YY/T 1465.6-2019 (YY/T1465.6-2019, YYT 1465.6-2019, YYT1465.6-2019) & related versions
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Immunogenic evaluation method of medical devices - Part 6: Determination of animal spleen lymphocyte subsets by flow cytometry
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YY/T 1465.6-2019
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YYT 1465.6-2019
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YY/T 1465.6-2019
YY
PHARMACEUTICAL INDUSTRY STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
ICS 11.120.20
C 30
Immunogenic Evaluation Method of Medical Devices - Part
6: Determination of Animal Spleen Lymphocyte Subsets by
Flow Cytometry
脏淋巴细胞亚群
ISSUED ON: JULY 24, 2019
IMPLEMENTED ON: AUGUST 1, 2020
Issued by: National Medical Products Administration
Table of Contents
Foreword ... 3
Introduction ... 4
1 Scope ... 5
2 Normative References ... 5
3 Terms and Definitions ... 5
4 Abbreviations ... 6
5 Reagents ... 6
6 Determination of Animal Spleen Lymphocyte Subsets by Flow Cytometry ... 7
7 Quality Control ... 11
8 Data Analysis ... 11
9 Test Report ... 11
Bibliography ... 12
Immunogenic Evaluation Method of Medical Devices - Part
6: Determination of Animal Spleen Lymphocyte Subsets by
Flow Cytometry
1 Scope
This Part of YY/T 1465 specifies the method for the determination of animal spleen lymphocyte
subsets by flow cytometry.
This Part is applicable to the evaluation of immune responses induced by medical devices /
materials.
2 Normative References
The following documents are indispensable to the application of this document. In terms of
references with a specified date, only versions with a specified date are applicable to this
document. In terms of references without a specified date, the latest version (including all the
modifications) is applicable to this document.
GB/T 16886.1 Biological Evaluation of Medical Devices - Part 1: Evaluation and Testing within
a Risk Management Process
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
What is defined in GB/T 16886.1 and GB/T 16886.20, and the following terms and definitions
are applicable to this document.
3.1 cluster of differentiation; CD
Cell surface markers that appear or disappear during the differentiation, maturation and
activation processes of cells of different lineages, which are usually identified with monoclonal
antibodies.
instrument, and operate in strict accordance with the instructions and precautions. If hemolytic
reagents without fixative effect (such as: ammonium chloride and hypotonic buffer, etc.) are
used, the specimens after fluorescent antibody staining need to be stored at 4 C, and the on-
machine detection must be completed within 1 hour.
5.5 Commercially available fluorescent microsphere optical path quality controls that match
the flow cytometer.
5.6 Fluorescein-labeled monoclonal antibodies. Antibody is a key step in lymphocyte subset
analysis, and the reactivity of the selected antibody to the cells should be considered. It is
recommended to use commercialized in vitro diagnostic reagents that combine antibodies. If
the laboratory prepares the combination of monoclonal antibodies by itself, it should be freshly
prepared before each test. Each antibody in the combination antibody needs to be separately
titrated to determine the optimal separation titer of noise and positive signal, and to detect the
average fluorescence intensity and positivity rate when used as a combination antibody and
when used alone as one component of the combination antibody, and the standard deviation of
comparability must be within 2 of the average value.
6 Determination of Animal Spleen Lymphocyte Subsets by
Flow Cytometry
6.1 Experimental Principle
FCM is a technology that utilizes flow cytometer as a means of detection, and can quickly,
accurately, objectively and simultaneously detect and quantify multiple characteristics of single
biological particles. Flow cytometer can be used to conduct multi-parameter and rapid
quantitative analysis of single spleen lymphocytes at the cellular and molecular level using
monoclonal antibodies.
6.2 Experimental Animals
6.2.1 General principle
All animal experiments shall be conducted in laboratories approved by national accreditation
institutions, comply with all applicable regulations on laboratory animal welfare, and shall also
comply with the requirements of GB/T 16886.2.
6.2.2 Animal species and requirements
Commonly used experimental animals are mice. In this Part, untested and healthy BALB/c mice,
SPF grade, 6 ~ 8 weeks old are recommended to be used. Before formal test, animals must be
kept for at least 5 days to adapt to the laboratory environment. Experimental animals should be
labeled with their species, strain, source, gender, weight and age. At the beginning of the test,
the weight difference of the animals should be controlled to a minimum range, and the weight
of each animal cannot exceed 20% of the average weight of the same gender. It is
recommended to use at least 3 mice of each gender.
If other species of animals are selected, their suitability should be explained.
6.3 Sample Preparation
In accordance with the principle of GB/T 16886.12, prepare the test samples.
NOTE: most of the immunogens that may exist in medical devices / materials are macromolecular
substances. The effectiveness of the selected test sample preparation method shall be
explained.
6.4 Test Grouping
Test grouping includes:
a) Negative control group (solvent control or sham operation group);
b) Positive control group (antigenic substances known to cause positive reactions): BSA
is recommended as a positive control.
NOTE: take 3 mg of BSA and evenly mix it with 9 mL of PBS (pH 7.4), then, evenly mix it with
CFA at a ratio of 1 : 1 (volume ratio) to form an emulsion. Subcutaneously inject 0.12 mL
on the back of each animal once a week. Other confirmed exposure doses and modes may
also be used.
c) Test sample group.
NOTE 1: existing immunological study data and information on the toxicokinetics of the test
substance or related substances can be used to select a corresponding dose range to avoid
excessive toxic reactions. The information can also be conductive to determine the
dosing frequency.
NOTE 2: the test dose, immunization frequency and test cycle can be selected in accordance with
the intended use of the product and human clinical use. Meanwhile, the tolerance of
experimental animals shall be taken into consideration. Test samples can be tested in
multiple dose groups. It is recommended to design three dose concentrations of high,
medium and low, and the dose group setting shall include the maximum dose clinically
used in humans.
6.5 Recommended Test Steps
6.5.1 Preparation of splenocyte suspension
Aseptically take the spleen of BALB/c mice, add culture medium and grind to obtain a cell
suspension; centrifuge to remove the culture medium. In accordance with the instructions for
the hemolysin used, lyse red blood cells. Add 2 mL of PBS to each tube, re-suspend the cells,
and centrifuge at 350 g for 5 minutes. After centrifugation, discard the supernatant and use PBS
to adjust the spleen cell concentration to 1 107 cells/mL.
6.5.2 Sample staining
In accordance with the combination of fluorescein-labeled monoclonal antibodies (hereinafter
referred to as “antibodies”) that comply with the FCM performance indicators, process the
sample to be tested and label it (see Table 1). Meanwhile, properly set the blank control,
antibody single-label control and isotype control tubes. Different types of FCM can set
appropriate sample staining steps in accordance with their performance indicators.
a) Blank control: add PBS to the flow tube instead of the antibody to adjust the
background voltage and florescence intensity during instrument analysis, and judge
the correctness of the chromosome system together with the isotype control;
b) Antibody single-label control: when analyzing a combination of two or more
antibodies, it is necessary to adjust the fluorescence compensation of the flow
cytometer through the antibody single-label control in accordance with the
instructions for the use of antibody;
c) Isotype control: add immunoglobulins of the same subtype, same source and same
label as the detection antibody to the flow cytometer instead of the antibody to
eliminate the non-specific staining part and judge the correctness of the chromosome
system together with the blank control. In addition, the boundary between negative
and positive groups can be determined in histograms and scatter plots;
d) Add the corresponding labeled antibodies to each flow tube: each antibody is prepared
in accordance with the mixing items set in the antibody staining scheme (see table 1)
that complies with the FCM performance indicators to form an antibody mixture
system that complies with the cell labeling requirements and is added to the flow tube;
NOTE: in accordance with the concentration and quantity of lymphocytes in the flow tube, strictly
follow the operating instructions to set the amount of antibody and adjust the optimal
proportion of lymphocytes and antibodies.
e) Add the spleen cell suspension with an adjusted concentration to the flow tube of each
sample, 100 L / tube, thoroughly mix it, and react at room temperature in the dark
for 20 minutes ~ 30 minutes; add 2 mL of PBS to each tube to re-suspend the cells,
then, centrifuge at 350 g for 5 minutes; after centrifugation, discard the supernatant;
f) After adding 0.5 mL of PBS to each tube to re-suspend the cells, place them in the
dark at 4 C ~ 10 C, until they are detected on the machine. Before the detection,
evenly mix the cells.
NOTE: if the antibody-labeled lymphocytes cannot be detected for a long time on the same day, 2%
serum can be added to the spleen cell suspension. However, it is advisable to centrifuge
and re-suspend the cells in serum-free PBS before it is put on the machine. If it cannot be
detected in time on the same day, it is advisable to use a cell fixative (for example, 0.1% ~
......
| Standard ID | YY/T 1465.6-2019 (YY/T1465.6-2019) | | Description (Translated English) | Immunogenic evaluation method of medical devices - Part 6: Determination of animal spleen lymphocyte subsets by flow cytometry | | Sector / Industry | Medical Device & Pharmaceutical Industry Standard (Recommended) | | Classification of Chinese Standard | C30 | | Classification of International Standard | 11.120.20 | | Word Count Estimation | 10,184 | | Date of Issue | 2019 | | Date of Implementation | 2020-08-01 | | Summary | This standard specifies a method for the determination of animal spleen lymphocyte subsets by flow cytometry. This standard applies to the evaluation of immune responses induced by medical devices/materials. |
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