Water in the Clinical Laboratories - yeec.com Water in the Clinical Laboratories...
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1 Water in the Clinical Laboratories 临床实验室纯水 演讲人： Mikael Cleverstam WW Application Expert Millipore Corp. 翻译： Charler Li Millipore China
Transcript of Water in the Clinical Laboratories - yeec.com Water in the Clinical Laboratories...
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Water in the Clinical Laboratories临床实验室纯水
演讲人： Mikael Cleverstam
WW Application ExpertMillipore Corp.
翻译： Charler Li
Millipore China
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临床实验室纯水
疾病诊断结果疾病诊断结果试剂试剂& & 水质水质
诊断设备诊断设备
医疗技术医疗技术
质量控制质量控制
水质是质量过程中的一个重要部分Water quality as part of the quality process
Diagnostic instruments
Assay developmentPatient results
Troubleshooting Your analyzer
CLSI 新标准的要求
Water purification
Quality system
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Normal seen Problems 常见问题
Frequent Calibrations 频繁的校正
High CV% 高CVFluctuation in quality results over the day/week/month 波动效应
Interfered assays 直接干扰分析
易干扰的分析– Calcium 钙 受矿物质，有机物几菌膜影响
– ALP 碱性磷酸酶 受菌膜，清洁剂，矿物质影响
– CK 肌酸激酶 受自来水水处理系统释放的杂质影响
– Amylase 淀粉酶 受柠檬酸盐，清洁剂，有机物（腐殖质和煤黄腐酸）
– LD 乳酸脱氢酶 受工业废水水源，有机物，H2O2的影响
– Phosphorus 磷 受柠檬酸盐，有机物的影响
– Iron 铁 受矿物质，有机物(腐殖质和煤黄腐酸)，清洁剂影响
– Magnesium 镁 受柠檬酸盐，矿物质影响
– Triglycerides 甘油三酸酯 受塑料,化学试剂影响
– Urea 尿素 受柠檬酸盐,自来水水处理系统影响
– Troponin I 肌钙蛋白Ｉ 受菌膜影响
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Water for Clinical Analysers分析仪中使用的纯水角色
Cuvette washing 比色杯冲洗
Tubing and probe rinsing 管道和加样探针的清洗
Reagent and buffer reconstitution 试剂或缓冲液的配置
Dilution 稀释 （标本）
Water Baths 水浴
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常见问题的处理过程
YES
YES
YES
NO
NO
Assay
Mechanical
Decontamination
Problem solved
Problem
WHY ??
Outside Source
Water Quality
Medical Medical TechnologistTechnologist
Diagnostic Diagnostic InstrumentsInstruments
Water Water QualityQuality
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检测工作中的困惑Diagnostic Dilemma越来越小的检测量和输送管路系统
Smaller sample size and reaction vessel are subjected to harsher environment
不可避免的菌膜问题，在仪器内部、液路分配和管道系统，需要更频繁的清洁和清洗来去除，但
是——由于工作量和强度的要求，能给予仪器充分清洁维护时间也越来越少
Inevitable build-up of biofilm in instruments, manifolds and tubing require more frequent decontaminations but Less and less time available for maintenance of the instruments
很多敏感的分析会被细菌和离子污染所影响
– 细菌释放外源性酶和离子，与检测试剂会发生被检测物相同的反应
Some sensitive assays can become contaminated with bacteria and ions– Bacteria release enzymes and ions whose behavior is similar to the enzymes targeted in the assay method
水质的要求和稳定监控，应与分析中其他质量控制要求同样重视
Increased need to monitor water quality as closely as any other instrument malfunction
Diagnostic Diagnostic InstrumentsInstruments
Water Water QualityQuality
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临床分析仪器中的微生物Bacteria in Clinical Analyzers
一些微生物产生耐受性，可以适应低营养度环境和在超纯水中生长– Some bacteria strains are able to adapt to stringent conditions and grow in
high purity water– 三种耐受性细菌经常在分析仪器中被发现
Three strains are commonly identified in clinical analyzers• Ralstonia pickettii皮氏拉斯通氏菌• Sphingomonas paucimobilis少动鞘氨醇单胞菌• Caulobacter crescentus新月柄杆菌
培养记数方法（CLSI） Count methods (CLSI)
– 总非自养细菌培养平板记数（ 20-28 °C, > 5 days, R2A 培养基）Total heterotrophic plate count (20-28 °C, > 5 days, R2A medium)
– 膜过滤或者涂布技术接种Membrane filtration or spread-plate techniques
– 荧光检测技术（更高细菌检出率）Epifluorescence more recently introduced (more bacteria detected)
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Biofilm Formation
Time
SurfaceParticles
Organic
Bacteria
Biofilm may shed bacteria, pyrogens etc
Diagnostic Diagnostic InstrumentsInstruments
Water Water QualityQuality
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Demonstration of ALP release from bacteria
Correlation between bacteria concentrations and levels of ALP in water
Bacteria Strain(identification by 16S rDNA sequencing)
Bacteria level(x 106 cfu/mL)
ALP concentration (μUnit/μL)
Sphingomonas paucimobilis
Caulobacter crescentus
Ralstonia pickettii
29.2
9.7
29.5
6.22
9.95
8.29
Diagnostic Diagnostic InstrumentsInstruments
Water Water QualityQuality
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Detection methods
ALP
+
Substrate-Phosphate
Pi
UV-VisiblepNPP
FluorescenceAttophosStarbrightMUPELF
ChemiluminescenceCDP-Star (dioxetane)CSPD (dioxetane)Lumigen PPDAMPPD
Diagnostic Diagnostic InstrumentsInstruments
Water Water QualityQuality
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什么干扰了钙分析结果？Interferences with Calcium Assay ?
水中存在的钙Calcium present in water– 产水在10 MΩ.cm左右
• 高钙浓度Maximum Calcium concentration: 10-15 ppb– 水箱中的水Water in a reservoir
• 水在水箱中水质会劣化: 1 MΩ.cm• 高钙浓度: 500 ppb
– 与钙检测试剂的剂量相比Compared to calcium dosed (100 ppm): • 如果电阻率正常，不太可能会是水中钙离子纯度影响检测结果（比
例仅占< 1% ) Unlikely to be the ionic purity of the water (contributes to < 1%)
那么，什么干扰了钙分析的结果？
So, what else can interfere with the assay?
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CLSI 指导原则Preparation and Testing of Reagent Water in the Clinical Laboratory
CLSI C3-A4, 2006 新版文件1997 C3-A3 NCCLS 文件修订版
Committee: 医院实验室医生和管理员，医院微生物学家，水纯化系统公司
hospital laboratory doctors and managers, hospital microbiologist, water purification systems companies
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CLSI 指导原则
CLSI指导原则的理念是使用者有责任检查所使用的水是否适合他的使用目的。
The philosophy of the CLSI guideline is that the user is responsible to check if
the water used is suited for the purpose
只定义了一种级别的水质One grade only of water quality is defined (CLRW)
其它级别的水质未定义：需要由使用者基于应用和水质需求来决定（与生产厂商
合作）Other water grades not specified: this needs to be decided by the user
(in collaboration with the manufacturer), based on the applications and the
water quality requirements
指导原则的目的: 为临床实验室提高纯水的质量系统提供支持。该标准定义了对
纯化方案的选择、系统维护和质量控制的推荐。to provide support to clinical laboratories to improve their quality system in regards to water. The guideline defines recommendations on the selection of water purification solutions, on system maintenance, and on quality control.
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CLSI 纯水分级
临床实验室试剂级水Clinical laboratory reagent water (CLRW)– 定义的参数Parameters defined
特殊试剂级水Special reagent water (SRW)
– 为需要其它参数以确保水质的特殊应用而设定，如：金属分析，PCR，DNA/RNA分析，细胞培
养，免疫分析
– May be specified for specific applications when additional parameter are needed to insure water quality ex: Metal analysis, PCR, DNA/RNA assays, cell cultures, immunoassays
仪器进水Instrument feed water (IFW)– 基于仪器厂商的推荐Based on manufacturer’s recommendations
由试剂厂商提供的用作稀释剂或试剂的水
Water Supplied by a Method Manufacturer for Use as a Diluent or Reagent– 不可替代ＣＬＲＷCannot be a substitute for CLRW
购买的瓶装纯水Commercially Bottled, Purified Water– 水的稳定性有待检验Water suitability needs to be checked
高压灭菌锅和冲洗用水Autoclave and Wash Water Applications
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指标 - CLRW
参数 标准值 检测目的 建议
电阻率 10 MΩ.cm @ 25 °C 离子纯度 在线分析监测
TOC < 500 ppb 有机物纯度 在线或离线监测
细菌 < 10 cfu/mL 细菌及其代谢物 总异养平板计数
Total heterotrophic plate count
颗粒 < 1，经0.2 µm 过滤
器过滤堵塞分析仪 终端过滤膜有挑战性
实验品质保证
• 目前临床实验室 多被引用和参考的标准Clinical labs reference this standard
• 多数分析仪器厂商也按照此标准推荐进水要求Analyzer manufacturers
• CAP认证也使用此标准作为参考
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IFW Specifications - Example
参数 标准值 检测目的
电阻率 离子纯度
TOC 有机物纯度
细菌 细菌及其代谢物
颗粒 堵塞分析仪
> 1 MΩ.cm @25 °C
< 500 ppb
< 10 cfu/mL
< 1，经0.2 µm 过滤
器过滤
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Example of Special Reagent Water (SRW)Water for Genetic assays - PCR
No ions to avoid interferences of ions such as Fe, Cd or Zn with enzymes
去除离子避免对酶有干扰（如Fe, Cd or Zn ）
Little organics (especially no humic or fulvic acids) that could bind to the DNA or interfere with the enzymes
非常小的有机物（特别是腐殖酸类），这些会影响DNA和酶的正常反应
No nucleases 无核酸酶
No bacteria 无菌
– Resistivity 18.2 MΩ.cm 电阻率
– TOC < 30 ppb 有机物
– Use of ultrafiltration – nuclease-free water 超滤手段去酶
– Microfiltration or ultrafiltration bacteria < 1 cfu/mL 微滤或超滤除菌
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质量: 验证 & 趋势监控 Trend Monitoring
“适用于使用目的”的水验证 “Fit for purpose” water Validation
纯水设备的验证 Water Equipment Validation
参数监控和发展趋势的记录 Documented parameters monitoring and trending“确保水继续达到指标要求” “Making sure water continues to meet specifications”
维护程序的开发 Maintenance Procedures development
进水质量要求 Feed water quality requirement
不应把CLSI当成一种束缚，而应把它看成为实验室需求和要求选择 合适的解决
方案的一种工具和参考指南。Rather than a constraint, the CLSI guideline should be considered as a tool and a reference guide to select the most appropriate solution to the laboratory needs and requirements
质量控制质量控制
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Conclusions
Pure Water is a Clinical Reagent!
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Thanks！谢谢！
Mikael Cleverstam
