CN111829940B

CN111829940B - Detection of Pig-a Gene Mutation in Rat Peripheral Blood by Flow Cytometry

Info

Publication number: CN111829940B
Application number: CN202010715818.7A
Authority: CN
Inventors: 陈锦瑶; 霍娇; 张立实; 刘运杰; 马思佳; 曾珠; 岳茜岚
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-07-23
Filing date: 2020-07-23
Publication date: 2022-03-22
Anticipated expiration: 2040-07-23
Also published as: CN111829940A

Abstract

Aiming at the technical problems existing in the prior art, the present invention provides a flow cytometry detection method for pig peripheral blood pig-a gene mutation test, which comprises the following steps: step 1: collecting target tissue cells; step 2: surface antigen Dyeing; Step 3: Dyeing with nucleic acid dye application solution; Step 4: After dyeing, use a dual-laser flow cytometer to detect the sample to obtain data. The assay uses forward scatter (FSC) and side scatter (SSC) to differentiate red blood cells, SYTO 13 to label reticulocytes, CD59-allophycocyanin (APC) to label mutant cells, Provided is a more convenient and economical flow cytometry detection method for pig peripheral blood pig-a gene mutation test in rats.

Description

Flow cytometry detection method for rat peripheral blood Pig-a gene mutation test

Technical Field

The invention relates to the technical field of toxicological safety evaluation genotoxicity tests, and in particular relates to a flow cytometry detection method for rat peripheral blood Pig-a gene mutation test.

Background

The Pig-a gene mutation test is a genotoxicity test reflecting the in vivo gene mutation end point established and developed in recent years, and the mutation level of a subject after being exposed to a mutagenic substance is determined mainly by detecting the expression level of a mutant cell surface marker. In mammals, the Pig-a gene is located on the X chromosome and encodes the N-acetylglucosaminyltransferase complex, which is primarily involved in the synthesis of Glycosylphosphatidylinositol (GPI). When the Pig-a gene is mutated, GPI cannot be normally synthesized, so that the expression of cell surface proteins (CD59, CD55 and the like) requiring GPI anchorage is lost, and in this case, monoclonal antibodies with fluorescent groups can be used for labeling and then detecting the protein by flow cytometry. The Pig-a gene mutation test has the advantages of small sampling amount, small damage to animals, capability of cross-species and cross-tissue detection and the like, and the application of the Pig-a gene mutation test can make up some defects of the existing genetic toxicology test and plays a vital role in the evaluation of genetic toxicity.

At present, two main methods for detecting the peripheral blood Pig-a gene mutation test of rats at home and abroad by flow cytometry are available. One is to use Forward Scatter (FSC) and Side Scatter (SSC) to distinguish erythrocytes, SYTO 13 to recognize reticulocytes, and CD 59-Phycoerythrin (PE) to recognize mutant cells. The other is that the mutant cell is identified by HIS49-biotin, reticulocyte by CD71-PE, and Fluorescein Isothiocyanate (FITC) by CD 59. The two methods involve complicated operation and high cost: the emission spectra of the fluorescent dye combination are overlapped, so that the operation steps of the method are increased, and the sensitivity is lower; some methods require destruction or removal of leukocytes to eliminate interference, making the procedure cumbersome; the existing two methods have high detection cost.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a flow cytometry detection method for rat peripheral blood Pig-a gene mutation test, which uses FSC and SSC to distinguish erythrocytes, SYTO 13 marks reticulocytes, and CD 59-Allophycocyanin (APC) marks mutant cells, and provides a simpler, more convenient and more economical flow cytometry detection method for rat peripheral blood Pig-a gene mutation test.

The technical scheme for solving the technical problems is as follows: a flow cytometry detection method for rat peripheral blood Pig-a gene mutation test comprises the following steps:

the method comprises the following steps: collecting target tissues, namely collecting peripheral blood of a tested rat; collecting peripheral blood which is naturally flowing venous blood or arterial blood, uniformly mixing with 1000U/mL heparin sodium solution to prevent blood coagulation, wherein the volume ratio of the peripheral blood to the 1000U/mL heparin sodium solution is 5:1, and then placing the mixed peripheral anticoagulated blood in a dark environment at 4 ℃ for preservation to be detected.

Step two: surface antigen staining, namely mixing and staining a sample to be detected and antibody application liquid; if a double-dyeing scheme is adopted for dyeing, taking the peripheral anticoagulation blood in the step one as a sample to be detected, mixing the sample to be detected with the antibody application liquid according to the volume ratio of 1:5, uniformly mixing, and placing in a dark environment at 4 ℃ for dyeing for 30 minutes; PBS containing 3. mu.g of CD59-APC and 2% fetal bovine serum per 100. mu.L of antibody application solution; if a three-dyeing scheme is adopted for dyeing, taking the peripheral anticoagulation blood in the step one as a sample to be detected, mixing the sample to be detected with the antibody application liquid according to the volume ratio of 1:5, uniformly mixing, and placing in a dark environment at 4 ℃ for dyeing for 30 minutes; each 100. mu.L of the antibody application solution contained 3. mu.g of CD59-APC, 1. mu.g of CD61-PE, and 2% fetal bovine serum in PBS.

Step three: mixing the sample with a nucleic acid dye application solution for dyeing; after the surface antigen staining is finished, centrifuging for 5 minutes by using PBS and cleaning once, wherein the rotating speed is 300 g/min; discarding the supernatant, collecting the bottom layer cells, uniformly mixing and dyeing by using a nucleic acid dye application solution, mixing the sample to be detected and the nucleic acid dye application solution according to the volume ratio of 1:50, wherein the nucleic acid dye application solution contains 150nmol/LSYTO 13 PBS, incubating for 30 minutes at 37 ℃ in a dark place, transferring the sample to a flow cell tube after incubation, and storing at 4 ℃ in the dark place for detection;

step four: after dyeing is finished, detecting the sample by using a double-laser flow cytometer to obtain data; if the double-staining scheme is used in the second step, the flow cytometer specifically comprises the following steps: a1, determining erythrocyte population by using FSC and SSC, and excluding thrombocyte, cell debris and adhesion; b1, gating by using a FITC channel signal to eliminate noise and leukocyte interference; c1, determining different cell populations by using an APC channel and a FITC channel, wherein the APC channel is marked with CD59, negative expression represents mutation, the FITC channel acquires a SYTO 13 fluorescent signal and marks RNA; d1, adjusting the position of the crossdoor by using the simulated mutant cell sample as a reference; if the third staining scheme is used in the second step, the flow cytometer specifically comprises the following steps: a2, determining erythrocyte population by using FSC and SSC, and excluding thrombocyte, cell debris and adhesion; b2, gating by using a FITC channel signal to eliminate noise and leukocyte interference; c2, setting a gate by using a PE channel to further eliminate the adhesion platelets with different degrees; d2, determining different cell populations by using an APC channel and a FITC channel, wherein the APC channel is marked with CD59, negative expression represents mutation, and the FITC channel acquires a SYTO 13 fluorescent signal and marks RNA; e2, adjusting the position of the crossdoor using the mock mutant cell sample as a reference.

After gating, APC +/SYTO 13+ is a wild reticulocyte group, APC +/SYTO 13-is a wild mature erythrocyte group, APC-/SYTO 13-is a mutant reticulocyte group, and APC-/SYTO 13-is a mutant mature erythrocyte group; the test analysis indexes are Reticulocyte (RET) mutation rate, mature erythrocyte group (RBC) mutation rate and RET ratio; detecting at least 1 × 10 per sample⁶RBC in mature Red blood cells^CD59-0.3X 10⁶RET in reticulocytes^CD59-The number of (2); wherein RET represents reticulocyte group, RBC represents mature erythrocyte group, RET^CD59-Represents a mutant reticulocyte population, RBC^CD59-Represents the mutant mature red blood cell group, and the specific calculation formula is as follows:

in the formula, UL, UR, LL and LR are the cell numbers of the upper left quadrant, the upper right quadrant, the lower left quadrant and the lower right quadrant in the cross gate respectively, and RBC and RET mutation rates are expressed by the number of mutant cells in each million cells.

Preferably, the minimum sample to be tested used in the second step is 20 μ L, so as to ensure that the number of target cells is sufficient.

Preferably, in the fourth step, a mock mutant cell control sample is prepared for setting a gate reference in each detection of the double staining scheme; the manufacturing method comprises the following steps: taking any negative control group of anticoagulated blood and PBS with the volume ratio of 1:5, wherein the PBS contains 2% fetal calf serum, uniformly mixing, and placing in a dark place at 4 ℃ for 30 minutes; centrifuging and washing the solution for 5 minutes by using 10mL PBS per 100 mu L PBS at the rotating speed of 300 g/min; discarding supernatant, collecting bottom layer cells, mixing a sample with a nucleic acid dye application solution according to a volume ratio of 1:50, incubating the nucleic acid dye application solution containing 150nmol/L of PBS (phosphate buffer solution) of SYTO 13 at 37 ℃ for 30 minutes in a dark place, sucking 90 mu L of the control sample and 210 mu L of the same animal source bichromal sample after incubation, mixing the samples uniformly, transferring the samples to a flow cell tube for storage at 4 ℃ in a dark place for detection, wherein the proportion of the control mutant cells is 30 percent, and the expected mutation rate is 300000 multiplied by 10^-6。

Preferably, the dual laser flow cytometer used in step four is a dual laser flow cytometer having 488nm and 633nm lasers.

The invention has the beneficial effects that: the invention provides a flow cytometry detection method for rat peripheral blood Pig-a gene mutation test. The method is simple and convenient to operate, low in cost, capable of identifying small-amplitude changes relative to the background, high in sensitivity and stability, and capable of providing a detection method which is high in efficiency, simple and convenient, high in stability and cost-saving for safety evaluation of food/health food, medicines, cosmetics, pesticides and the like.

Drawings

FIG. 1 is a schematic diagram of the flow gating method and the results (double-staining) of the Pig-a gene mutation test of the present invention;

FIG. 2 is a schematic diagram of the flow gating method and the results (triple staining) of the Pig-a gene mutation test of the present invention;

FIG. 3 is a double staining protocol of the invention RBC^CD59-Detecting a linear regression trend schematic diagram;

FIG. 4 is a drawing showingDouble staining protocol RET of the invention^CD59-And detecting a linear regression trend schematic diagram.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

The embodiment discloses a flow cytometry detection method for rat peripheral blood Pig-a gene mutation test, which comprises the following steps:

Step three: mixing the sample with a nucleic acid dye application solution for dyeing; after the surface antigen staining is finished, centrifuging for 5 minutes by using PBS and cleaning once, wherein the rotating speed is 300 g/min; discarding the supernatant, collecting the bottom layer cells, uniformly mixing and dyeing by using a nucleic acid dye application solution, mixing the sample to be detected and the nucleic acid dye application solution according to the volume ratio of 1:50, wherein the nucleic acid dye application solution contains 150nmol/LSYTO 13 PBS, incubating for 30 minutes at 37 ℃ in a dark place, transferring the sample to a flow cell tube after incubation, and storing at 4 ℃ in the dark place for detection.

Step four: after dyeing is finished, detecting the sample by using a double-laser flow cytometer to obtain data; if the double staining scheme is used in the second step, as shown in fig. 1, the flow cytometer specifically comprises the following steps: a1, determining erythrocyte population by using FSC and SSC, and excluding thrombocyte, cell debris and adhesion; b1, gating by using a FITC channel signal to eliminate noise and leukocyte interference; c1, determining different cell populations by using an APC channel and a FITC channel, wherein the APC channel is marked with CD59, negative expression represents mutation, the FITC channel acquires a SYTO 13 fluorescent signal and marks RNA; d1, adjusting the position of the crossdoor by using the simulated mutant cell sample as a reference; in FIG. 1, A-D are gating processes, wherein A, B is gated according to FSC and SSC with red blood cells inside the gate and excluding adhesion, cell debris, etc.; c is cells that exclude SYTO 13 hyper-staining, mainly leukocytes; d is CD59-APC and SYTO 13 scatter after gating. E is a positive result chart, and a large number of mutant cells are seen in the lower left and lower right quadrants. F is a simulated mutant cell map, which provides a specific range of negative cell populations. If the third staining scheme is used in the second step, as shown in fig. 2, the flow cytometer specifically comprises the following steps: a2, determining erythrocyte population by using FSC and SSC, and excluding thrombocyte, cell debris and adhesion; b2, gating by using a FITC channel signal to eliminate noise and leukocyte interference; c2, setting a gate by using a PE channel to further eliminate the adhesion platelets with different degrees; d2, determining different cell populations by using an APC channel and a FITC channel, wherein the APC channel is marked with CD59, negative expression represents mutation, and the FITC channel acquires a SYTO 13 fluorescent signal and marks RNA; e2, adjusting the position of the crosshatch using the mock mutant cell sample as a reference, in fig. 2, a-E are gating processes, wherein A, B is gating according to FSC and SSC, red blood cells are in the gate, and adhesion, cell debris, etc. are excluded; c is to eliminate strong positive and weak positive cell groups of PE, mainly adherent platelets of different degrees; d is cells that exclude SYTO 13 high-staining, mainly leukocytes; e is a post-gated CD59-APC and SYTO 13 scattergram. F is a positive result chart, and a large number of mutant cells are seen in the lower left and lower right quadrants. G is a simulated mutant cell map, which provides a specific range of negative cell populations.

After gating, APC +/SYTO 13+ is wild reticulocyte population, APC +/SYTO 13-is wild mature erythrocyte population, and APC-/SYTO 13-is mutantThe reticulocyte group is changed, APC-/SYTO 13-is a mutant mature erythrocyte group; the experimental analysis indexes are RET mutation rate, RBC mutation rate and RET proportion; detecting at least 1 × 10 per sample⁶RBC in mature Red blood cells^CD59-0.3X 10⁶RET in reticulocytes^CD59-The number of (2); wherein RET represents reticulocyte group, RBC represents mature erythrocyte group, RET^CD59-Represents a mutant reticulocyte population, RBC^CD59-Represents the mutant mature red blood cell group, and the specific calculation formula is as follows:

wherein UL, UR, LL, LR are the cell numbers of the upper left, upper right, lower left, lower right quadrants of the phylum crossroads of FIGS. 1D and 2E, respectively, and the RBC and RET mutation rates are expressed as the number of mutant cells per million cells.

Example 1

Sample collection

A5-week-old SPF SD male rat is taken, and the weight of the rat is 150-160 g. The toxicant exposure dose of the test object A is 0, 20, 40, 80 and 160mg/kg.bw/d, the solvent is pure water, the stomach is drenched and toxicant exposure is continuously carried out for 28 days, and the peripheral blood is collected for detection respectively on 1 day, 14 days and 28 days before the test.

When blood is collected, the tail of the patient is wiped by an alcohol cotton ball to fill the blood vessel at the tail part, and the blood vessel is punctured by a syringe needle in the direction parallel to the blood vessel. 50 μ L of the naturally flowing blood was collected in a centrifuge tube (containing 10 μ L of heparin sodium solution) and immediately mixed to prevent clotting. After blood collection, animals should be disinfected and stopped bleeding. And placing the collected peripheral anticoagulation blood in a refrigerator at 4 ℃ in a dark place for testing.

Reagent preparation

APC mouse anti-rat CD59 was purchased from BD, usa; PE mice anti-rat CD61, SYTO 13 were purchased from Thermo Fisher Scientific, USA; fetal bovine serum was purchased from Shanghai Simon Gene Biotechnology, Inc.; PBS was purchased from Hyclone, USA; heparin sodium was purchased from Sigma-Aldrich, usa.

Cell staining

(1) 20 mu L of sample to be detected is added into 100 mu L of staining system (the staining system contains 1 mu g of CD61-PE, 3 mu g of CD59-APC and 2 mu L of fetal calf serum), mixed evenly and placed at 4 ℃ for shading and staining for 30 minutes.

(2) After the surface antigen staining was complete, the cells were transferred to a 15mL centrifuge tube (containing 10mL PBS) and washed once by centrifugation at 300g/min for 5 minutes. The supernatant was discarded, the bottom layer cells (about 20. mu.L) were collected, resuspended in a centrifuge tube containing 1mL of SYTO 13 application solution (150nmol/L), mixed well and incubated at 37 ℃ for 30 minutes in the dark.

(3) After incubation, the samples were transferred to a flow cell tube and stored at 4 ℃ in the dark for examination.

Flow cytometry detection

Data collection and analysis was performed using a flow cytometer FACSVerse, BD company, usa, using the Software BD facsuie Software Bundle v 1.0. A blank control, a mock mutation control, PE and SYTO 13 single stain controls were prepared prior to detection. The specific door setting steps are as follows: determining erythrocyte population by using FSC and SSC, and removing platelets, cell fragments and adhesion; setting a gate by using an FITC channel signal to eliminate noise and leukocyte interference; thirdly, a PE channel is used for arranging a door to further remove the adhesion platelets with different degrees; determining different cell populations by using an APC channel (marked CD59, negative expression represents mutation) and a FITC channel (obtaining SYTO 13 fluorescent signal and marking RNA); and fifthly, using the simulated mutant cell sample as a reference to adjust the position of the cross gate. The experimental analysis indexes are RET mutation rate, RBC mutation rate and RET ratio. Detecting at least 1 × 10 per sample⁶RBC in mature Red blood cells^CD59-0.3X 10⁶RET in reticulocytes^CD59-The number of the cells.

The result of the detection

The results are shown in table 1:

table 1 test article a Pig-a gene mutation test results (n ═ 6,

)

note: statistical differences compared to the solvent control group, p < 0.05.

Evaluation of results

In this example, the test group had significantly increased RBC and RET mutation rates compared to the control group, and had an obvious dose-response relationship, which could be confirmed as a positive result. In this example, a small change in mutation rate from background levels was observed at lower exposure doses, indicating a higher sensitivity of the method of the invention.

Example 2

Sample collection

A5-week-old SPF SD male rat is taken, and the weight of the rat is 150-160 g. The toxicant dose of the test object B is 20, 40 and 80mg/kg. The stomach is infused and infected continuously for 3 days, and the peripheral blood is collected for detection on the 28 th day of the test.

Reagent preparation

APC mouse anti-rat CD59 was purchased from BD, usa; SYTO 13 was purchased from Thermo Fisher Scientific, USA; fetal bovine serum was purchased from Shanghai Simon Gene Biotechnology, Inc.; PBS was purchased from Hyclone, USA; heparin sodium was purchased from Sigma-Aldrich, usa.

Cell staining

(1) 20 mu L of sample to be detected is added into a 100 mu L staining system (the staining system contains 3 mu g of CD59-APC and 2 mu L of fetal calf serum), mixed evenly and placed at 4 ℃ for shading and staining for 30 minutes.

Flow cytometry detection

Data collection and analysis was performed using a flow cytometer FACSVerse, BD company, usa, using the Software BD facsuie Software Bundle v 1.0. A blank control and a mock mutation control were prepared prior to testing. The specific door setting steps are as follows: determining erythrocyte population by using FSC and SSC, and removing platelets, cell fragments and adhesion; setting a gate by using an FITC channel signal to eliminate noise and leukocyte interference; ③ use APC channel (labeled CD59, negative expression represents mutation) and FITC channel (acquisition of SYTO 13 fluorescence signal, labeled RNA) to identify different cell populations; and fourthly, using the simulated mutant cell sample as a reference to adjust the position of the crossdoor. The experimental analysis indexes are RET mutation rate, RBC mutation rate and RET ratio. Detecting at least 1 × 10 per sample⁶RBC in mature Red blood cells^CD59-0.3X 10⁶RET in reticulocytes^CD59-The number of the cells.

The result of the detection

The results are shown in table 2:

table 2 test article B Pig-a gene mutation test results (n-5,

)

note: statistical differences compared to the solvent control group, p < 0.05.

Evaluation of results

In this example, the test group had significantly increased RBC and RET mutation rates compared to the control group, and had an obvious dose-response relationship, which could be confirmed as a positive result. The double-dyeing scheme is adopted in the embodiment, the detection cost is low, the adjustment and compensation of a single-dyeing sample are not required, and the operation is simpler and more convenient.

The detection results of the above examples show that: the method is simple and convenient to operate and high in detection sensitivity. When the double-dyeing scheme is used, the cost is low; when the triple-staining scheme is used, small-amplitude mutation rate increase relative to the background can be identified, the detection sensitivity can be further improved, and the genetic toxicity of the tested object under low-dose exposure can be identified.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. The flow cytometry detection method of the Pig-a gene mutation test in rat peripheral blood, is characterized in that, it comprises the following steps:

Step 1: Collect the target tissue, collect the peripheral blood of the test rats; the collected peripheral blood is the natural flow venous blood or arterial blood, and is mixed with 1000 U/mL heparin sodium solution to prevent coagulation. The volume ratio of mL heparin sodium solution was 5:1, and then the mixed peripheral anticoagulant was stored at 4°C in a dark environment until testing;

Step 2: For surface antigen staining, mix the sample to be tested and the antibody application solution for staining; if a double staining scheme is used for staining, take the peripheral anticoagulant in step 1 as the sample to be tested and the antibody application solution at a volume ratio of 1:5 Mix, mix well and place in a dark environment at 4°C for staining for 30 minutes; each 100 μL of antibody application solution includes 3 μg of CD59-APC and 2% fetal bovine serum in PBS; if the three-staining scheme is used for staining, take the sample from step 1. Peripheral anticoagulation as the sample to be tested and the antibody application solution were mixed at a volume ratio of 1:5, and then placed in a dark environment at 4°C for staining for 30 minutes; each 100 μL of the antibody application solution included 3 μg of CD59-APC, 1 μg of CD61-PE and 2% fetal bovine serum in PBS;

Step 3: Mix and stain the sample with the nucleic acid dye application solution; after the surface antigen staining is completed, use PBS to centrifuge once for 5 minutes, and the speed is 300g/min; discard the supernatant, collect the underlying cells, and mix with the nucleic acid dye application solution for staining. The test sample and the nucleic acid dye application solution were mixed at a volume ratio of 1:50. The nucleic acid dye application solution contained 150 nmol/L SYTO13 in PBS, and incubated at 37 °C for 30 minutes in the dark. After incubation, the samples were transferred to a flow cytometry tube. and stored at 4 °C in the dark until inspection;

Step 4: After the staining, use the dual-laser flow cytometer to detect the sample to obtain data; if the double-staining scheme is used in Step 2, the specific gating steps of the flow cytometer are: a1. Use FSC and SSC to determine the red blood cell population , exclude platelets, cell debris, and adhesion; b1, use FITC channel signal gate to exclude noise and leukocyte interference; c1, use APC channel and FITC channel to determine different cell populations, where APC channel marks CD59, negative expression represents mutation, FITC channel acquisition SYTO13 fluorescence signal, labeled RNA; d1. Use the simulated mutant cell sample as a reference to adjust the position of the cross gate; if the three-staining scheme is used in step 2, the specific gate setting steps of the flow cytometer are: a2. Use FSC and SSC to determine the red blood cell population, Exclude platelets, cell debris and adhesion; b2. Use FITC channel signal gate to exclude noise and leukocyte interference; c2. Use PE channel to further exclude platelets with different degrees of adhesion; d2. Use APC channel and FITC channel to determine different cell populations, The APC channel marks CD59, negative expression represents mutation, and the FITC channel obtains the SYTO 13 fluorescent signal to label RNA; e2, use the simulated mutant cell sample as a reference to adjust the position of the cross gate;

For each test of the double staining protocol, a mock mutant cell control sample needs to be prepared for gate reference; the preparation method is: take any negative control group anticoagulation and PBS with a volume ratio of 1:5, in which PBS contains 2% Fetal bovine serum, mixed well and placed in the dark at 4 °C for 30 minutes; centrifuged every 100 μL of PBS with 10 mL of PBS for 5 minutes at a speed of 300 g/min; discarded the supernatant and collected the underlying cells. The nucleic acid dye application solution contains 150 nmol/L SYTO 13 in PBS, and incubate at 37 °C for 30 minutes in the dark. After the incubation, draw 90 μL of the control sample and mix it with 210 μL of the double-stained sample from the same animal. The cells were homogenized, transferred to a flow cytometer tube and stored at 4 °C in the dark until detection. The proportion of mutant cells in the control was 30%, and the expected mutation rate was 300000´10 ^-6 ;

After setting the gate, APC+/SYTO 13+ is the wild reticulocyte population, APC+/SYTO 13- is the wild mature red blood cell population, APC-/SYTO 13- is the mutant reticulocyte population, APC-/SYTO 13- is the mutant mature red blood cell population Red blood cell population; the test analysis indicators are reticulocyte mutation rate, mature red blood cell mutation rate and reticulocyte ratio; the number of mutant mature red blood cells in 1×10 ⁶ mature red blood cells is detected at least in each sample, and the number of mutant mature red blood cells in 0.3×10 ⁶ reticulocytes is detected. The number of mutant reticulocytes; the specific calculation formula is: mature red blood cell mutation rate × 10 ^-6 = mutant mature red blood cells / total mature red blood cells × 10 ⁶ ; reticulocyte mutation rate × 10 ^-6 = mutant reticulocytes / total reticulocytes Red blood cells×10 ⁶ ; reticulocyte ratio %=total reticulocytes/total red blood cells×100; the mutation rate of mature red blood cells and reticulocytes is expressed as the number of mutant cells per million cells.

2. The rat peripheral blood Pig-a gene mutation test flow cytometry detection method according to claim 1, wherein the sample to be tested adopted in the step 2 is at least 20 μL, to ensure the number of target cells adequate.

3. rat peripheral blood Pig-a gene mutation test flow cytometry detection method according to claim 2, is characterized in that, the double laser flow cytometer used in described step 4 is to have 488nm and 633nm laser dual-laser flow cytometer.