Method for counting CD34-positive cells in hematopoietic stem cells

Abstract

Disclosed in the present invention is a method for counting CD34-positive cells in hematopoietic stem cells, which method is characterized by comprising the following steps: extracting hematopoietic stem cells, adding a red blood cell lysis buffer for lysis, centrifuging and washing same, and resuspending same to obtain a cell suspension; putting the cell suspension into a flow tube, adding a CD34 antibody and a CD45 antibody, uniformly mixing same, then adding a fluorescent nucleic acid dye, centrifuging and washing same, and then counting the CD34-positive cells by means of a flow cytometer. In the method for counting CD34-positive cells in hematopoietic stem cells of the present invention, the extracted hematopoietic stem cells are subjected to red blood cell lysis removal and then nucleic acid dyeing, and are prepared into a cell suspension, so that the interference of impurities such as red blood cells can be reduced, and the accuracy of flow cytometry counting is improved. The method has the advantages of simple operation, easy operation, and strong consistency in repeated operations. Furthermore, the provided CD34-positive-cell counting gating method has strong universality, and effectively screens out CD34-positive cells for counting.

Description

Method for counting CD34 positive cells in hematopoietic stem cells Technical Field

[0001] The invention belongs to the technical field of cell analysis, in particular to a method for counting CD34 positive cells in hematopoietic stem cells. Background Art

[0002] Hematopoietic stem cells are an important tool for treating blood system diseases and certain genetic defects. In the early days, hematopoietic stem / progenitor cell numbers were calculated using nucleated cell counts and colony-forming unit (CFU) counts. The former had poor consistency with HSC numbers, while the latter was time-consuming and subject to significant variability.

[0003] The discovery that CD34 is expressed on hematopoietic progenitor cells indicates strong hematopoietic activity. Clinical studies have also confirmed that transplantation of CD34+-enriched cell fractions can safely and durably achieve multilineage hematopoietic reconstitution. Flow cytometry is used to count and sort tiny particles suspended in a fluid. This technique allows for continuous analysis of multiple parameters of individual cells passing through optical or electronic detectors. Flow cytometry (FCM) is a technique that enables high-speed, individual cell quantitative analysis and sorting of single cells or other biological particles in suspension by detecting fluorescent signals from labeled cells. Currently, flow cytometry is the preferred method for CD34+ cell counting, but analytical parameters vary significantly between laboratories, and this lack of consistency significantly reduces its practicality. A highly accurate and consistent method for counting CD34-positive cells is urgently needed. Summary of the Invention

[0004] Based on this, the present invention provides a method for counting CD34-positive cells in hematopoietic stem cells, which has high accuracy and strong consistency in repeated operations.

[0005] The present invention provides a method for counting CD34-positive cells in hematopoietic stem cells, comprising the following steps:

[0006] S1, extract hematopoietic stem cells, add red blood cell lysis buffer to lyse, centrifuge and wash, and resuspend to obtain a cell suspension;

[0007] S2, placing the cell suspension in a flow cytometer, adding CD34 antibody and CD45 antibody, mixing evenly, adding fluorescent nucleic acid dye, centrifuging and washing, and then counting CD34 positive cells by flow cytometry.

[0008] In some embodiments, the fluorescent nucleic acid dye comprises 7-AAD (7-aminoactinomycin D).

[0009] In some embodiments, the method for setting a gating for CD34 positive cell count comprises the following steps:

[0010] 1) setting the region of all 7-AAD-labeled living cells in the fluorescence scattergram obtained by the flow cytometer as the 7-AAD gate;

[0011] 2) Among the cells in the 7-AAD gate, set the CD45-positive cell area, which is recorded as the WBC gate, and set the lymphocyte area, which is recorded as the Lymphatic gate;

[0012] 3) Perform angular scatter analysis in the WBC gate, set the P1 gate to determine the location of the mononuclear cell population, identify cells with low to medium S intensity, and remove the influence of cell debris and aggregated platelets;

[0013] 4) Perform side scatter analysis in the WBC gate and set the area of ​​all CD34-positive cells as the CD34+ gate;

[0014] 5) Among the cells within the CD34+ gate, set the area of ​​CD34-positive cells and CD45-weakly positive cells, recorded as the CD34+CD45dim gate, and set the Lymphatic-1 gate at the position of the Lymphatic gate to remove the CD45-strongly positive cell cluster;

[0015] 6) Identify cells with low to moderate S intensity within the CD34+CD45dim gate to obtain CD34 positive cells.

[0016] In some embodiments, in step S1, the specific operations include: extracting hematopoietic stem cells, washing with a biological buffer, centrifuging, adding the centrifugal precipitate to a red blood cell lysis buffer, mixing evenly, and suspending in the dark at room temperature to fully lyse the red blood cells in the sample, centrifuging at room temperature, removing the supernatant, and then adding a biological buffer to wash and centrifuge to remove the red blood cells, and resuspending in a biological buffer to obtain a cell suspension.

[0017] In some embodiments, the mass ratio of the centrifugal pellet to the red blood cell lysate is 1:(0.8-2).

[0018] In some embodiments, the biological buffer comprises any one of DPBS buffer, Hepes buffer or Tris buffer.

[0019] In some embodiments, the light protection time is 10-20 minutes, and the mixture is shaken and mixed every 4-6 minutes.

[0020] In some embodiments, the concentration of the cell suspension is (8-12)×10 5 cells / 100μl.

[0021] In some embodiments, in step S2, the CD34 antibody is a PE-labeled mouse anti-human CD34 antibody; and / or the CD45 antibody is a FITC-labeled mouse anti-human CD45 antibody.

[0022] In some embodiments, in step S2, the operation of adding the fluorescent nucleic acid dye includes: incubating the sample in the dark at room temperature for 15-25 minutes, adding 7-AAD, mixing, and continuing to incubate in the dark at room temperature for 8-12 minutes.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a method for counting CD34-positive cells in hematopoietic stem cells. Extracted hematopoietic stem cells are lysed to remove red blood cells, followed by nucleic acid staining to prepare a cell suspension. This method reduces interference from impurities such as red blood cells and improves the accuracy of flow cytometry counting. The method is simple and easy to operate, with high consistency across repeated operations. Furthermore, the proposed gating method for CD34-positive cell counting is highly universal and effectively selects CD34-positive cells for counting. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG1 is a flow cytometry scattergram of a method for counting CD34 positive cells in hematopoietic stem cells according to the present invention; wherein, the cell adhesion bodies are removed.

[0026] Figure 1A shows a scatter plot of the FSC-A and FSC-H parameters of the cells; Figure 1B shows all cells, and cells within the 7-AAD gate are live cells; Figure 1C shows cells within the 7-AAD gate, CD45-positive cells within the WBC gate, and lymphocytes within the Lymphatic gate; Figure 1D shows cells within the WBC gate under angular scatter (FSC) / SSC, and mononuclear cells within the P1 gate; Figure 1E shows cells within the WBC gate under SSC, and the CD34+ gate is set to include all CD34+ cells; Figure 1F shows cells within the CD34+ gate, the CD34+CD45dim gate includes CD34+ cells and CD45 weakly positive cells, and the Lymphatic-1 gate is at the same position as the Lymphatic gate; Figure 1G shows CD34+ cells that meet the requirements in the CD34+CD45dim gate. Implementation Method

[0027] The technical solutions of the present invention are further illustrated by specific examples below. Those skilled in the art should understand that the examples are only for helping to understand the present invention and should not be regarded as specific limitations of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0029] A method for counting CD34-positive cells in hematopoietic stem cells comprises the following steps:

[0030] S1, extract hematopoietic stem cells, add red blood cell lysis buffer to lyse, centrifuge and wash, and resuspend to obtain a cell suspension;

[0031] S2, placing the cell suspension in a flow cytometer, adding CD34 antibody and CD45 antibody, mixing evenly, adding fluorescent nucleic acid dye, centrifuging and washing, and then counting CD34 positive cells by flow cytometry.

[0032] In some embodiments, the fluorescent nucleic acid dye comprises 7-AAD.

[0033] In some embodiments, the method for setting a gating for CD34 positive cell count comprises the following steps:

[0034] 1) setting the region of all 7-AAD-labeled living cells in the fluorescence scattergram obtained by the flow cytometer as the 7-AAD gate;

[0035] 2) Among the cells in the 7-AAD gate, set the CD45-positive cell area, which is recorded as the WBC gate, and set the lymphocyte area, which is recorded as the Lymphatic gate;

[0036] 3) Perform angular scatter analysis in the WBC gate, set the P1 gate to determine the location of the mononuclear cell population, identify cells with low to medium S intensity, and exclude cell debris and platelet aggregates;

[0037] 4) Perform side scatter analysis in the WBC gate and set the area of ​​all CD34-positive cells as the CD34+ gate;

[0038] 5) Among the cells within the CD34+ gate, set the area of ​​CD34-positive cells and CD45-weakly positive cells, recorded as the CD34+CD45dim gate, and set the Lymphatic-1 gate at the position of the Lymphatic gate to remove the CD45-strongly positive cell cluster;

[0039] 6) Identify cells with low to moderate S intensity within the CD34+CD45dim gate to obtain CD34 positive cells.

[0040] In some embodiments, in step S1, the specific operations include: extracting hematopoietic stem cells, washing with a biological buffer, centrifuging, adding the centrifugal precipitate to a red blood cell lysis buffer, mixing evenly, and suspending in the dark at room temperature to fully lyse the red blood cells in the sample, centrifuging at room temperature, removing the supernatant, and then adding a biological buffer to wash and centrifuge to remove the red blood cells, and resuspending in a biological buffer to obtain a cell suspension.

[0041] In some embodiments, the mass ratio of the centrifugal pellet to the red blood cell lysate is 1:(0.8-2).

[0042] In some embodiments, the biological buffer comprises any one of DPBS buffer, Hepes buffer or Tris buffer.

[0043] In some embodiments, the light protection time is 10-20 minutes, and the mixture is shaken and mixed every 4-6 minutes.

[0044] In some embodiments, the concentration of the cell suspension is (8-12)×10 5 cells / 100μl.

[0045] In some embodiments, in step S2, the CD34 antibody is a PE-labeled mouse anti-human CD34 antibody; and / or the CD45 antibody is a FITC-labeled mouse anti-human CD45 antibody.

[0046] In some embodiments, in step S2, the operation of adding the fluorescent nucleic acid dye includes: incubating the sample in the dark at room temperature for 15-25 minutes, adding 7-AAD, mixing, and continuing to incubate in the dark at room temperature for 8-12 minutes. Example 1

[0047] 1. Sample screening

[0048] Medical history collection-level testing of donors for placenta- or umbilical cord blood-derived hematopoietic stem cell trials: including the donor's basic information, past medical history, family history, etc.

[0049] The donor is screened for infectious diseases (infectious diseases include HIV, HBV, HCV, syphilis, EB, etc.). If necessary, the donor's blood routine, liver and kidney function, four coagulation tests, blood type, etc. need to be checked.

[0050] 2. Method for counting CD34-positive cells in hematopoietic stem cells

[0051] The counting method comprises the following steps:

[0052] 1) Extract hematopoietic stem cells from placenta or umbilical cord blood, wash with 10 ml of DPBS, and then centrifuge to remove the supernatant to obtain a crude hematopoietic stem cell sample;

[0053] 2) Dilute an appropriate amount of crude hematopoietic stem cell sample at a 1:1 ratio into a centrifuge tube containing 1× Pharm Lyse Lysing Buffer (red blood cell lysis buffer). Mix thoroughly and incubate at room temperature in the dark for 15 minutes. Remove the tube and shake it back and forth every 5 minutes to fully lyse the red blood cells in the sample. Centrifuge at room temperature for 5 minutes and remove the supernatant.

[0054] 3) Take an appropriate amount of DPBS to centrifuge and wash the sample, remove the supernatant, and remove the red blood cells for later use;

[0055] 4) According to the actual total number of cells in the sample to be tested, add appropriate amount of DPBS to prepare 1×10 6 cells / 100 μl of cell suspension;

[0056] 5) Take three flow-through tubes and label them NEG, ISO, and CD34 / CD45 / 7-AAD, respectively. NEG is the negative control tube; ISO is the isotype control tube; and CD34 / CD45 / 7-AAD is the sample tube.

[0057] 6) Add 10ul of PE-labeled mouse anti-human CD34 antibody and 10ul of FITC-labeled mouse anti-human CD45 antibody to the tube labeled CD34 / CD45 / 7-AAD; add 10ul of PE-labeled mouse IgG1 and flow cytometry isotype control antibody to the tube labeled ISO; do not add any antibody reagents to the NEG tube;

[0058] 7) Mix all the above tubes evenly and incubate in the dark at room temperature for 20 minutes. Then add 5 μl of 7-AAD to the tube labeled with CD34 / CD45 / 7-AAD, mix the tube evenly, and incubate in the dark at room temperature for another 10 minutes.

[0059] 8) Take an appropriate amount of DPBS for centrifugation and wash, remove the supernatant, add an appropriate amount of DPBS to each tube, mix well, and prepare a cell suspension;

[0060] 9) On-machine testing within 2 hours.

[0061] The CD34-positive cell counting gating method, as shown in FIG1 , includes the following steps:

[0062] 1) In the flow cytometry fluorescence scattergram obtained by the flow cytometer, the FSC-A and FSC-H parameters are used to remove cell adhesions and reduce the interference signal of flow detection;

[0063] 2) In the side scatter (SSC) analysis, the area of ​​all 7-AAD-labeled live cells was set as the 7-AAD gate;

[0064] 3) Among the cells in the 7-AAD gate, set the CD45-positive cell area, which is recorded as the WBC gate, and set the lymphocyte area, which is recorded as the Lymphatic gate;

[0065] 4) Perform angular scatter analysis in the WBC gate, set the P1 gate to determine the location of the mononuclear cell population, identify cells with low to medium S intensity, and exclude cell debris and platelet aggregates;

[0066] 5) Perform side scatter analysis in the WBC gate and set the area of ​​all CD34-positive cells as the CD34+ gate;

[0067] 6) Among the cells within the CD34+ gate, set the area of ​​CD34-positive cells and CD45-weakly positive cells, recorded as the CD34+CD45dim gate, and set the Lymphatic-1 gate at the position of the Lymphatic gate to remove the CD45-strongly positive cell cluster;

[0068] 7) Identify cells with low to moderate S intensity within the CD34+CD45dim gate to obtain CD34 positive cells.

[0069] The method for counting CD34-positive cells in hematopoietic stem cells of the present invention improves the accuracy of flow cytometry counting, is simple and easy to operate, and has high consistency in repeated operations.

[0070] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for counting CD34 positive cells in hematopoietic stem cells, characterized in that: The following steps are involved: S1, extracting hematopoietic stem cells, adding red blood cell lysis buffer to lyse, centrifuging and washing, and resuspending to obtain a cell suspension; S2, placing the cell suspension in a flow cytometer, adding CD34 antibody and CD45 antibody and mixing evenly, adding fluorescent nucleic acid dye, centrifuging and washing, and then counting CD34 positive cells by flow cytometry.

2. The method for counting CD34 positive cells in hematopoietic stem cells according to claim 1, characterized in that: The fluorescent nucleic acid dye includes 7-AAD.

3. The method for counting CD34 positive cells in hematopoietic stem cells according to claim 2, characterized in that: The CD34 positive cell counting gating method includes the following steps: 1) setting all 7-AAD-labeled living cell regions in the fluorescence scattergram obtained by the flow cytometer, and recording them as the 7-AAD gate; 2) In the cells of the 7-AAD gate, set the CD45 positive cell area, recorded as the WBC gate, and set the lymphocyte area, recorded as the Lymphatic gate; 3) Perform angular scatter analysis in the WBC gate, set the P1 gate to determine the location of the mononuclear cell population, identify cells with low to medium S intensity, and remove the influence of cell debris and aggregated platelets; 4) performing side scatter analysis in the WBC gate and setting the area of ​​all CD34-positive cells as the CD34+ gate; 5) Among the cells in the CD34+ gate, set the CD34 positive cells and CD45 weakly positive cell areas, recorded as the CD34+CD45dim gate, and set the Lymphatic-1 gate at the position of the Lymphatic gate to remove the CD45 strongly positive cell clusters; 6) Identify cells with low to medium intensity within the CD34+CD45dim gate to obtain CD34 positive cells.

4. The method for counting CD34 positive cells in hematopoietic stem cells according to claim 1, characterized in that: In step S1, the specific operations include: extracting hematopoietic stem cells, washing with biological buffer, centrifuging, adding the centrifugal precipitate to red blood cell lysis solution, mixing evenly, protecting from light at room temperature to fully lyse the red blood cells in the sample, centrifuging at room temperature, removing the supernatant, then adding biological buffer to centrifuge and wash to remove the red blood cells, and resuspending in biological buffer to obtain a cell suspension.

5. The method for counting CD34 positive cells in hematopoietic stem cells according to claim 4, characterized in that: The mass ratio of the centrifugal sediment to the red blood cell lysate is 1:(0.8-2).

6. The method for counting CD34 positive cells in hematopoietic stem cells according to claim 4, characterized in that: The biological buffer includes any one of DPBS buffer, Hepes buffer or Tris buffer.

7. The method for counting CD34 positive cells in hematopoietic stem cells according to claim 4, characterized in that: The light-shielding time is 10-20 minutes, during which the mixture is shaken and mixed every 4-6 minutes.

8. The method for counting CD34 positive cells in hematopoietic stem cells according to claim 4, characterized in that: The concentration of the cell suspension is (8-12)×10 5 cells / 100μl.

9. The method for counting CD34 positive cells in hematopoietic stem cells according to claim 1, characterized in that: In step S2, the CD34 antibody is a PE-labeled mouse anti-human CD34 antibody; and / or the CD45 antibody is a FITC-labeled mouse anti-human CD45 antibody.

10. The method for counting CD34 positive cells in hematopoietic stem cells according to claim 1 or 2, characterized in that: In step S2, the operation of adding the fluorescent nucleic acid dye includes: keeping the sample in the dark at room temperature for 15-25 minutes, adding 7-AAD, mixing evenly, and keeping the sample in the dark at room temperature for 8-12 minutes.

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