YY/T 1465.4-2017 English PDF (YY/T1465.4-2017)

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YY/T 1465.4-2017: Immunogenic evaluation method of medical devices - Part 4: Phagocytosis of mouse peritoneal macrophages on chicken erythrocytes - Ex-vivo method
YY/T 1465.4-2017
Immunogenic evaluation method of medical devices—Part 4. Phagocytosis of mouse peritoneal macrophages on chicken erythrocytes—Ex-vivo method
ICS 11.040.01
C30
People's Republic of China Pharmaceutical Industry Standard
Medical device immunogenicity evaluation method
Part 4. Mouse peritoneal macrophage phagocytosis
Chicken red blood cell test
Part 4.Phagocytosisofmouseperitonealmacrophagesonchickenerythrocytes-
Ex-vivomethod
Released on.2017-03-28
2018-04-01 implementation
State Food and Drug Administration issued
Foreword
YY/T 1465 "Methods for Evaluation of Immunogenicity of Medical Devices" is divided into the following sections.
--- Part 1. In vitro T lymphocyte transformation test;
--- Part 2. Determination of serum immunoglobulin and complement components (ELISA method);
--- Part 3. Determination of plaque forming cells by agar solid phase method;
--- Part 4. Half-in vivo method for phagocytosis of chicken red blood cells by mouse peritoneal macrophages;
--- Part 5. Determination of alpha-Gal antigen clearance in animal-derived medical devices using the M86 antibody.
This part is the fourth part of YY/T 1465.
There are other criteria for other methods of evaluating the immunogenicity of medical devices.
This part is drafted in accordance with the rules given in GB/T 1.1-2009.
Please note that some of the contents of this document may involve patents. The issuing organization of this document is not responsible for identifying these patents.
This part is proposed by the State Food and Drug Administration.
This part is under the jurisdiction of the National Technical Committee for Standardization of Medical Device Biology Evaluation (SAC/TC248).
This section drafted by. Sichuan Medical Device Biomaterials and Products Inspection Center, Shandong Province Medical Device Product Quality Inspection Center.
Drafters of this section. Yuan Wei, Liang Jie, Qiao Chunxia, Zheng Xiuzhen.
introduction
The immune response is an important defense mechanism of the body. As an exogenous substance, medical devices can pass through various kinds after contact with the human body.
The pathway affects the body's immune system. At present, it is unclear whether the immune response generated by medical devices or materials is beneficial or harmful to the host.
Especially for animal-derived medical devices, allogeneic instruments and tissue engineering instruments. Therefore, applying medical devices, material components or dips
It is very important that the extract is subjected to an immune response study to obtain relevant information.
The immunotoxicity caused by medical devices may involve various aspects of the immune response, including irritation/acute inflammation, chronic inflammation, immunity
Inhibition, immune stimulation, hypersensitivity and autoimmunity. These processes may involve a variety of immune cells, especially lymphocytes, granulocytes and
Macrophages, etc. Macrophages are an important class of immune cells that directly participate in multiple aspects of innate and adaptive immune responses.
surface. Guidance on the possible immune response and potential immunotoxicity of medical devices in contact with humans is given in GB/T 16886.20.
However, there is a lack of specific test methods. This part of YY/T 1465 is expected to provide specific test methods for the implementation of GB/T 16886.20.
This section provides a method for determining the ability of mouse macrophages to phagocytose chicken red blood cells, which is a non-specific immunity and antigen for medical devices/materials.
Provides detection methods for the impact of the presentation process and can be used as an alternative method in immunotoxicology tests in GB/T 16886.20.
quasi. However, this method has certain limitations, and the applicability of the method should be confirmed before use. Other confirmed methods are also available
use.
Medical device immunogenicity evaluation method
Part 4. Mouse peritoneal macrophage phagocytosis
Chicken red blood cell test
1 Scope
This part of YY/T 1465 gives a semi-in vivo method for determining the phagocytosis of chicken red blood cells by mouse peritoneal macrophages.
This section applies to the evaluation of the impact of medical devices/materials on macrophage phagocytosis.
2 Normative references
The following documents are indispensable for the application of this document. For dated references, only dated versions apply to this article.
Pieces. For undated references, the latest edition (including all amendments) applies to this document.
GB/T 16886.1 Biological evaluation of medical devices - Part 1. Evaluation and testing in the process of risk management (GB/T 16886.1-
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. Systemic toxicity test (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 samples (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 immunological toxicology of medical devices
(GB/T 16886.20-2015, ISO /T S10993-20.2006, IDT)
3 Terms and definitions
The terms and definitions defined in GB/T 16886.1, GB/T 16886.2, GB/T 16886.12 and GB/T 16886.20 apply to this
file.
4 Test principle
After immunizing mice with chicken red blood cells, mouse macrophages can recognize and phagocytose them as foreign bodies. Swallowed chicken red by staining count
The number of macrophages in the cells reflects the phagocytic function of macrophages. When the medical device/material acts on the body, by detecting macrophage
The effect of the cell on the phagocytosis of chicken red blood cells can be evaluated by the influence of medical devices/materials on the phagocytic function of macrophages.
5 test animals
5.1 Selection of animal species
The animal species selected in this experiment are mice, male or female. Balb/c mice are the preferred line of recommendation. Should use weight between 18g~22g
Adults who have not given birth and are not pregnant. At the beginning of the trial, the animal's body weight difference should be minimal and does not exceed ±20% of the average body weight. when
Other strain animals can also be selected when sufficient data is available.
5.2 Animal preparation
All animal testing should be conducted in a laboratory approved by a national accreditation body and in compliance with all applicable regulations for laboratory animal welfare.
And should also meet the requirements of GB/T 16886.2. Animals were randomly selected for individual labeling and adapted to laboratory conditions prior to testing
At least 5d.
6 Sample preparation and route of exposure
6.1 For insoluble non-degradable substances commonly found in medical devices, sample extracts can be prepared according to the principles of GB/T 16886.12. should
Scaling with a suitable solvent to dissolve all extractable components. The extract should be prepared on the same day unless there is stability data
Acceptability of storage. According to the intended use of the sample, it is advisable to determine the animal contact of the sample/leaching solution according to the requirements of GB/T 16886.11.
the way. Common animal contact methods include gavage, intraperitoneal injection, and intravenous injection. Contact dose and contact time can be referred to
The requirements for acute, subacute, subchronic and chronic systemic toxicity tests in GB/T 16886.11 are designed and reported in the final test report.
Write it.
6.2 For biodegradable medical devices, the device/animal contact/immunization method should be designed according to the intended clinical application. When available
When studying the literature, it is possible to consider the experimental design using the methods reported in the literature to compare with the existing data. When there is no reference
When considering the animal exposure/immunization method, an implantation route similar to the intended clinical application may be considered.
6.3 Liquid test substances can be used directly or diluted.
Note. The immunogens in medical devices are mostly macromolecular substances. For the preparation of sample extracts, the effectiveness of the selective extraction method should be explained.
7 Selection of control samples
7.1 Positive control
Positive control substances that promote macrophage phagocytosis are recommended. 100 IU/g of gamma interferon (IFN-γ) or
10 mg/kg phytohemagglutinin A (PHA); a positive control substance capable of inhibiting macrophage phagocytosis. 5 mg/kg of dexamethasone
Loose or.200 mg/kg cyclophosphamide. Other well-tested positive control test substances can also be used. Selection of positive control substances
The choice and application method should be determined according to the intended use and test purpose of the test sample.
7.2 Negative control
The leaching medium (such as physiological saline) recommended in GB/T 16886.12 was used in the same treatment as the test sample.
Note. When there is a known long-term clinical history, the level of immunotoxicity is considered acceptable and the test sample composition is the same as expected.
When it is listed, it can also be used as a negative control, but its application needs to be fully demonstrated.
8 test steps
8.1 Test grouping
The experimental animals were grouped according to random principles, with at least 5 animals per group.
a) test sample set;
b) a positive control group;
c) Negative control group.
Note. Gradient dilution of the material or its extract can help to derive the dose-response relationship of the immune response. The recommended test sample group is treated with different doses.
test.
8.2 Preparation of chicken red blood cell suspension
Fresh anticoagulated chicken blood, centrifuged at 1000 g for 5 min, discard the supernatant, centrifuge 3 times with sterile physiological saline, and discard the supernatant. Physiological salt
The water was formulated into a 5% (volume fraction) chicken red cell suspension.
8.3 Sample and Control Contact
Sample contact phase. Each experimental group of animals was exposed to the sample according to the dose, route and cycle selected in Chapter 6 of this standard. Negative control group
Animals were exposed in the same manner as a negative control (leaching medium).
Macrophage phagocytosis test phase. on the first day and the second day after the end of the sample contact period, each positive control group was set as above.
A positive control substance was injected at 1/4 of the dose, and no operation was performed in the test group and the negative control group. On the third day of the test, each group was only
The animals were intraperitoneally injected with 0.5 mL of 0.5% starch physiological saline solution, and the mice were gently licked to make them evenly dispersed after the injection. Start of experiment
On the 4th and 7th day, the positive control group was injected with the positive control substance again at the 1/4 of the above set dose, and the test sample group and
The negative control group did not perform any operation.
8.4 Chicken red blood cell immunity
On day 8 of the experiment, each animal was intraperitoneally injected with 0.5 mL of a 5% chicken red blood cell suspension. After 8h~12h, the animals were sacrificed and the laparotomy was cut in the middle.
Wall skin, 2 mL of normal saline was injected into the abdominal cavity, the abdomen of the mouse was gently rubbed for 1 min, and 1 mL of the peritoneal washing solution was aspirated and dropped on 2 slides.
On top, put in a covered test box with wet gauze and transfer to a 37 ° C incubator for 30 min. The incubation is completed and rinsed in physiological saline to
Remove unattached cells. After drying, fix it in 1.1 acetone-methanol solution for 5 min, 4% (volume fraction) Giemsa-phosphate buffer
The solution was stained for 3 min, rinsed with running water, rinsed with distilled water and dried.
9 Calculation of results
Macrophages were counted under a microscope, and each piece was randomly counted at.200. The percentage of phagocytosis and swallowing of each animal were calculated according to formula (1) and formula (2).
Phage index.
Percentage of phagocytosis =
Number of macrophages that phagocytose chicken red blood cells
Total number of macrophages counted × 100%
(1)
Phagocytic index =
Total number of chicken red blood cells that are swallowed
Total number of macrophages counted
(2)
Note 1. If the drop is too dark, it can be decolorized with 1% HCl; if it is too light, it can be counterstained.
Note 2. If the liquid in the abdominal cavity is a bloody exudate, it should not be used.
10 result determination
When the test group and the negative control group showed statistically significant differences in the percentage of phagocytosis and the phagocytic index, the test substance was considered to be considered.
The quality has an effect on the phagocytic function of mouse macrophages. If the material is diluted with a gradient or its extract, the percentage of phagocytosis occurs.
The relationship between the rate and the dose effect on the phagocytic index suggests that there may be an effect on macrophage phagocytosis.
11 Reliability check
11.1 A positive control is to confirm the suitability of the test by using a substance that has been shown to promote or inhibit macrophage phagocytosis.
Suitability. When it is necessary to verify that the laboratory has the ability to perform this test and be able to carry out in-lab reproducibility and interlaboratory reproducibility evaluation
It is recommended to use a positive control for each test.
11.2 For data that is frequently performed on this test (frequency not less than once a month) and has a long-term positive control, it is confirmed that it has
For laboratories with the ability to repeat and accurately test positive results, positive control trials can be performed periodically (interval ≤ 6 months).
12 test report
The test report should contain the following information.