YY/T 0588-2017 English PDF (YYT0588-2017)

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YY/T 0588-2017: Flow Cytometer
YY/T 0588-2017
Flow cytometer
ICS 11.100
C44
People's Republic of China Pharmaceutical Industry Standard
Replacing YY/T 0588-2005
Flow cytometry
Released on.2017-12-05
2018-12-01 implementation
State Food and Drug Administration issued
Foreword
This standard was drafted in accordance with the rules given in GB/T 1.1-2009.
This standard is revised on the basis of YY/T 0588-2005 "Flow Cytometry", compared with YY/T 0588-2005, except for editing
The main technical changes outside the sexual modification are as follows.
---Modified normative references;
--- Modified the definition of "ploid";
--- Modified the "fluorescence sensitivity" name and requirements, changed to "fluorescence detection limit", increased the fluorescence of the corresponding channel fluorescein of other lasers
Light detection limit requirements;
--- Modified the name of "forward angular scattered light sensitivity" and changed to "forward angular scattered light detection limit";
---Modified the "instrument resolution" requirements, remove the half-width requirement, and specify the requirements of FSC, FITC, PE, other fluorescein meet
Manufacturer's requirements (see 4.5 Instrument Resolution Requirements);
--- Revised the "Surface Marker Detection Accuracy" requirement and increased the requirements of CD16/CD56 and CD19;
--- Modified the "repetition of surface marker detection" requirements, the coefficient of variation of CD3, CD4, CD8 positive percentage results
Subsection requirements and increase the requirements for measuring CD16/CD56 and CD19 (see 4.9 Repeatability requirements for surface marker detection);
--- Revised the "carrying pollution rate" requirement and changed it to no more than 0.5%;
--- Removed "instrument function";
--- Added GB 4793.9, YY 0648 security requirements (see 4.14 security);
--- Added GB/T 18268.1, GB/T 18268.26 electromagnetic compatibility requirements (see 4.15 electromagnetic compatibility);
--- Modified the "fluorescence detection limit" test method (see 5.2 fluorescence detection limit);
--- Modified the "fluorescence linear" test method (see 5.3 fluorescence linearity);
--- Modified the "forward angular scattered light detection limit" test method (see 5.4 forward angular scatter light detection limit);
--- Modified the "instrument resolution" test method (see 5.5 instrument resolution);
--- Revised the test method for carrying pollution rate (see 5.10 carrying pollution rate).
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 standard was proposed by the State Food and Drug Administration.
This standard is under the jurisdiction of the National Medical Clinical Laboratory and the In vitro Diagnostic System Standardization Technical Committee (SAC/TC136).
This standard is mainly drafted by. Beijing Medical Device Inspection Institute, Beckman Coulter Trade (China) Co., Ltd., BD Medical Devices
(Shanghai) Co., Ltd., Shenzhen Mindray Biomedical Electronics Co., Ltd., Aisen Bio (Hangzhou) Co., Ltd.
The main drafters of this standard. Song Wei, Liu Qiuyue, Li Weigong, Wu Hao, Wu Jian.
This standard replaces YY/T 0588-2005.
Flow cytometry
1 Scope
Logos, labels and instructions for use, packaging, transportation and storage.
This standard applies to the biochemical and biophysical properties of the surface and internal surfaces of single cells or other non-biological particles used in clinical practice.
Flow cytometry for quantitative analysis and sorting of components (only for flow cytometry with sorting function).
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 191 packaging storage and transportation icon
Safety of electrical equipment for measurement, control and laboratory use - Part 1. General requirements
GB 4793.9 Safety requirements for electrical equipment for measurement, control and laboratory use - Part 9. Laboratory analysis and other purposes
Special requirements for moving and semi-automatic equipment
GB/T 14710 Medical electrical equipment environmental requirements and test methods
GB/T 18268.1 Electromagnetic compatibility requirements for electrical equipment - Part 1 . General requirements
GB/T 18268.26 Electromagnetic compatibility requirements for electrical equipment for measurement, control and laboratory - Part 26. Particular requirements
External diagnostic (IVD) medical equipment
GB/T 29791.3 Information provided by in vitro diagnostic medical device manufacturers (labeling) Part 3. Professional in vitro diagnostic equipment
Safety of electrical equipment for measurement, control and laboratory - Part 2-101. In vitro diagnostic (IVD) medical equipment
Special requirements
3 Terms and definitions
The following terms and definitions apply to this document.
3.1
Resolution resolution
The maximum precision that flow cytometry can achieve when measuring.
3.2
Fluorescence detection limit sensitivityoffluorescence
The minimum number of fluorescent molecules that can be detected by flow cytometry. Flow cytometry fluorescence detection limit with MESF (moleculesofequiva-
Lentsolublefluorochrome) means that the same amount of soluble fluorescent molecules.
3.3
Scattered light scatter
When the cell meets the laser in the liquid stream, it scatters light in all directions of the 360° solid angle of the space, called scattered light.
3.4
Forward scatter light forwardscatter, FSC
The scattered light detected directly in front of the incident light is called forward-angled scattered light, and the intensity of the forward-angled scattered light is related to the size of the cell.
3.5
Lateral angular scattered light sidescatter, SSC
Also called 90° scattered light. Lateral angular scattered light is more sensitive to the refractive index of cell membrane, cytoplasm and nuclear membrane, and also for larger particles in cytoplasm.
There will be reactions that give information about the fine structure and particle properties inside the cell.
3.6
Forward angular scatter light detection limit FSCsensitivity
The minimum particle size that can be detected by a flow cytometer is the smallest particle size that can be detected by previously scattered angular light.
3.7
Ploid polity
The genetic material content of biological cells (including animal cells, plant cells, microorganisms).
3.8
Carrying pollution rate carry-over
The analyte is carried by the instrument from one test sample to the next, thereby erroneously causing the second sample to be analyzed.
The increase in degrees.
3.9
Standard microsphere standardparticle
Microspheres of uniform size and/or labeled with consistent, constant fluorescein for calibration by flow cytometry.
4 Technical requirements
4.1 Normal working conditions
The normal working conditions of the flow cytometer should meet the following requirements.
a) Ambient temperature. as specified in the flow cytometry manual;
b) Relative humidity. as specified in the flow cytometry manual;
c) Power supply voltage. AC 220V ± 22V, 50Hz ± 1Hz;
d) Atmospheric pressure. as specified in the flow cytometer instructions;
e) Protect from direct sunlight and heat sources.
4.2 Fluorescence detection limit
The fluorescence detection limit of flow cytometry should meet the following requirements.
a) The fluorescence detection limit of flow cytometry for fluorescein isothiocyanate (FITC) should be no more than.200MESF;
b) The fluorescence detection limit of phycoerythrin (PE) by flow cytometry should be no more than 100MESF;
c) Flow cytometry for the channel corresponding to other lasers (such as red laser, violet laser, ultraviolet laser, green laser)
The fluorescence detection limit of at least one fluorescein in the optics should be in accordance with the manufacturer's stated requirements.
4.3 Fluorescence linearity
The linear correlation coefficient (r) of the fluorescence intensity of the flow cytometer should not be lower than 0.98.
4.4 Forward scatter light detection limit
The detection limit of forward scatter light by flow cytometry should be no more than 1 μm.
4.5 Instrument resolution
The full peak width of the fluorescence channel of the forward angulating light and fluorescent signal of the flow cytometer should meet the requirements of Table 1.
Table 1 Instrument resolution requirements
Fluorescein requirement (CV)
FSC ≤3.0%
FITC ≤3.0%
PE ≤3.0%
Other fluorescein meets manufacturer's requirements
4.6 Forward angle scattered light and side angle scattered light resolution
4.6.1 It should be possible to separate red blood cells and platelets from peripheral blood.
4.6.2 It should be possible to separate the three groups of peripheral blood leukocytes (lymphocytes, monocytes, granulocytes).
4.7 ploidy analysis linearity
When the flow cytometry is used for diploid cell cycle analysis, the ratio of the average fluorescence intensity of G2/M to G0/G1 should be 1.95~2.05.
Inside.
4.8 Surface marker detection accuracy
Percentage of CD3, CD4, CD8, CD16/CD56 and CD19 positive percentages expressed on the surface of lymphocytes when tested by flow cytometry
Should be within the given range.
4.9 Repeatability of surface marker detection
The coefficient of variation (CV) for repeated detection of CD3, CD4, CD8, CD16/CD56 and CD19 positive percentage results should be consistent with.
a) When the positive percentage is greater than or equal to 30%, the CV value should be no more than 8%; or
b) When the percentage of positive is less than 30%, the CV value should be no more than 15%.
4.10 Carrying pollution rate
The carrying contamination rate of the flow cytometer should be no more than 0.5%.
4.11 Instrument stability
When the ambient temperature does not change by more than 5% of the set temperature, the forward scattered light (FSC) and all fluorescent channel peaks are detected within 8 hours.
The fluctuation of the number of tracks should not exceed ±10%.
4.12 Appearance
The appearance should meet the following requirements.
a) The appearance of the instrument should be neat, without scratches, and the text and logo should be clear;
b) The fastener connection should be firm and reliable and must not be loose.
4.13 Environmental test
It should meet the requirements of Group I of the climate environment in GB/T 14710 and Group I of the mechanical environment.
4.14 Security
Should meet the requirements of the applicable provisions of GB 4793.1, GB 4793.9, YY 0648.
4.15 Electromagnetic Compatibility
Should comply with the provisions of the applicable provisions of GB/T 18268.1, GB/T 18268.26.
5 Test methods
5.1 Test conditions
Perform the test conditions according to 4.1, using reagents, controls and standard microspheres adapted to the flow cytometer, and follow the test before the test.
The manufacturer's instructions for proper operation and calibration of the flow cytometer.
5.2 Fluorescence detection limit
The standard microspheres are thoroughly mixed and tested on the machine, and no less than 10,000 standard microspheres are collected, and the test results are subjected to histograms.
Analysis, the average fluorescence intensity of each peak is obtained; the equivalent amount of MESF of each peak provided according to the standard microsphere specification, and the analysis
The average fluorescence intensity of each peak is determined by linear regression using the common logarithm (Lg value), and the average fluorescence intensity value at the blank microsphere corresponds to the MESF.
The number of objects is the fluorescence detection limit.
5.3 Fluorescence linearity
The standard microspheres are thoroughly mixed and then tested on the machine to collect not less than 10,000 microspheres. The flow cytometry uses a histogram to test
Analysis of the results, the average fluorescence intensity of each peak was obtained; the equivalent amount of MESF of each peak provided according to the standard microsphere specification, and analysis
The average fluorescence intensity of each peak obtained was linearly regressed by the MESF number (y) and the average fluorescence intensity (x), and the correlation coefficient (r) was calculated.
5.4 Forward scatter light detection limit
The standard microspheres are thoroughly mixed and then tested on the machine to check the peak signal displayed on the histogram and the standard microspheres showing the peak signal.
diameter.
5.5 Instrument resolution
The standard microspheres were thoroughly mixed and tested on the machine to calculate the CV value of the full peak width of the standard microspheres of each fluorescent channel. The results should be consistent with
4.5 requirements.
5.6 Forward angle scattered light and side angle scattered light resolution
5.6.1 Add 5 μL of sodium citrate anticoagulated whole blood to the test tube containing 1 mL of sheath fluid, mix it, and check it on the machine to check the forward scatter light.
And whether the lateral angle scattering spot map can separate platelets from red blood cells.
5.6.2 Take 100μLEDTA anticoagulated whole blood, dissolve the red blood cells and check on the machine. Check the forward scatter light and the side scatter light spot map.
Whether the three groups of white blood cells (lymphocytes, monocytes, granulocytes) can be separated.
5.7 ploidy analysis linearity
The average fluorescence intensity of G0/G1 phase and G2/M phase was detected by standard fluorescent cell staining or standard cell nucleus.
Calculate the ratio of the two average fluorescence intensities.
5.8 Surface marker detection accuracy
Using a quality control cell-on-board test with labeled CD3, CD4, CD8, CD16/CD56, and CD19, repeat the assay 5 times, recording each
Percentages of CD3, CD4, CD8, CD16/CD56, and CD19 positive values were tested and the mean values were calculated separately.
5.9 Repeatability of surface marker detection
Test according to the method of 5.8, repeat the test 10 times, and calc...