YY/T 1465.6-2019Immunogenic evaluation method of medical devices - Part 6: Determination of animal spleen lymphocyte subsets by flow cytometry (English PDF)

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.

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 x 10^7 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 µL / 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% ~