Quality control product for flow detection of leukocyte differentiation antigen CD34 and preparation method thereof

Abstract

The invention discloses a quality control product for flow detection of a leukocyte differentiation antigen CD34 and a preparation method, and relates to the technical field of quality control products for medical detection. According to the preparation method of the quality control product for flow cytometry of the leukocyte differentiation antigen CD34, provided by the invention, by adopting the microsphere coupled CD34 and CD45 proteins to replace genetically engineered cells and utilizing the physical stability and uniform particle size distribution of a microsphere material, the batch consistency of the quality control product is remarkably improved, and meanwhile, the efficacy period of the product is prolonged. Furthermore, in a peripheral blood treatment link, through sequence optimization of firstly fixing peripheral blood cells, then adding back plasma and finally mixing with the microspheres, stabilization of a cell membrane structure is facilitated, and release of endogenous interfering substances due to cell rupture is effectively avoided. The optimization of the sequence enables the quality control product to be closer to the real processing condition of a clinical sample, so that the specificity and the signal-to-noise ratio of a flow detection signal are improved, and the correlation between a quality control result and clinical actual detection is enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of quality control products for medical detection, and particularly to a quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 and a preparation method thereof. Background Art

[0002] Hematopoietic stem cell transplantation can reconstruct hematopoietic function, and has less tumor cell contamination. It can be widely used in the treatment of various malignant hematological diseases, solid tumors, genetic diseases, severe combined immunodeficiency diseases, etc. The sources of hematopoietic stem cells mainly include peripheral blood, bone marrow, umbilical cord blood, etc. Among them, the content of hematopoietic stem cells in umbilical cord blood is the most abundant. Due to its rich source, convenient collection, and increasingly simple storage and transplantation operation techniques, it has been increasingly valued and recognized by the majority of medical staff and patients.

[0003] Currently, the important indicators for evaluating hematopoietic stem cell transplantation include: the total number of nucleated cells and the total number of CD34 + cells. Among them, the number and functional status of CD34 + cells directly determine the transplantation effect, and its detection is mainly carried out by flow cytometry single-platform (absolute counting) or double-platform (counting the percentage and then combining with whole blood cell count) methods. The current detection system needs to rely on standardized quality control products for quality control to ensure the accuracy of experimental data and the comparability of results across laboratories.

[0004] Currently, the mainstream quality control products mainly rely on imports, and the manufacturers are mainly BD Company in the United States, Beckman Coulter Company in the United States, R&D Company in the United States, and Streck Company in the United States. However, the high cost of imported quality control products, complex transportation processes, and short remaining expiration dates after transportation limit their wide application, resulting in some laboratories being difficult to achieve normalized quality control management. The comparability of results across laboratories is poor and the quality is uncontrollable, thus affecting the judgment and curative effect of clinical hematopoietic stem cell transplantation. Although there have been domestic technologies attempting to develop quality control products by constructing cell lines through genetic engineering, there are deficiencies such as high dispersion of CD34 + cell population distribution and deviation of side scatter signal (SSC), further highlighting the technical bottleneck of the existing quality control system.

[0005] Technical specifications at home and abroad have put forward strict requirements for the stem cell detection process and quality control, especially setting clear standards for cell counting accuracy and stability. However, the performance limitations of existing quality control products not only increase the risk of fluctuations in detection results, but also may affect the scientific evaluation of transplantation effects in clinical practice. Therefore, developing a quality control product that can optimize cell distribution characteristics and improve detection stability has become a key requirement for improving the hematopoietic stem cell detection system. Summary of the Invention

[0006] The technical problem to be solved by the present invention is the problem of high dispersion of the CD34 cell population distribution and large side scatter signal (SSC) in existing domestic quality control products, and a quality control product, a preparation method and an application for flow cytometry detection of leukocyte differentiation antigen CD34 are provided. + To solve the above problems, the present invention proposes the following technical solutions:

[0007] In the first aspect, the present invention provides a preparation method for a quality control product for flow cytometry detection of leukocyte differentiation antigen CD34, comprising the following steps: S1. Take 20 μL - 200 μL of microspheres, after washing and activation, add 10 - 500 µL of CD34 protein and 10 - 500 µL of CD45 protein for incubation. After the incubation is completed, add a blocking agent. After washing the blocked microspheres, store them at 4°C to 8°C to obtain CD34 microspheres; S2. Collect peripheral blood from healthy adults and separate plasma and blood cells; S3. Fix the blood cells and add the plasma to obtain fixed peripheral blood; S4. Add the CD34 microspheres obtained in S1 to the peripheral blood obtained in S3 according to a preset ratio, and mix well to obtain a quality control product; S5. Determine the absolute number of CD34 microspheres and white blood cells in the quality control product obtained in S4.

[0008] A further technical solution thereof is that in the step S3, it further includes counting the white blood cells in the fixed peripheral blood so that the white blood cell concentration in the fixed peripheral blood is 4.0×10 9 ~ 10.0×10 9 cells / L.

[0009] A further technical solution thereof is that the operation of fixing the blood cells includes: Prepare 0.005 - 0.75% (w / v) CrCl3 solution and 0.1 - 0.5% (w / v) paraformaldehyde solution with a solvent in advance; Mix the CrCl3 solution and the blood cells at a volume ratio of 1:1, fix at 0 - 8°C for 5 min to 18 h, centrifuge to remove the supernatant, and wash with PBS to remove the CrCl3 solution; then mix the paraformaldehyde solution and the blood cells at a volume ratio of 1:1, fix at 0°C to 8°C for 16 - 22 h, centrifuge to remove the supernatant, and wash with PBS to remove the paraformaldehyde solution to obtain fixed blood cells.

[0010] A further technical solution thereof is that in the step S4, the preset ratio includes: in CD34 +In the low-value quality control product, the proportion of CD34 microspheres in peripheral blood white blood cells is 0.1% - 0.3%; CD34 + In the high-value quality control product, the proportion of CD34 microspheres in peripheral blood white blood cells is 0.3% - 0.6%.

[0011] A further technical solution thereof is that the operation of washing the microspheres includes: taking 20 μL - 200 μL of microspheres and adding them to 1 - 5 mL of MES coupling buffer, mixing evenly, rotating and incubating for 5 - 30 min, centrifuging to remove the supernatant, repeating the washing 1 - 3 times, and resuspending the microspheres in the MES coupling buffer.

[0012] A further technical solution thereof is that the operation of activating the microspheres includes: adding a reaction solution containing 10 μL - 200 μL of EDC and 10 μL - 200 μL of NHS to the washed microspheres, mixing evenly, and rotating and incubating at 37 °C for 10 - 30 min.

[0013] A further technical solution thereof is that in step S5, a flow cytometer is used to measure the proportion of CD34 microspheres in the quality control product and determine the target value, and a five-class blood cell counter is used to measure the absolute number of white blood cells in the quality control product and determine the target value.

[0014] A further technical solution thereof is that the microspheres are selected from silica carboxyl microspheres, polystyrene microspheres or magnetic microspheres, the particle size of the microspheres is 3 - 20 μm, preferably 5 - 15 μm, and more preferably 5 - 13 μm.

[0015] In a second aspect, the present invention provides a quality control product for flow cytometry detection of leukocyte differentiation antigen CD34, which is prepared by the method described in the first aspect.

[0016] The present invention also provides the quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 described above, or the quality control product prepared by the preparation method of the quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 described in the first aspect in the preparation of a kit for detecting CD34 + cells.

[0017] Compared with the prior art, the technical effects that the present invention can achieve include: The preparation method of the quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 provided by the present invention uses microsphere-coupled CD34 and CD45 proteins to replace genetically engineered cells, avoiding the limitations of the traditional technology that rely on complex cell culture and gene modification. The directional coupling process of the proteins on the microsphere surface not only simplifies the preparation process and reduces the biosafety risk, but also significantly improves the batch-to-batch consistency of the quality control product through the physical stability of the microsphere material and the uniform particle size distribution, and at the same time extends the product shelf life, solving the problem of performance instability caused by the attenuation of the activity or the variation of the epitope of traditional genetically engineered cells.

[0018] In addition, in the peripheral blood treatment step of the preparation method of the present invention, by optimizing the order of first fixing peripheral blood cells, then adding back plasma, and finally mixing with microspheres, it breaks through the defect in the prior art that direct mixing and then fixing easily damages cells. Fixing blood cells before mixing can reduce the influence of the solution and operation used in the mixing process on the protein on the surface of the microspheres, and avoid protein denaturation caused by pH changes or frequent operations reducing the amount of protein on the surface of the microspheres. The optimization of this order maintains the stability of the quality control product state, not only improving the specificity and signal-to-noise ratio of the flow cytometry detection signal, but also enhancing the correlation between the quality control result and the actual clinical detection.

[0019] Generally speaking, through the collaborative innovation of microsphere coupling and processing order, this method overcomes the technical bottlenecks such as cell dispersion, large SSC, and short shelf life of traditional CD34 + cells while retaining the biomimetic characteristics of clinical samples, and provides a standardized quality control product with stronger stability, simpler operation, and wider clinical applicability for CD34 flow cytometry detection. Brief Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It is a flowchart of the preparation method of the quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 provided by the embodiment of the present invention; Figure 2 It is the stability test results of low-value quality control and high-value quality control prepared by the embodiment of the present invention; Figure 3 It is the test results of the control group of the low-value quality control product produced by BD Company; Figure 4 It is the test results of the experimental group of the low-value quality control product produced by BD Company; Figure 5 It is the test results of the control group of the high-value quality control product produced by BD Company; Figure 6 It is the test results of the experimental group of the high-value quality control product produced by BD Company; Figure 7 It is the test results of the control group of the KRC2201 low-value quality control product; Figure 8 It is the test results of the experimental group of the KRC2201 low-value quality control product; Figure 9 It is the test results of the control group of the KRC2201 high-value quality control product; Figure 10 The test results of the experimental group of the KRC2201 high-value quality control product; Figure 11 The test results of the control group of the low-value quality control product prepared in this embodiment; Figure 12 The test results of the experimental group of the low-value quality control product prepared in this embodiment; Figure 13 The test results of the control group of the high-value quality control product prepared in this embodiment; Figure 14 The test results of the experimental group of the high-value quality control product prepared in this embodiment. Specific implementation manners

[0022] The present invention will be further described below by way of specific application examples, but the present invention is not limited to the specific application example scope described herein. For the experimental methods without specific conditions in the following specific application examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications. If the temperature is not particularly emphasized, the reaction is usually carried out at room temperature, and the room temperature in the present invention refers to 16°C to 30°C.

[0023] Unless otherwise specified, the experimental methods used in the following application examples are all conventional methods.

[0024] Unless otherwise specified, the materials and reagents used in the following application examples can all be obtained commercially. The CD34 protein and CD45 protein used in the examples are both commercially available human recombinant CD34 protein and human recombinant CD45 protein, and are both His-tagged proteins expressed in HEK293 cells.

[0025] The embodiment of the present invention provides a preparation method of a quality control product for flow cytometry detection of leukocyte differentiation antigen CD34, including the following steps: S1. Take 20 μL - 200 μL of microspheres, after washing and activation, add 10 - 300 μL of CD34 protein and 10 - 300 μL of CD45 protein for incubation. After the incubation is completed, add a blocking agent. After washing the blocked microspheres, store them at 4°C to 8°C to obtain CD34 microspheres; S2. Collect peripheral blood from healthy adults and separate plasma and blood cells; S3. Fix the blood cells and add the plasma to obtain fixed peripheral blood; S4. Add the CD34 microspheres obtained in S1 to the peripheral blood obtained in S3 according to a preset ratio, and mix well to obtain a quality control product; S5. Determine the values of the CD34 microspheres and the absolute number of white blood cells in the quality control product obtained in S4.

[0026] For further referenceFigure 1 , which is a flowchart of a preparation method of a quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 provided by an embodiment of the present invention.

[0027] The following details each step: S11. Cleaning of silica carboxyl microspheres Take 40 μL of silica carboxyl microspheres with a diameter of 5 μm and add them to 1 mL of coupling buffer MES (50 mM, pH 6.0). Pipette and mix well, incubate with rotation at room temperature for 10 min, centrifuge to remove the supernatant (6000 rpm, 10 min), repeat the cleaning once, and finally resuspend the silica carboxyl microspheres in 500 μL of coupling buffer.

[0028] S12. Activation of silica carboxyl microspheres Slowly add 20 μL of freshly prepared 50 mg / mL EDC and 20 μL of 50 mg / mL NHS reaction solution to the centrifuge tube. Pipette and mix well, incubate with rotation at 37 °C for 15 min.

[0029] S13. Coupling of microspheres with proteins After the incubation, centrifuge to remove the supernatant, wash once with 1 mL of buffer MES, add 80 μL of 1 mg / mL CD34 protein and 40 μL of 1 mg / mL CD45 protein, and incubate with rotation at 37 °C for 2 hours.

[0030] S14. Blocking of surface groups of microspheres After the coupling incubation, centrifuge to remove the supernatant, then add 500 μL of blocking solution 2% BSA to the centrifuge tube for blocking, and incubate with rotation at 37 °C for 1 hour.

[0031] S15. Cleaning and preservation of microspheres Centrifuge the blocked microspheres to remove the supernatant, wash once with 1 mL of blocking solution (2% BSA), and then resuspend the microspheres in 500 μL of PBS buffer, and store them refrigerated at 4 °C to 8 °C.

[0032] S21. Collection of human peripheral blood Use a blood collection tube with EDTA as an anticoagulant to collect 90 mL of adult peripheral blood, and transfer the peripheral blood to a 250 mL dispensing bottle that is mold-free, sterile, and pyrogen-free for mixing.

[0033] S22. Centrifugal separation of plasma and blood cells After mixing the peripheral blood, take 40 mL and transfer it to two 50 mL centrifuge tubes respectively, label them as low value and high value, centrifuge at 4 °C, 2000g for 10 min, aspirate the plasma corresponding to the low value and high value labels, and store them refrigerated at 2 to 8 °C for standby.

[0034] S31. Fixation of blood cells Twenty-four hours before fixation, prepare 0.1% (w / v) CrCl3 solution and 0.35% (w / v) paraformaldehyde solution using DPBS respectively.

[0035] Mix the CrCl3 solution with the centrifuged peripheral blood cells at a volume ratio of 1:1 and fix at 2 - 8°C for 1 h. Centrifuge at 4°C and 2000g for 10 min, discard the supernatant, wash the blood cells with DPBS twice and discard the supernatant; then mix the paraformaldehyde solution with the centrifuged peripheral blood cells at a volume ratio of 1:1 and fix at 2 - 8°C for 18 h. Centrifuge at 4°C and 2000g for 10 min, discard the supernatant, wash the blood cells with DPBS twice and discard the supernatant to obtain fixed blood cells.

[0036] Add back the low-value and high-value plasma to the corresponding fixed blood cells and mix well to obtain fixed peripheral blood (low-value, high-value).

[0037] S32. Counting of human peripheral blood leukocytes Take 2 mL each of the fixed high- and low-value peripheral blood, and use a five-part differential hematology analyzer to continuously count the white blood cells 15 times. The average white blood cell density of the 15 results is calculated as follows: low-value: 7.734×10 9 cells / L, CV: 1.59%; high-value: 7.681×10 9 cells / L, CV: 1.70%.

[0038] Take 30 µL of the microspheres conjugated with protein into 1 mL of PBS solution. Without adding cell dyes, the average microsphere concentration measured by the trypan blue counting method is 3.900 ×10 6 cells / mL.

[0039] S41. Mixing of microspheres and peripheral blood cells According to the low-value average white blood cell density of 7.734×10 9 cells / L and the microsphere density of 3.900×10 6 cells / mL, with the remaining volume of peripheral blood being 38 mL and the percentage of CD34 microspheres in total white blood cells in low-value quality control being 0.100%; for the high-value average white blood cell density of 7.681×10 9 cells / L and the microsphere density of 3.900×10 6 cells / mL, with the remaining volume of peripheral blood being 38 mL and the percentage of CD34 microspheres in total white blood cells in high-value quality control being 0.450%, calculate the microsphere feeding amounts in low-value and high-value quality controls respectively and conduct feeding. After thorough mixing, store at 2 - 8°C to obtain low-value quality control products and high-value quality control products.

[0040] S51, CD34 microspheres and absolute quantification of white blood cell count Take 2.5 mL of the above-mentioned CD34 low-value and high-value quality controls respectively + Perform 20 consecutive counts of white blood cells using a five-part differential hematology analyzer. The average white blood cell density of the 20 results is calculated as follows: low-value quality control: 7760 cells / μL, CV: 1.59%; high-value quality control: 7681 cells / μL, CV: 1.70%.

[0041] Perform 20 consecutive tests on the proportion of CD34 microspheres in the CD34 low-value and high-value quality controls using a flow cytometer. The average proportion of CD34 microspheres in the 20 results is calculated as follows: low-value quality control: 0.111% (0.089 - 0.132%); high-value quality control: 0.490% (0.447 - 0.532%).

[0042] Taking the silica carboxyl microspheres in the above examples as an example, the quality control product and its preparation method for flow cytometry detection of the leukocyte differentiation antigen CD34 of the present invention are introduced. In other examples, those skilled in the art can also select polystyrene microspheres or magnetic microspheres, etc., and the present invention does not limit this.

[0043] Experiment 1: Stability test Adopt the 120-day continuous test method (day 0, day 3, day 10, day 14, day 18, day 24, day 31, day 38, day 45, day 50, day 65, day 70, day 75, day 80, day 85, day 90, day 95, day 100, day 105, day 111, day 115, day 120) to test the changes in the proportion of CD34 microspheres in the low-value and high-value quality controls prepared in this example. The test results are shown in Figure 2 , it can be seen that from day 0 to day 120, all are within the target values (low-value quality control: 0.089 - 0.132%, high-value quality control: 0.447 - 0.532%). Therefore, the quality control product prepared in the examples of the present invention can be stably stored within 120 days, and the quality control validity period can reach 120 days.

[0044] Test method: a. Prepare 2 flow cytometry tubes for each of the low-value and high-value CD34 quality control blood samples. Mark one tube as the isotype control tube and the other as the experimental tube. Add 100 μL of CD34 quality control blood to each tube; b. Add 10 μL of FITC-CD45 antibody and 10 μL of PE-ISO antibody to the control tube, and add 10 μL of FITC-CD45 antibody and 10 μL of PE-CD34 antibody to the experimental tube. Gently shake and mix well, then place in a 4°C refrigerator and incubate in the dark for 30 min; c. After incubation, take out the quality control blood, add 1 mL of 1× hemolysin to each tube, mix well. Place in a 4°C refrigerator and hemolyze in the dark for 10 min; d. Take out the quality control blood, centrifuge at 500 g for 3 min, discard the supernatant, add 1 mL of 1× PBS to resuspend and mix well, centrifuge at 500 g for 3 min, and repeat 2 times; e. Discard the supernatant, add 300 - 500 μL of 1× PBS to resuspend and mix well, and wait for on-machine detection.

[0045] The quality control product prepared in the embodiment of the present invention is used for performance comparison with the quality control product produced by BD Company (Product Name: Stem CellControl CD34 + Whole Blood Process Control) and the product of Shenzhen Kenuo Medical Laboratory (KRC2201).

[0046] The test results of the quality control product produced by BD Company are shown in Figures 3 - 6 ; The test results of the KRC2201 quality control product are shown in Figures 7 - 10 ; The quality control product prepared in the embodiment of the present invention is shown in Figures 11 - 14 .

[0047] It can be seen from the flow cytometry diagram that in the low-value experimental group of the previous-generation product KRC2201 of our company, the distribution of CD34 + cells is significantly dispersed, and the SSC is significantly larger, indicating that the stability of its low-value quality control product is insufficient and the performance does not meet expectations.

[0048] In the high-value experimental group of KRC2201, the aggregation of the CD34 + cell population is slightly better than that of the low-value group, but the SSC is still large, indicating that the performance of the high-value quality control product is still unstable.

[0049] The low-value experimental group of the quality control product of the embodiment of the present invention shows that the distribution of the CD34 + cell population is highly concentrated, and the SSC value is significantly reduced, indicating that the performance control is better than that of the KRC2201 product, and the SSC value remains within the ideal range.

[0050] In the high-value experimental group, the CD34 + cell population of the high-value quality control product provided in the embodiment of the present invention still maintains tight aggregation, and the SSC value is also significantly reduced, indicating that its high-value quality control product has excellent stability.

[0051] The low-value quality control product and high-value quality control product provided by the embodiments of the present invention have a concentrated and uniform distribution of CD34 + cells, and the SSC value is stable. Compared with the quality control products of imported BD company and KRC2201 product, the CD34 + cell population in the embodiments of the present invention is significantly separated, making it easier to gate, which can significantly reduce the gating differences among different experimenters. The comprehensive performance completely exceeds that of the KRC2201 product, and it is also superior to the quality control products of imported BD company in terms of the validity period.

[0052] The above results show that the quality control products provided by the present invention significantly improve the concentration of CD34 + cell distribution under both low-value and high-value conditions, effectively reduce the SSC value, solve the key problems of the previous generation product (KRC2201), and have better performance. In addition, the validity period of the quality control products provided by the present invention can reach 120 days, and the stability is significantly improved.

[0053] In the above embodiments, the descriptions of each embodiment have their own focuses. For the parts not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0054] The above is the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of various equivalent modifications or substitutions, and these modifications or substitutions should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A preparation method of a quality control product for flow cytometry detection of leukocyte differentiation antigen CD34, characterized in that, It includes the following steps: S1. Take 20 μL - 200 μL of microspheres. After washing and activation, add 10 - 500 µL of CD34 protein and 10 - 500 µL of CD45 protein for incubation. After the incubation is completed, add a blocking agent. Wash the blocked microspheres and store them at 4°C - 8°C to obtain CD34 microspheres; S2. Collect peripheral blood from healthy adults and separate plasma and blood cells; S3. Fix the blood cells and add the plasma to obtain fixed peripheral blood; S4. Add the CD34 microspheres obtained in S1 to the peripheral blood obtained in S3 according to a preset ratio, and mix well to obtain a quality control product; S5. Determine the absolute number of CD34 microspheres and white blood cells in the quality control product obtained in S4.

2. The preparation method of the quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 according to claim 1, characterized in that, In the step S3, it further includes counting the white blood cells in the fixed peripheral blood to make the concentration of white blood cells in the fixed peripheral blood be 4.0×10 9 ~ 10.0×10 9 cells / L.

3. The preparation method of the quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 according to claim 1, characterized in that, The operation of fixing the blood cells includes: Prepare 0.005 - 0.75 % (w / v) CrCl3 solution and 0.1 - 0.5 % (w / v) paraformaldehyde solution with a solvent in advance; Mix the CrCl3 solution and the blood cells at a volume ratio of 1:1, fix at 0 - 8°C for 5 min - 18 h, centrifuge to remove the supernatant, and wash with PBS to remove the CrCl3 solution; then mix the paraformaldehyde solution and the blood cells at a volume ratio of 1:1, fix at 0°C - 8°C for 16 - 22 h, centrifuge to remove the supernatant, and wash with PBS to remove the paraformaldehyde solution to obtain fixed blood cells.

4. The preparation method of the quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 according to claim 1, characterized in that, In the step S4, the preset ratio includes: in the CD34 + low-value quality control product, the proportion of CD34 microspheres in peripheral blood white blood cells is 0.1% - 0.3%; CD34 + In the high-value quality control product, the proportion of CD34 microspheres in peripheral blood white blood cells is 0.3% - 0.6%.

5. The preparation method of the quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 according to claim 1, characterized in that, The operation of washing the microspheres includes: Take 20 μL - 200 μL of microspheres and add 1 - 5 mL of MES coupling buffer, mix well, rotate and incubate for 5 - 30 min, centrifuge to remove the supernatant, repeat the washing 1 - 3 times, and resuspend the microspheres in MES coupling buffer.

6. The preparation method of the quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 according to claim 1, characterized in that, The operation of activating the microspheres includes: Add a reaction solution containing 10 μL - 200μL of EDC and 10 μL - 200μL of NHS to the washed microspheres, mix well, and rotate and incubate at 37°C for 10 - 30 min.

7. The preparation method of the quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 according to claim 1, wherein, In the step S5, use a flow cytometer to measure the proportion of CD34 microspheres in the quality control product and determine the target value, and use a five - classification blood cell counter to measure the absolute number of white blood cells in the quality control product and determine the target value.

8. The preparation method of the quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 as described in claim 1, characterized in that, The microspheres are selected from silica carboxyl microspheres, polystyrene microspheres or magnetic microspheres, and the particle size of the microspheres is 3 - 20 μm.

9. A quality control product for flow cytometry detection of leukocyte differentiation antigen CD34, characterized in that, Prepared by the method according to any one of claims 1 - 8.

10. The quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 as described in claim 9, or the quality control product prepared by the preparation method of the quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 as described in any one of claims 1-8, in the preparation of a kit for detecting CD34 + cells

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