检测。结果在常温(25℃)条件下,在污染有HIV的人凝血因子Ⅷ种混入体积分数9%S/D灭活剂,作用60 min,HIV灭活对数值为6.83。重复处理3批人凝血因子Ⅷ制品,对HIV均达到完全灭活,经细胞培养盲传三代,均未出现细胞病变。结论 S/D法能够完全灭活人凝血因子Ⅷ产品中污染的人免疫缺陷病毒,灭活效果可靠。     

80℃,72h干热处理FⅧ制剂中指示病毒灭活效果的考察

以水泡性口炎病毒（ＶＳＶ）、Ｓｉｎｄｂｉｓ和ＰｏｌｉｏＩ型疫苗病毒为指示病毒，考察８０℃，７２ｈ干热处理对ＦⅧ制剂中的病毒灭活效果。结果表明，该法对加入制品中的ＶＳＶ、Ｓｉｎｄｂｉｓ和ＰｏｌｉｏＩ型疫苗病毒的灭活能力分别为８．３、９．１和７．７ｌｇＴＣＩＤ５０／ｍｌ；证实８０℃，７２ｈ干热处理，对模拟脂包膜和非包膜病毒的指示病毒ＶＳＶ、Ｓｉｎｄｂｉｓ和ＰｏｌｉｏＩ型疫苗病毒，均可起到有效的灭活作用。     

Two pathogen reduction technologies—methylene blue plus light and shortwave ultraviolet light—effectively inactivate hepatitis C virus in blood products

BACKGROUND: Contamination of blood products with hepatitis C virus (HCV) can cause infections resulting in acute and chronic liver diseases. Pathogen reduction methods such as photodynamic treatment with methylene blue (MB) plus visible light as well as irradiation with shortwave ultraviolet (UVC) light were developed to inactivate viruses and other pathogens in plasma and platelet concentrates (PCs), respectively. So far, their inactivation capacities for HCV have only been tested in inactivation studies using model viruses for HCV. Recently, a HCV infection system for the propagation of infectious HCV in cell culture was developed. STUDY DESIGN AND METHODS: Inactivation studies were performed with cell culture–derived HCV and bovine viral diarrhea virus (BVDV), a model for HCV. Plasma units or PCs were spiked with high titers of cell culture–grown viruses. After treatment of the blood units with MB plus light (Theraflex MB‐Plasma system, MacoPharma) or UVC (Theraflex UV‐Platelets system, MacoPharma), residual viral infectivity was assessed using sensitive cell culture systems. RESULTS: HCV was sensitive to inactivation by both pathogen reduction procedures. HCV in plasma was efficiently inactivated by MB plus light below the detection limit already by 1/12 of the full light dose. HCV in PCs was inactivated by UVC irradiation with a reduction factor of more than 5 log. BVDV was less sensitive to the two pathogen reduction methods. CONCLUSIONS: Functional assays with human HCV offer an efficient tool to directly assess the inactivation capacity of pathogen reduction procedures. Pathogen reduction technologies such as MB plus light treatment and UVC irradiation have the potential to significantly reduce transfusion‐transmitted HCV infections.     

短波紫外线与交联淀粉碘对血浆中辛德比斯病毒灭活的研究

对ＵＶＣ与ＣＳＩ分别对血浆中病毒灭活的效果和对血浆中ＦⅧ凝血活性的影响进行了观察。ＣＳＩ按１００ｍｇ／ｍｌ加入血浆，作用３０ｍｉｎ与６０ｍｉｎ，能使ＳＶ分别下降２５５与３６１个对数级，但血浆ＦⅧ凝血活性亦分别降至５７３３％和０；继续延长作用时间至１２０ｍｉｎ，灭活病毒的效果增加不明显。用剂量为７１７０～５７３６０Ｊ／ｍ２的ＵＶＣ照射血浆，可使ＳＶ下降４２６～６７１个对数级。照射剂量为７１７０Ｊ／ｍ２时，病毒灭活速度较快，ＦⅧ凝血活性也无损害，但超过此剂量后的病毒下降速度减缓，而对ＦⅧ凝血活性的损害明显加重。     

Chikungunya virus and the safety of plasma products

BACKGROUND: Chikungunya virus (CHIKV) outbreaks were previously restricted to parts of Africa, Indian Ocean Islands, South Asia, and Southeast Asia. In 2007, however, the first autochthonous CHIKV transmission was reported in Europe. High‐level viremia, a mosquito vector that is also present in large urban areas of Europe and America, and uncertainty around the resistance of this Alphavirus toward physiochemical inactivation processes raised concerns about the safety of plasma derivatives. To verify the safety margins of plasma products with respect to CHIKV, commonly used virus inactivation steps were investigated for their effectiveness to inactivate this newly emerging virus. STUDY DESIGN AND METHODS: Pasteurization for human serum albumin (HSA), vapor heating for Factor VIII inhibitor bypassing activity, solvent/detergent (S/D) treatment for intravenous immunoglobulin (IVIG), and incubation at low pH for IVIG were investigated for their capacity to inactivate CHIKV and the closely related Sindbis virus (SINV). The obtained results were compared to previous studies with West Nile virus and the commonly used model virus bovine viral diarrhea virus. RESULTS: The data generated demonstrate the effective inactivation of CHIKV as well as SINV by the inactivation steps investigated and thereby support results from earlier validation studies in which model viruses were used. CONCLUSION: High inactivation capacities with respect to CHIKV were demonstrated. This provides solid reassurance for the safety of plasma products and the results verify that the use of model viruses is appropriate to predict the inactivation characteristics of newly emerging viruses when their physicochemical properties are well characterized.     

亚甲兰光化学法对FFP中凝血因子及总蛋白含量的影响

目的探讨亚甲蓝光化学法(MB-P)对新鲜冰冻血浆(FFP)中Fg、FⅡ∶C、FⅤ∶C、FⅧ∶C、FⅩ∶C、FⅫ∶C及总蛋白含量的影响,为制定病毒灭活血浆标准及其临床应用提供数据参考。方法随机抽取88份FFP分成灭活组(n=40)及过滤组(n=48),分别对2组样本处理前后的FⅡ∶C、FⅤ∶C、FⅧ∶C、FⅩ∶C、FⅫ∶C、Fg及总蛋白含量进行检测。结果灭活组FⅤ∶C、FⅧ∶C处理后含量显著低于灭活前(分别为t=3.40,P<0.01;t=2.57,P<0.01),但Fg、FⅡ∶C、FⅩ∶C、FⅫ∶C及总蛋白灭活前后的含量无明显差异(P>0.05),过滤组处理前后FⅤ∶C、FⅧ∶C无明显差异(P>0.05)。结论 MB-P对FFP中不稳定的凝血因子FⅤ∶C和FⅧ∶C含量有明显影响,均在可接受的范围内,对其他凝血因子及总蛋白的含量无影响。     

静脉注射人免疫球蛋白酸性孵放法灭活艾滋病病毒的效果研究

目的研究静脉注射人免疫球蛋白在生产过程中酸性孵放法灭活艾滋病病毒的效果。方法采用细胞培养法,对含有艾滋病病毒的静脉注射人免疫球蛋酸性孵化法灭活效果进行了实验室检测。结果将含有艾滋病病毒的静脉注射人免疫球蛋白酸度调整为pH4,于21～25℃条件下放置4d,其中艾滋病病毒的滴度(TCID50)由9.50log下降至2.00log以下,下降值达到7.00log以上。相同的样品在pH值7.0条件下放置14d,其中艾滋病病毒滴度仍在3.3log以上;经放置21d,所有样品中艾滋病病毒滴度均在2.0log以下。经过对3批样品重复测定,均未出现细胞病变;经过将样品盲传三代仍未出现细胞病变。结论采用酸性孵放法常温下放置21d,可使静脉注射人免疫球蛋白中艾滋病病毒完全失去活性。     

亚甲蓝光化学法灭活病毒的效果观察

目的研究亚甲蓝光化学法对病毒的灭活效果,探讨其实际应用的可能性。方法用细胞培养法和分子生物学技术,对亚甲蓝光化学法灭活病毒的效果进行了实验室观察。结果在含胎牛血清的悬液内加入3.0μmol/L亚甲蓝,经15 000LUX的光照强度下照射5 min,可使滴度6.50 lg TCID50辛德毕斯病毒下降至检测限以下。在上述条件下处理后的标本经定量RT-PCR方法检测,随亚甲蓝浓度的增加,病毒核酸拷贝数逐渐降低。结论亚甲蓝光化学灭活法对含血清的悬液内辛德毕斯病毒具有明显破坏作用,病毒核酸受损程度随着亚甲蓝浓度的增加而增加并与病毒感染滴度的降低程度有相关性。     

Plasma transfusion in liver transplantation: a randomized,double‐blind,multicenter clinical comparison of three virally secured plasmas

BACKGROUND: The clinical equivalence of plasma treated to reduce pathogen transmission and untreated plasma has not been extensively studied. A clinical trial was conducted in liver transplant recipients to compare the efficacy of three plasmas. STUDY DESIGN AND METHODS: A randomized, equivalence, blinded trial was performed in four French liver transplantation centers. The three studied (fresh‐frozen) plasmas were quarantine (Q‐FFP), methylene blue (MB‐FFP), and solvent/detergent (S/D‐FFP) plasmas. The primary outcome was the volume of plasma transfused during transplantation. Secondary outcomes included intraoperative blood loss, hemostasis variables corrections, and adverse events. RESULTS: One‐hundred patients were randomly assigned in the MB‐FFP, 96 in the S/D‐FFP, and 97 in the Q‐FFP groups, respectively. The median volumes of plasma transfused were 2254, 1905, and 1798 mL with MB‐FFP, S/D‐FFP, and Q‐FFP, respectively. The three plasmas were not equivalent. MB‐FFP was not equivalent to the two other plasmas, but S/D‐FFP and Q‐FFP were equivalent. The median numbers of transfused plasma units were 10, 10, and 8 units with MB‐FFP, S/D‐FFP, and Q‐FFP, respectively. Adjustment on bleeding risk factors diminished the difference between groups: the excess plasma volume transfused with MB‐FFP compared to Q‐FFP was reduced from 24% to 14%. Blood loss and coagulation factors corrections were not significantly different between the three arms. CONCLUSION: Compared to both Q‐FFP and S/D‐FFP, use of MB‐FFP was associated with a moderate increase in volume transfused, partly explained by a difference in unit volume and bleeding risk factors. Q‐FFP was associated with fewer units transfused than either S/D‐FFP or MB‐FFP.