一种新的实时荧光定量PCR方法对血小板制品细菌污染检测效果的评价

目的建立一种自主研发的针对细菌16 S r DNA基因的实时荧光定量PCR(Real-time PCR)方法并评价该方法对浓缩血小板制品中细菌污染检测的效果。方法设计16S r DNA基因保守区引物,构建SYBR Green Real-time PCR反应体系;然后分别将大肠杆菌、金黄色葡萄球菌、表皮葡萄球菌、蜡样芽孢杆菌和绿脓杆菌以初始浓度为1CFU/m L、10 CFU/m L和100 CFU/m L接种到浓缩血小板中,经22℃保存7 d后,用实时定量荧光PCR方法进行细菌检测。结果细菌污染后的血小板在常规保存条件下,最长保存期7 d,不同接种浓度的细菌生长情况的变化趋势基本一致,所有种类的细菌均在d 1、2表现出迅猛的增殖高峰,d 3以后增殖趋于平缓。结论该Real-time PCR检测体系可定量地检测出血小板的细菌污染的情况,可适用于血小板输注前的快速细菌污染检测。     

Evaluation of the 3D BacT/ALERT automated culture system for the detection of microbial contamination of platelet concentrates

Bacterial transmission remains the major component of morbidity and mortality associated with transfusion-transmitted infections. Platelet concentrates are the most common cause of bacterial transmission. The BacT/ALERT 3D automated blood culture system has the potential to screen platelet concentrates for the presence of bacteria. Evaluation of this system was performed by spiking day 2 apheresis platelet units with individual bacterial isolates at final concentrations of 10 and 100 colony-forming units (cfu) mL-1. Fifteen organisms were used which had been cited in platelet transmission and monitoring studies. BacT/ALERT times to detection were compared with thioglycollate broth cultures, and the performance of five types of BacT/ALERT culture bottles was evaluated. Sampling was performed immediately after the inoculation of the units, and 10 replicates were performed per organism concentration for each of the five types of BacT/ALERT bottles. The mean times for the detection of these 15 organisms by BacT/ALERT, with the exception of Propionibacterium acnes, ranged from 9.1 to 48.1 h (all 10 replicates were positive). In comparison, the time range found using thioglycollate was 12.0-32.3 h (all 10 replicates were positive). P. acnes' BacT/ALERT mean detection times ranged from 89.0 to 177.6 h compared with 75.6-86.4 h for the thioglycollate broth. BacT/ALERT, with the exception of P. acnes, which has dubious clinical significance, gave equivalent or shorter detection times when compared with the thioglycollate broth system. The BacT/ALERT system detected a range of organisms at levels of 10 and 100 cfu mL-1. This study validates the BacT/ALERT microbial detection system for screening platelets. Currently, the system is the only practically viable option available for routinely screening platelet concentrates to prevent bacterial transmission.     

基于微流控芯片的实时荧光定量 ＰＣＲ 技术快速检测血小板制剂细菌 污染

摘要：目的 通过微流控芯片检测平台应用 Ｔａｑｍａｎ 探针的实时定量 ＰＣＲ 技术对血小板制剂中的细菌１６Ｓ ｒＤＮＡ 进行检测，探 讨该体系在血小板细菌污染的快速检测的应用。方法 向机采血小板中人为添加一定浓度的金黄色葡萄球菌或铜绿假单胞 菌，模拟成１０２ ～１０８ ＣＦＵ／ ｍＬ 细菌污染的血液标本，经１０倍梯度稀释后进行细菌计数及微流控芯片 ＦＱ ＰＣＲ 检测细菌１６Ｓ ｒＤＮＡ，并 对检测体系进行特异性、检出限和重复性评价。结果 微流控芯片 ＦＱ ＰＣＲ 体系特异性较强，探针和引物对阳性标本均有反 应，与空白对照无反应。对污染血小板的金黄色葡萄球菌，该方法的最低检出限为 ８６５ ＣＦＵ／ ｍＬ，而对铜绿假单胞菌的最低检 出限为 ８８５ ＣＦＵ／ ｍＬ。当金黄色葡萄球菌与铜绿假单胞菌浓度分别达到最低检测限时，空白对照与各细菌组的 Ｃｔ 值之差分别 为 ４．３５±１．０１ 和 ２．０３±０．６１。各浓度菌液提取的 ＤＮＡ 进行微流控芯片 ＦＱ ＰＣＲ 扩增后的 Ｃｔ 值计算重复性指数在 ０．０１２～ ０．０５２ 范围内。结论 微流控芯片 ＦＱ ＰＣＲ 检测平台可特异、有效地检出血小板中的细菌污染，为确保输血安全提供帮助。     

应用实时荧光定量PCR方法检测血小板制品细菌污染

目的应用实时荧光定量PCR方法检测血小板制品中细菌的探讨。方法选取金黄色葡萄球菌及大肠埃希氏杆菌,用改良的Chelex-100法抽提细菌基因组DNA,进行荧光定量PCR检测。并应用过滤法去除反应体系中潜在的细菌及其基因组DNA污染。结果针对16srRNA基因保守序列进行扩增的荧光定量PCR方法具有较高的特异性和灵敏度,与人类淋巴细胞及病毒等的基因组无交叉反应。在金黄色葡萄球菌检测中,应用过滤法后最低含菌量组与阴性对照组Ct值有极显著差异(P<0.001)。该方法对金黄色葡萄球菌的最低检出量为0.3CFUs/PCR,与阴性对照Ct值有显著差异(P<0.01),在大肠埃希氏杆菌中该法可检测出0.1CFUs/PCR,与阴性对照Ct值有显著性差异(P<0.01)。结论改良Chelex-100法抽提细菌基因组及后续的荧光定量PCR分析用于检测血小板制品中的细菌污染,检测实验缩短到3-4h,操作简单,灵敏性高,特异性好,为在临床标本大规模检测中的应用提供理论基础和实验数据。     

Blood surveillance and detection on platelet bacterial contamination associated with septic events

The objective of this study was to evaluate the risk of transfusion-associated septic events in Taiwan. In Taiwan, most blood components are provided from blood centres; so platelet (PLT) bacterial contaminations are rarely reported. The study's aim is to investigate the prevalence of PLT bacterial contamination in Kaoshiung Armed Forces General Hospital by using BacT/ALERT system for routine screening.
A total of 82 apheresis and 2256 whole blood-derived PLT units were tested. A measured quantity of 1 mL aliquots were taken as samplings from all blood bag tubing of PLT units, and then further incubated in a bacterial detection system (BacT/ALERT). The subcultures of true-positive bottles underwent bacterial identification by using Vitek system, microscopic observation and culture-based methods. Eight units (0·34%, 8 of 2338) were found to have bacterial contamination. The true-positive rate of the whole blood-derived and apheresis PLTs was 0·31% (7 of 2256) and 1·22% (1 of 82), respectively. Six microorganisms were identified with the most dominate being Staphylococcus epidermidis . One case of transfusion-associated sepsis was confirmed; in addition, the holding period of PLTs ( F = 4·522, P = 0·034) and positive detection of PLT bacterial contamination ( F = 46·605 ,P < 0·001) were associated with post-transfusion sepsis. Thus, in this study, although the transfusion-associated septic event was rarely found and PLT units were provided from blood centres, bacterial screening was necessary to safely quarantine the transfusion. The holding period of PLT units should be no more than 4 days in order to avoid possible bacterial contamination.     

Variation of pH-measurement in platelet concentrates

To measure pH in platelet concentrates, blood gas analysers with different calibration principles may be used. In this study, variances observed in pH measurements with two types of blood gas analysers were investigated. pH was measured in crystalloid solutions (platelet additive solution (PAS-II), phosphate-buffered solutions) and two types of platelet concentrates (containing 100% plasma, or 65% PAS-II/35% plasma) with two blood gas analysers: either using liquid and gas calibration (AVL 945), or only liquid calibration (AVL OMNI). These measurements were compared with a reference method. Especially for PAS-II, large variation in pH was observed between AVL 945, AVL OMNI and the reference method: 6.91 +/- 0.02, 7.35 +/- 0.02 and 7.188 +/- 0.010, respectively (mean +/- SD; n = 12, P < 0.0001, paired t-test). A significant difference in pH was also found for platelet concentrates in 65% PAS/35% plasma (6.88 +/- 0.09 on AVL 945 and 7.02 +/- 0.09 on AVL OMNI, n = 134, P < 0.0001). Comparison with the reference method revealed minor differences with AVL 945, whereas AVL OMNI gave a mean difference in pH of + 0.17. Platelets in 100% plasma revealed smaller differences (6.93 +/- 0.13 for AVL 945 and 6.99 +/- 0.13 for AVL OMNI, n = 95, P < 0.0001). We conclude that different blood gas analysers can yield different pH values, especially in weak buffered solutions such as platelet concentrates in PAS-II. Validation of blood gas analysers for pH measurement of these solutions is therefore mandatory.     

Problem of bacterial contamination in platelet concentrates

Bacterial contamination of blood is being recognized more frequently now and is one of the serious complications of transfusion. Use of integrally attached collection systems and strict standards for skin preparation, collection and storage of blood and components have reduced but not eliminated the risk of bacterial contamination. As bacteraemia may be part of acute or sub acute infections, strict donor selection is warranted. The longer the storage time, the greater is the number of organisms and amount of endotoxin present in the unit and associated with transfusion reactions. Importance of haemovigilance system and awareness among clinicians on the potential complications will go a long way in reducing patient morbidity. New approaches for detection of bacterial contamination, pathogen reduction and developments in the field of platelet biology will increase blood safety.     

BacT／ALERT 3D血液样本细菌检测中假阳性反应的分析

本研究通过分析BacT／ALERT 3D系统血液样本细菌检测中的假阳性反应结果，评估该系统的特异性，以减少假阳性反应的产生。对5年间BacT／ALERT 3D所有阳性反应瓶进行细菌分离和鉴定，并对留样和可获得的相关血袋进行重复试验，共检测11395份血液样本。结果表明：初次培养阳性反应122份（1．07％），其中107份（88．7％）从阳性反应的培养瓶中分离出细菌，另15份（12．3％）中未分离出任何细菌。细菌生长引起的阳性反应的监测曲线显示明显的急剧上升。结论：在检测过程中维持孵箱温度的稳定、避免孵育箱的温度急剧降低可减少假阳性信号的产生；对报告阳性的培养瓶进行进一步的孵育，观察生长曲线是否有急剧上升的对数生长期有助于鉴别假阳性反应，从而提高BacT／ALERT 3D系统的特异性。     

Improved in-vitro quality of platelet concentrates stored in a dextrose-free synthetic medium

Summary. The licenced balanced salt solution Plasma-Lyte, buffered with a clinical solution of sodium bicarbonate, was evaluated as a suspending fluid for platelet concentrates. Platelets suspended in this medium showed better pH maintenance over 5 days of storage compared to platelets stored in plasma (7·0 vs 6·45, P < 0·001). This was reflected in improvements in in-vitro indicators of platelet viability hypotonic shock response (79 vs 48%, P < 0·05), aggregation to paired agonists (86 vs 62%, P < 0·05); and platelet size distribution (104 vs 119%, P < 0·001). Dissolved bicarbonate measurement showed less depletion of bicarbonate in the synthetic medium compared to plasma, which suggests a lower rate of lactate formation. A synthetic medium containing dextrose showed inferior platelet storage characteristics when compared to the plasmalyte/bicarbonate medium in a paired study (Day 5, pH 6·53 vs 6·9, P < 0·05). The results suggest that utilization of substrates other than dextrose allows platelets to metabolize without the accumulation of lactate that leads to pH drops during storage in plasma, and continue to support the feasibility of storing platelets in a non-plasma environment.     

Amotosalen photochemical inactivation of severe acute respiratory syndrome coronavirus in human platelet concentrates

A novel human coronavirus causing severe acute respiratory syndrome (SARS) emerged in epidemic form in early 2003 in China and spread worldwide in a few months. Every newly emerging human pathogen is of concern for the safety of the blood supply during and after an epidemic crisis. For this purpose, we have evaluated the inactivation of SARS-coronavirus (CoV) in platelet concentrates using an approved pathogen inactivation device, the INTERCEPT Blood System. Apheresis platelet concentrates (APCs) were inoculated with approximately 10(6) pfu mL(-1) of either Urbani or HSR1 isolates of SARS-CoV. The inoculated units were mixed with 150 microm amotosalen and illuminated with 3 J cm(-2) UV-A light. The viral titres were determined by plaque formation in Vero E6 cells. Mixing SARS-CoV with APC in the absence of any treatment decreased viral infectivity by approximately 0.5-1 log10. Following photochemical treatment, SARS-CoV was consistently inactivated to the limit of detection in seven independent APC units. No infectious virus was detected after treatment when up to one-third of the APC unit was assayed, demonstrating a mean log10-reduction of >6.2. Potent inactivation of SARS-CoV therefore extends the capability of the INTERCEPT Blood System in inactivating a broad spectrum of human pathogens including recently emerging respiratory viruses.     

Evaluation of apheresis platelet concentrates stored for 5 days in PL 732 bags

To assess the functional viability of platelets collected by standard apheresis techniques using the Fenwal CS-3000 "closed system" and stored in Fenwal PL 732 plastic bags for 5 days at room temperature with agitation, a number of in vitro parameters (pH, morphology, platelet volume distribution, osmotic recovery, aggregation, and platelet-associated IgG) were examined as a function of storage time. During the first 24 hours of storage, minimal changes were observed in the test parameters with the exception of ADP-induced aggregation (75% decrease [10 uM], 84% decrease [5 microM]). Significant differences were observed between fresh (day 0) and 5-day-old platelet concentrates in all parameters except median platelet volume. These observed changes in in vitro test parameters with storage time are similar to those previously observed for comparably stored random single-donor platelet concentrates. Thus, the "closed-system" PL 732 apheresis platelet concentrates would be expected to be as effective in vivo as random single-donor platelet concentrates, while minimizing recipient exposure to transmissible agents of infectious disease.     

外周血基因转录本检测中2种标本处理方法的比较

目的　选择合理的标本处理方法 ,提高外周血基因转录本检测的灵敏度。方法　 5 3例胃癌患者外周血标本分别以淋巴细胞分离液 (FICOLL)和红细胞裂解液 (RLS)处理后提取RNA ,以荧光定量RT PCR技术检测 β actin基因和CEA基因转录本。 结果　RLS法和FICOLL法定量检测 5 3例外周血标本的 β actin基因转录本的平均浓度分别为 1.19× 10 6和 2 .4 0× 10 5拷贝 /ml,2组测定值差异有显著性 (P <0 .0 1)。RLS法检测 5 3例中 13例为CEAmRNA阳性 ,阳性率为 2 4 .5 % ;FICOLL法检测到 5例阳性 ,阳性率 9.4 % ,RLS法的阳性检出率高于FICOLL法 (P <0 .0 5 )。结论　RLS法分离外周血有核细胞的效果优于FICOLL法 ,有利于提高基因表达检测的灵敏度。     

Differences in time to detection and recovery of Mycobacterium spp. between the MGIT 960 and the BacT/ALERT MB Automated Culture Systems

Mycobacterium sp. recovery and time to detection were compared in the MGIT 960 and BacT/ALERT MB automated broth culture systems. The MGIT 960 demonstrated shorter time to detection (13.5 versus 25.2 days) and greater sensitivity (100% versus 66.6%) for recovery of the Mycobacterium tuberculosis complex than the BacT/ALERT MB system.     

医用电解质溶液作添加液制备汇集少白细胞血小板

目的采用医用电解质溶液作添加液(PAS),建立1种白膜法制备添加液汇集少白细胞血小板(PAS汇集BC-PCs)的方法。方法从400ml全血中分离白膜层,容量35—40ml,放(22±2)℃静置过夜,将ABO同型的6袋白膜汇集,加220g添加液(90%复方电解质注射液、8%ACD-A血液保养液和2%含量为50g/L的碳酸氢钠注射液的混合液)稀释白膜,汇集后的白膜在(22±2)℃中,以300×g离心10min,将上层的富含血小板悬液再经白细胞滤除器过滤去除白细胞,并转移到血小板保存袋内,即制备成1个成人治疗量的PAS汇集BC-PCs,制备过程在一个特制的密闭系统内完成。结果共制备30个成人治疗量的PAS汇集BC-PCs,其容量为(270±32)ml、血小板含量为(2.96±0.31)×1011、WBC混入量为(1.3±0.2)×106、RBC混入量为(5.8±1.1)×109、CD62P表达率为(22.5±10.6)%。保存8d后的pH为7.14±0.04、低渗休克反应率(HSR)为(54.0±8.2)%、CD62P表达率为(45.7±13.8)%。结论由复方电解质注射液、ACD-A保养液和碳酸氢钠注射液的混合液为添加液汇集血小板的方法可行。     

Optimization of the PAXgene blood RNA extraction system for gene expression analysis of clinical samples

One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene to stabilize RNA in whole blood; however, RNA extracted by the PAXgene method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.