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1、临床分子诊断方法学验证要求关 明复旦大学附属华山医院检验医学科分子诊断面临的挑战 涉及方法较多，如PCR、荧光定量PCR、测序等； 实验步骤较多，包括标本的分离保存、处理、检测； 手工程度较高，自动化程度普遍偏低，较多依赖手工，不同的实验室条件和操作者等因素对实验结果的影响较大； 尚未建立公认的质量管理标准等； 更高的准确性要求； 结果报告缺乏标准化； SUMMARY OF CHANGES UNDERSTANDING THE 2010 CAP ACCREDITATION CHECKLIST COMPONENTS HOW TO INSPECT USING R.O.A.D INSPECTION T
2、ECHNIQUE ADDITIONAL INFORMATION APPLICABILITY PROFICIENCY TESTING QUALITY MANAGEMENT AND QUALITY CONTROL GENERAL ISSUES PROCEDURE MANUAL ASSAY VALIDATION REQUISITIONS SPECIMEN HANDLING SPECIMEN PROCESSING QUANTITATIVE ASSAYS: CALIBRATION AND STANDARD REAGENTS CONTROLSANALYSIS Extraction Amplificatio
3、n Restriction Endonucleases Sequencing Detection Methods Agarose and Polyacrylamide Gel Electrophoresis Real-Time Polymerase Chain Reaction (PCR) ArraysCONTENTS POST ANALYSIS Results Reporting Records PARENTAGE AND FORENSIC IDENTITY TESTING IN SITU HYBRIDIZATION (ISH) Fluorescence In Situ Hybridizat
4、ion (FISH) Brightfield In Situ Hybridization PERSONNEL PHYSICAL FACILITIES AND EQUIPMENT INSTRUMENTS Spectrophotometers Signal Detection Instruments Film Processing/Photographic Equipment Electrophoresis Equipment and Power Supplies Pipettes Thermometers and Temperature-Dependent EquipmentSpace LABO
5、RATORY SAFETY RADIATION SAFETY SUMMARY OF CHANGES UNDERSTANDING THE 2010 CAP ACCREDITATION CHECKLIST COMPONENTS HOW TO INSPECT USING R.O.A.D INSPECTION TECHNIQUE ADDITIONAL INFORMATION APPLICABILITY PROFICIENCY TESTING QUALITY MANAGEMENT AND QUALITY CONTROL GENERAL ISSUES PROCEDURE MANUAL ASSAY VALI
6、DATION REQUISITIONS SPECIMEN HANDLING SPECIMEN PROCESSING QUANTITATIVE ASSAYS: CALIBRATION AND STANDARD REAGENTS CONTROLSANALYSIS Extraction Amplification Restriction Endonucleases Sequencing Detection Methods Agarose and Polyacrylamide Gel Electrophoresis Real-Time Polymerase Chain Reaction (PCR) A
7、rraysCONTENTS不同认可体系对方法学验证的要求 差异: CAP的“方法学验证（assay validation）”和ISO15189中的“诊断性验证（diagnostic validation）” 共同点：新的检测方法应用到病人临床诊断之前必须进行方法学验证，以了解该检测的临床表现，保证实验结果可信度5-7。 CAP提出了明确的要求：“实验室必须进行分析验证各项检测的性能参数”，还对验证试验覆盖的标本范围（如不同的基因型等）、标本类型（如血液、尿液、新鲜/冷冻组织、石蜡包埋组织等）、验证的参数（如参考值、报告范围、准确度、分析灵敏度、分析特异性和精确度等）做了相应的规定。5Guzel
8、 O, Guner EI. ISO 15189 accreditation: Requirements for quality and competence of medical laboratories, experience of a laboratory I. Clin Biochem,2009,42: 274-278.6Yanikkaya-Demirel G. ISO 15189 accreditation: Requirements for quality and competence of medical laboratories, experience of a laborato
9、ry II. Clin Biochem,2009,42: 279-283.7Jennings L, Van DV, Gulley ML. Recommended principles and practices for validating clinical molecular pathology tests. Arch Pathol Lab Med,2009,133: 743-755.方法学验证的要求的修正和改进 ISO15189目前的版本是ISO技术委员会ISO/TC212 (临床实验室检验及体外诊断实验系统)标准，新版本会对方法学验证提出更多、更高的要求。 实验室自建的方法的验证； 经过
10、验证的方法在超出原验证范围之外 经过验证的方法被修改 即使是生产商已经验证的方法，实验室也必须重新验证 或者确证试验的分析特性 方法学验证的要求的修正和改进 “verification”和“validation” “确证试验-verification” “验证试验-validation”IVDs. And LDTs. In vitro diagnostics (IVDs):商业化生产；通过FDA 510(k) clearance or PMA (premarket approval)。 Laboratory-developed tests (LDTs): 许多新的分子诊断实验局限于实验室内使用
11、（科研开展）。 Many of these clinically important LDTs are unlikely to ever become FDA cleared or approved because low test volumes make it economically unfeasible to go through the current FDA 510(k) or PMA approval process. FDA cleared: In vitro diagnostics that have gone through and passed a 510(k) tria
12、l. FDA approved: An IVD that has gone through and passed a PMA trial. 510(k) trials are designed to show that an IVD is substantially equivalent to a legally marketed predicate device. 510(k) trials are less involved and less expensive than PMA trials. Premarket approval trials are required for clas
13、s III medical devices or if there is no predicate device available for comparison. They are significantly more expensive to perform than 510(k) trials.Verification/确证试验 使用范围：主要用于体外诊断（in vitro diagnostics，IVDs）项目，指已经商业化生产，并已经通过美国FDA 510(k) clearance 或approval过程的分子诊断项目。 主要目的：确证厂家提供的数据在本实验室的的实际效果 主要指标：准确性、精确性、参考范围和可报告范围 （这些指标通常在厂商的说明书中提及）。 PARR (precision, accuracy, reportable range, and reference range).Validation/验证试验 使用范围：LDTs, and modified FDA
