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YY/T 1465.1-2016: Immunogenic Evaluation Method of Medical Devices - Part 1: T Lymphocyte Transformation Test in Vitro Delivery: 9 seconds. True-PDF full-copy in English & invoice will be downloaded + auto-delivered via email. See step-by-step procedure Status: Valid
Similar standardsYY/T 1465.1-2016: Immunogenic Evaluation Method of Medical Devices - Part 1: T Lymphocyte Transformation Test in Vitro---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/YYT1465.1-2016 YY PHARMACEUTICAL INDUSTRY STANDARD ICS 11.040.01 C 30 Immunogenic Evaluation Method of Medical Devices - Part 1.T Lymphocyte Transformation Test in Vitro Issued on. JANUARY 26, 2016 Implemented on. JANUARY 01, 2017 Issued by. China Food and Drug Administration Table of ContentsForeword... 3 Introduction... 4 1 Scope... 5 2 Normative References... 5 3 Terms and Definitions... 5 4 Abbreviation... 5 5 Test Animal... 6 6 Sample Preparation... 6 7 MTT Method... 7 8 CFSE Method... 8 9 Test Report... 9 Bibliography... 10ForewordYY/T 1465 Immunogenic Evaluation Method of Medical Devices consists of the following parts. --- Part 1.T Lymphocyte Transformation Test In Vitro; --- Part 2.Serum Immunoglobulin and Complement Component Detection (ELISA Method). This Part belongs to Part 1 of YY/T 1465. This Part was drafted as per the rules specified in GB/T 1.1-2009. Please note that some contents of this document may involve patents. The issuing organization of this document shall not assume the responsibility for identifying these patents. This Part was proposed by China Food and Drug Administration. This Part shall be under the jurisdiction of National Technical Committee for Standardization of Biological Evaluation on Medical Devices (SAC/TC 248). Drafting organizations of this Part. Jinan Quality Supervision and Inspection Center for Medical Devices of China Food and Drug Administration; Sichuan Testing Center for Biomaterials and Medical Devices; and Shanghai Biomaterials Research & Testing Center. Chief drafting staffs of this Part. Liu Chenghu, Wang Xianmei, Hou Li, Yuan Tun, Liang Jie, Xue Yang, and Sun Jiao.1 ScopeThis Part gives the MTT method and CFSE method for the T lymphocyte transformation test in vitro; it is applicable to evaluate the effect of medical devices/materials on the T lymphocyte immune function.2 Normative ReferencesThe following documents are essential to the application of this document. For the dated documents, only the versions with the dates indicated are applicable to this document; for the undated documents, only the latest version (including all the amendments) are applicable to this document. GB/T 16886.1 Biological Evaluation of Medical Devices - Part 1.Evaluation and Testing GB/T 16886.2 Biological Evaluation of Medical Devices - Part 2.Animal Welfare Requirements3 Terms and DefinitionsFor the purposes of this document, the terms and definitions given in GB/T 16886.1 and GB/T 16886.20 apply.4 AbbreviationThe following abbreviations are applicable to this document.5 Test Animal5.1 General All animal tests shall be carried out in the laboratories approved by the national accreditation agency and in compliance with all applicable regulations for laboratory animal welfare; and shall also comply with the requirements of GB/T 16886.2. 5.2 Animal species and requirements The commonly used experimental animal is a mouse. This Part recommended to use the healthy Grade-SPF, 6-8 weeks of age BALB/c mice that have not been tested. If other strains of mice are used, the suitability shall be explained.6 Sample PreparationThe test sample preparation shall be carried out in accordance with the principles of GB/T 16886.12.7 MTT Method7.1 Principle MTT is a pale-yellow cell dye; it enters into the living cells and forms water-insoluble blue-purple crystalline formazan particles in mitochondria under the role of succinate dehydrogenase and deposits in the cells; 7.2 Lymphocyte preparation The BALB/c mice were sacrificed by taking the cervical vertebrae off; the single cell suspension was prepared by grinding; collect the cell suspension; take 400g to centrifuge for 5min; discharge the supernatant; add 5mL of red blood cell lysate; mix up; incubate at 4°C for 5min; add RPMI1640 nutrient solution containing 10% FCS to stop the reaction; take 400g to centrifuge for 5min; discharge the supernatant; add RPMI1640 nutrient solution containing 10% FCS to adjust the cell concentration into 2×106 cells/mL. 7.3 Test grouping 7.4 Test procedures Add the cell suspension into a 96-well plate; 200µL per well; set six replicate wells. Add ConA to positive control group till the final concentration is 5µg/mL. 7.5 Data analysis Record the absorbance values, A, for each group; use appropriate statistical method to deal with the test results. Use mitogen to confirm whether the test system is working properly; meanwhile, it can compare the immune response from the cells to antigen and mitogen.8 CFSE Method8.1 Principle Carboxyfluorescein diacetate succinimidyl ester (CFSE) is a living fluorescent dye that can penetrate the living cell membrane and irreversible binds to intracellular proteins. When the cells divide, CFSE-labeled fluorescence is evenly distributed into the two daughter cells; its fluorescence intensity is half of that of the parental cells. 8.2 Test procedures 8.2.1 Aseptically prepare the mouse spleen lymphocyte suspension according to 7.2; wash at least 2 times by PBS; discharge the supernatant; resuspend the cells by 2mL of PBS containing 0.1% BSA; separately add CFSE till the final concentration is 5µM; mix up. Incubate at 37°C for 15min to 30min in the dark. 8.2.2 Add 3mL of pre-cooled RPMI1640 nutrient solution containing 2% FCS to stop the staining reaction; mix gently; after placing on the ice for 5min; take 400g to centrifuge for 5min; discharge the supernatant; add 2mL of RPMI1640 nutrient solution containing 10% FCS; mix up. 8.3 Data analysis The test results shall be recorded by using the mean fluorescence intensity combined with the percentage of proliferating cells; appropriate statistical methods shall be used to deal with the test results.9 Test ReportThe test report shall contain the following information. ......Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al. 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