YY/T 1465.1-2016 (YY/T1465.1-2016, YYT 1465.1-2016, YYT1465.1-2016)
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Immunogenic evaluation method of medical devices. Part 1: T Lymphocyte transformation test in vitro
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YY/T 1465.1-2016
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| Standard ID | YY/T 1465.1-2016 (YY/T1465.1-2016) | | Description (Translated English) | Immunogenic evaluation method of medical devices. Part 1: T Lymphocyte transformation test in vitro | | Sector / Industry | Medical Device & Pharmaceutical Industry Standard (Recommended) | | Classification of Chinese Standard | C30 | | Classification of International Standard | 11.040.01 | | Word Count Estimation | 7,726 | | Date of Issue | 2016-01-26 | | Date of Implementation | 2017-01-01 | | Quoted Standard | GB/T 16886.1; GB/T 16886.2; GB/T 16886.12; GB/T 16886.20 | | Drafting Organization | State Food and Drug Administration Jinan Medical Device Quality Supervision and Inspection Center | | Administrative Organization | National Medical Device Biology Evaluation Standardization Technical Committee | | Regulation (derived from) | China Food and Drug Administration Bulletin 2016 No.25 | | Proposing organization | State Administration of Food and Drug Administration | | Issuing agency(ies) | State Administration of Food and Drug Administration | | Summary | This standard specifies the MTT assay and CFSE method for in vitro T lymphocyte transformation assays, which are useful for assessing the effect of medical devices/materials on T lymphocyte immune function. |
YY/T 1465.1-2016
YY
PHARMACEUTICAL INDUSTRY STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
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 Contents
Foreword ... 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 ... 10
Immunogenic Evaluation Method of Medical Devices -
Part 1. T Lymphocyte Transformation Test in Vitro
1 Scope
This 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 References
The 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
Requirements
GB/T 16886.12 Biological Evaluation of Medical Devices - Part 12. Sample
Preparation and Reference Materials
GB/T 16886.20 Biological Evaluation of Medical Devices - Part 20. Principles and
Methods for Immunotoxicology Testing of Medical Devices
3 Terms and Definitions
For the purposes of this document, the terms and definitions given in GB/T 16886.1
and GB/T 16886.20 apply.
4 Abbreviation
The following abbreviations are applicable to this document.
NOTE. The immunogens that may be present in the medical devices/materials are mostly
macromolecular substances; therefore, the effectiveness of the preparation method for the
selected test samples shall be explained.
7 MTT Method
7.1 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; dissolve the formazan in the cells, and
determine its absorbance value by an enzyme-linked immunosorbent assay; then it
can indirectly reflect the number and/or metabolic activity of the living cells. When
stimulated by an antigen or mitogen, the lymphocytes may undergo a specific or non-
specific immune response, proliferate and differentiate; the number or metabolic
activity of the cells may increase. Inspect the ability of lymphocytes to proliferate by
MTT dyes, then it can reflect the effects of immunogens in the medical
devices/materials on the lymphocyte immune function.
NOTE 1. MTT is a commonly used staining marker for detecting the cell proliferation. Other
suitable staining markers such as BrdU, MTS and CCK-8 can also be used. 3H-TdR is a highly
sensitive marker for detecting the cell proliferation; but the laboratory to use it shall have the
qualification to use radioactive materials and the testing staffs shall be trained and qualified.
NTOE 2. This Method is suitable for detecting the proliferation of T lymphocytes in vitro; does
not rule out the interference of the proliferation of other types of cell subsets on the results of
this experiment.
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
a) Control group for cell nutrient solution (blank control);
b) Negative control group (cell control);
c) Positive control group (cell + ConA, 5µg/mL);
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