YY/T 1465.3-2016Immunogenic evaluation method of medical devices - Part 3: Plaque forming cells assay-Agar gel solid-phase method (English PDF)

1 Scope

This Part of YY/T 1465 provides a method for the determination of plaque-forming cells by agar gel solid-phase method.

This Part is suitable for evaluating the impact of medical devices/materials on the body's humoral 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) 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 16886.2-2011, ISO 10993-1:2009, IDT)

GB/T 16886.2 Biological evaluation of medical devices - Part 2: Animal welfare requirements (GB/T 16886.2-2011, ISO 10993-2:2006, IDT)

GB/T 16886.11 Biological evaluation of medical devices - Part 11: Tests for systemic toxicity (GB/T 16886.11-2011, ISO 10993-6:2006, IDT)

GB/T 16886.12 Biological evaluation of medical devices - Part 12: Sample preparation and reference materials (GB/T 16886.12-2005, ISO 10993-12:2002, IDT)

GB/T 16886.20 Biological evaluation of medical devices - Part 20: Principles and methods for immunotoxicology testing of medical devices (GB/T 16886.20-2015, ISO/TS 10993-20:2006, IDT)

3 Terms and Definitions

For the purposes of this Document, the terms and definitions given in GB/T 16886.1, GB/T 16886.2, GB/T 16886.12, and GB/T 16886.20 apply.

4 Test Principle

After sensitizing mice with sheep red blood cells (SRBC), B lymphocytes (plasma cells) capable of producing anti-SRBC antibodies will appear in the immune organs of the mice, especially the spleen. The number and function of these cells can reflect the status of the body's humoral immune function. When these cells that can secrete anti-SRBC antibodies are incubated with SRBCs, the released antibodies will bind to the SRBCs to form antigen-antibody complexes. With the participation of complement, the SRBCs around these B lymphocytes will dissolve and plaque will appear. When medical devices/materials act on the human body, the impact of medical devices/materials on the body's humoral immune function can be evaluated by detecting changes in the number of these plaque-forming cells.

5 Test Animals

6 Sample Preparation and Exposure Routes

6.1 For common insoluble non-degradable substances in medical devices, sample extracts can be prepared according to the principles of GB/T 16886.12. A suitable solvent shall be used for rigorous extraction to dissolve all extractable components. Extracts should be prepared on the same day unless stability data are available to demonstrate acceptability of storage. According to the intended use of the sample, refer to the requirements of GB/T 16886.11 to determine the animal contact mode of the sample/extract. Commonly used methods of animal contact include intragastric administration, intraperitoneal injection, and intravenous injection. The exposure dose and exposure time can be designed with reference to the requirements for acute, subacute, sub-chronic and chronic systemic toxicity tests in GB/T 16886.11, and shall be stated in the final test report.

6.2 For degradable medical devices, the contact method between the device and animals shall

NOTE: Gradient dilution of the material or its extract will help to derive the dose-effect relationship of the immune response. It is recommended that the test sample group be tested with different doses.

8 Test Procedures

9 Calculation of Results

9.1 Express the results as the number of plaque-forming cells per 106 spleen cells.

9.2 At least 3 agar plates shall be prepared for each animal; the mean and standard deviation of plaque-forming cells of each animal shall be reported; and appropriate statistical methods shall be selected for comparison between groups.

10 Judgment of Result

When there is a statistically significant difference in the number of plaque-forming cells between the test group and the negative control group, the test substance can be considered to have an impact on the plaque-forming cell measurement test. This effect may indicate that the test substance shall interfere with the body's humoral immunity. If a gradient dilution of the sample/extract is used for the test, and a dose-effect relationship appears on the number of plaque-forming cells, it indicates that there may be an impact on the plaque-forming cell assay.

11 Inspection of Reliability

11.1 The inhibitory effect of interfering substances on humoral immunity is the primary immunotoxicity that needs to be considered. Therefore, the positive control in this test is to confirm the suitability of the test by using substances that have been shown to have inhibitory effects on humoral immune function. When it is necessary to confirm that the laboratory has the ability to conduct this test and can carry out intra-laboratory repeatability and inter-laboratory reproducibility evaluation, it shall recommend to use a positive control for each test.

11.2 For laboratories that frequently conduct this test (at a frequency of no less than once a month) and have long-term positive control data to prove their ability to obtain reproducible and accurate positive results, the positive control test can be performed periodically (with an interval of <= 6 months).

12 Test Report

The test report should contain the following information:

a) Test sample name, specification model and batch number;

b) Test and control sample preparation methods;

c) The strain, age and weight of the test animals;

d) Test conditions and test procedures;

e) Test results;

f) Result evaluation; […]