分子诊断检测的标准化与自动化 2011-08-18王兆强
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2010年3月8日，SFDA发出《关于加快血源筛查用核酸诊断试剂技术审评等工作 的通知》，要求中国药品生物制品检定所、药品审评中心、药品认证管理中心、 行受理服务中心对血源筛查用核酸检测试剂进行加快审评审批。 NAAT血液筛检方法总是从血浆制品的原料浆的筛检开始的,积累了充分的经验后, 才逐步应用到血站的采供血系统。例如,早在1994年FDA就曾提出血浆蛋白生产的 原料浆必须进行NAT筛检；日本从97年11月起在原料浆中进行NAAT筛检。自 1999年7月，美国、日本、欧洲开始在血站系统鼓励、进行NAAT检测
2
Since 1992
中国历程：临床分子诊断检测
九十年代中国，在我国由于部分医部机构受利益的驱动，在缺乏技术、设备以及 规范化管理的情况下，PCR技术临床应用泛滥，出现大量假阳性和假阴性结果， 甚至出现虚假检测报告，造成检验结果和临床意义应用十分混乱，严重扰乱正常 医疗秩序，特别在性病基因检测方面，甚至还带来一系列道德伦理、家庭纠纷以 及社会和法律问题。 PCR技术临床应用所出现的一系列问题引起卫生部管理层高度重视，卫生部医政 司于1998年下发了卫医发[1998]9号文件宣布PCR技术暂停应用于临床诊断。 卫生部2002年1月14日发布“临床基因扩增检验实验室管理暂行办法”，同年2月 20日卫生部临床检验中心发布“临床基因扩增实验室工作规范”。 两个文件宣布了PCR技术临床应用解冻，明确规定临床基因扩增检验实验室开展 PCR技术必需具备的基本条件：①规范的PCR实验室②编写适合本实验室的质量 手册③经PCR专业知识培训的技术人员（PCR上岗证）④使用有生产批文的PCR 试剂。具备条件的二级以上医院可向卫生部或省临床检验中心申请技术验收。 3
9பைடு நூலகம்
Since 1992
Validation panels for NAAT
3000 copies/ml to below 0.3 copies/ml with half log dilutions. They are suitable for analyzing the analytical sensitivity of qualitative or quantitative NAT assays. The WHO standards are expressed in international units (IU)/ml. These are arbitrary measurement units assigned to ampoules of the lyophilised standard by the expert committee of the WHO. The unitage in IU/ml is unique for every virus, but not harmonised between viruses. The EUROHEP standards are expressed in Eurohep units/ml (EU/ml). The Italian ISS standards are calibrated against the WHO standards and therefore expressed in IU/ml. Our internal DDL standards are quantified in copies/ml as determined in multiple bDNA 3.0 assays. The HIV-RNA group O and HIV2-RNA subtype A standards are quantified against the HIV-1 subtype B standard by comparing dilution series in the Murex HIV-Ag assay. All standards mentioned above have also been calibrated in multiple bDNA 3.0 assays. One IU of HBV-DNA was found to be equivalent to 5.33 copies; one IU HCV-RNA equals 2.73 copies and one IU HIV-1 RNA is equivalent to 0.48 copies in the bDNA 3.0 assay. One copy should be equal to one DNA or RNA molecule or one virion. HIV is a diploid virus and contains two RNA copies per virus. The panels are grouped according to different standards. The different dilutions are filled off with 4.3 ml in 5.0 ml vials. Each panel contains one vial per dilution
-TrendControl: will result in an S/CO ratio different from the negative
population, but not always positive. Minor changes in sensitivity yield lower S/CO ratios. Analysis of field results on TrendControls reveals the proportion positive results is changing over time indicating the sensitivity of the assay varies over time.
Since 1992
分子诊断检测：标准化与自动化
Standardization and Automation in Molecular Diagnostics
王兆强 博士
澳斯邦生物工程有限公司 jzwang@ausbio.com
1
Since 1992
The history of the Polymerase Chain Reaction (PCR)
Since 1992
中国历程：血液筛查分子诊断检测
2009年11月26日，卫生部医政司举办血液筛查核酸检测推广会
《卫生部办公厅关于印发〈2010年血站核酸检测项目管理方案〉的通知》（卫办 医政函﹝2010﹞1118号），全国第一批(12个省份、15家单位)核酸检测试点单位， 已于2010年5月31日起全面开展核酸检测试点工作。
7
Since 1992
External Controls for Blood Screening NAAT
Run controls -CheckControl: should be found positive. Assays that express their
qualitative result in S/CO ratio can use run controls 5 times the 95 % hit rate. The latter will yield a "saturated" S/CO ratio. Monitoring this ratio will not identify small changes in sensitivity.
1984年11月15日，Cetus公司的Kary Mullis 的PCR实验获得了成功
On March 28, 1985 the entire development group (including Mullis) filed an application申请有关PCR的第一个专利。
December 1985 a joint venture between Cetus and Perkin-Elmer was established to develop instruments and reagents for PCR In the Spring of 1985 John Sninsky at Cetus began to use PCR for the difficult task of measuring the amount of HIV circulating in blood. on November 19, 1987 a press release announces the commercial availability of the "PCR-1000 Thermal Cycler" and "AmpliTaq DNA Polymerase" On July 23, 1991 Cetus announced that its sale to the neighboring biotechnology company Chiron，As part of the sale, rights to the PCR patents were sold for USD $300 million to Hoffman-La Roche (who in 1989 had bought limited rights to PCR). Many of the Cetus PCR researchers moved to the Roche subsidiary, Roche Molecular Systems. On October 13, 1993 Kary Mullis, who had left Cetus in 1986, was awarded the Nobel Prize in Chemistry
Validation panels
Proficiency panels
8
Since 1992
Proficiency panels for NAAT
EDCNet (Electronic Data Collection – internet-based reporting) provides a subscriber the ability to directly enter and then graph data generated from quality control (QC) results used to monitor serological or nucleic acid tests.
4
Since 1992
主要内容
分子诊断检测的标准化
Standardization in Molecular Diagnostics
分子诊断检测的自动化
Automation in Molecular Diagnostics
5
Since 1992
分子诊断检测的标准化
Standardization in Molecular Diagnostics 参考品标准化International Standard
试剂标准化standardization in kit system 质控品标准化run controls, validation panels and proficiency panels
6
Since 1992
External Controls for Blood Screening NAAT
The use of external controls, calibrated in copies/ml (and IU/ml), is essential for independent monitoring of the performance and sensitivity of nucleic acid test runs and IVD reagent batches in blood banks.
2
Since 1992
中国历程：临床分子诊断检测
九十年代中国，在我国由于部分医部机构受利益的驱动，在缺乏技术、设备以及 规范化管理的情况下，PCR技术临床应用泛滥，出现大量假阳性和假阴性结果， 甚至出现虚假检测报告，造成检验结果和临床意义应用十分混乱，严重扰乱正常 医疗秩序，特别在性病基因检测方面，甚至还带来一系列道德伦理、家庭纠纷以 及社会和法律问题。 PCR技术临床应用所出现的一系列问题引起卫生部管理层高度重视，卫生部医政 司于1998年下发了卫医发[1998]9号文件宣布PCR技术暂停应用于临床诊断。 卫生部2002年1月14日发布“临床基因扩增检验实验室管理暂行办法”，同年2月 20日卫生部临床检验中心发布“临床基因扩增实验室工作规范”。 两个文件宣布了PCR技术临床应用解冻，明确规定临床基因扩增检验实验室开展 PCR技术必需具备的基本条件：①规范的PCR实验室②编写适合本实验室的质量 手册③经PCR专业知识培训的技术人员（PCR上岗证）④使用有生产批文的PCR 试剂。具备条件的二级以上医院可向卫生部或省临床检验中心申请技术验收。 3
9பைடு நூலகம்
Since 1992
Validation panels for NAAT
3000 copies/ml to below 0.3 copies/ml with half log dilutions. They are suitable for analyzing the analytical sensitivity of qualitative or quantitative NAT assays. The WHO standards are expressed in international units (IU)/ml. These are arbitrary measurement units assigned to ampoules of the lyophilised standard by the expert committee of the WHO. The unitage in IU/ml is unique for every virus, but not harmonised between viruses. The EUROHEP standards are expressed in Eurohep units/ml (EU/ml). The Italian ISS standards are calibrated against the WHO standards and therefore expressed in IU/ml. Our internal DDL standards are quantified in copies/ml as determined in multiple bDNA 3.0 assays. The HIV-RNA group O and HIV2-RNA subtype A standards are quantified against the HIV-1 subtype B standard by comparing dilution series in the Murex HIV-Ag assay. All standards mentioned above have also been calibrated in multiple bDNA 3.0 assays. One IU of HBV-DNA was found to be equivalent to 5.33 copies; one IU HCV-RNA equals 2.73 copies and one IU HIV-1 RNA is equivalent to 0.48 copies in the bDNA 3.0 assay. One copy should be equal to one DNA or RNA molecule or one virion. HIV is a diploid virus and contains two RNA copies per virus. The panels are grouped according to different standards. The different dilutions are filled off with 4.3 ml in 5.0 ml vials. Each panel contains one vial per dilution
-TrendControl: will result in an S/CO ratio different from the negative
population, but not always positive. Minor changes in sensitivity yield lower S/CO ratios. Analysis of field results on TrendControls reveals the proportion positive results is changing over time indicating the sensitivity of the assay varies over time.
Since 1992
分子诊断检测：标准化与自动化
Standardization and Automation in Molecular Diagnostics
王兆强 博士
澳斯邦生物工程有限公司 jzwang@ausbio.com
1
Since 1992
The history of the Polymerase Chain Reaction (PCR)
Since 1992
中国历程：血液筛查分子诊断检测
2009年11月26日，卫生部医政司举办血液筛查核酸检测推广会
《卫生部办公厅关于印发〈2010年血站核酸检测项目管理方案〉的通知》（卫办 医政函﹝2010﹞1118号），全国第一批(12个省份、15家单位)核酸检测试点单位， 已于2010年5月31日起全面开展核酸检测试点工作。
7
Since 1992
External Controls for Blood Screening NAAT
Run controls -CheckControl: should be found positive. Assays that express their
qualitative result in S/CO ratio can use run controls 5 times the 95 % hit rate. The latter will yield a "saturated" S/CO ratio. Monitoring this ratio will not identify small changes in sensitivity.
1984年11月15日，Cetus公司的Kary Mullis 的PCR实验获得了成功
On March 28, 1985 the entire development group (including Mullis) filed an application申请有关PCR的第一个专利。
December 1985 a joint venture between Cetus and Perkin-Elmer was established to develop instruments and reagents for PCR In the Spring of 1985 John Sninsky at Cetus began to use PCR for the difficult task of measuring the amount of HIV circulating in blood. on November 19, 1987 a press release announces the commercial availability of the "PCR-1000 Thermal Cycler" and "AmpliTaq DNA Polymerase" On July 23, 1991 Cetus announced that its sale to the neighboring biotechnology company Chiron，As part of the sale, rights to the PCR patents were sold for USD $300 million to Hoffman-La Roche (who in 1989 had bought limited rights to PCR). Many of the Cetus PCR researchers moved to the Roche subsidiary, Roche Molecular Systems. On October 13, 1993 Kary Mullis, who had left Cetus in 1986, was awarded the Nobel Prize in Chemistry
Validation panels
Proficiency panels
8
Since 1992
Proficiency panels for NAAT
EDCNet (Electronic Data Collection – internet-based reporting) provides a subscriber the ability to directly enter and then graph data generated from quality control (QC) results used to monitor serological or nucleic acid tests.
4
Since 1992
主要内容
分子诊断检测的标准化
Standardization in Molecular Diagnostics
分子诊断检测的自动化
Automation in Molecular Diagnostics
5
Since 1992
分子诊断检测的标准化
Standardization in Molecular Diagnostics 参考品标准化International Standard
试剂标准化standardization in kit system 质控品标准化run controls, validation panels and proficiency panels
6
Since 1992
External Controls for Blood Screening NAAT
The use of external controls, calibrated in copies/ml (and IU/ml), is essential for independent monitoring of the performance and sensitivity of nucleic acid test runs and IVD reagent batches in blood banks.