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

Abstract

This invention discloses a quality control sample for flow cytometry detection of leukocyte differentiation antigen CD34 and its preparation method, relating to the field of flow cytometry quality control technology. The preparation method of this invention successfully prepared a quality control sample for flow cytometry detection of leukocyte differentiation antigen CD34. The quality control sample for flow cytometry detection of leukocyte differentiation antigen CD34 provided by this invention, as the first domestic quality control sample for CD34 flow cytometry counting, fills a gap in this product field in China. This quality control sample has performance close to imported reagents, uses the same operating method, and does not incur additional learning costs; furthermore, the product's shelf life can be stably maintained at 45–50 days, and its usage cost is far lower than imported quality control samples.

Description

Technical Field

[0001] This invention relates to the field of cell flow cytometry quality control technology, and in particular to a quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 and its preparation method. Background Technology

[0002] Hematopoietic stem cell transplantation can restore hematopoietic function and has a lower incidence of tumor cell contamination. It can be widely used to treat various malignant hematological diseases, solid tumors, genetic diseases, and severe combined immunodeficiency diseases. The main sources of hematopoietic stem cells are peripheral blood, bone marrow, and umbilical cord blood, with umbilical cord blood having the richest content. Due to its abundant source, convenient collection, and increasingly simplified storage and transplantation techniques, it is receiving increasing attention and recognition from both doctors and patients.

[0003] Currently, the important indicators for evaluating hematopoietic stem cell transplantation are the total number of nucleated cells and the total number of CD34+ cells. Among them, CD34+ cells are recognized as the key indicator of whether cells have hematopoietic function. Their detection is mainly carried out by flow cytometry single-platform (absolute count) or dual-platform (percentage count combined with whole blood cell count) methods.

[0004] Domestic regulations, such as the "Management Standards for Unrelated Hematopoietic Stem Cell Consistency Technology," "Management Standards for Unrelated Hematopoietic Stem Cell Collection Technology," "Technical Specifications for Umbilical Cord Blood Hematopoietic Stem Cell Banks (Trial)," and the "Management Standards for Hematopoietic Stem Cell Transplantation Technology (2017 Edition)," along with 15 other "restricted clinical application" medical technology management standards and quality control indicators, all stipulate corresponding requirements for stem cell CD34+ cell counting and quality control. To ensure the accuracy of test results and monitor the stability of the testing process, internal quality control materials are used in the testing system for internal quality control. Summary of the Invention

[0005] This invention addresses the problems mentioned in the background section by providing a quality control product for flow cytometry detection of leukocyte differentiation antigen CD34. This quality control product fills a gap in the domestic market and solves the problem of short shelf life of imported quality control products after reaching the user, thus reducing the user's usage cost. Simultaneously, a method for preparing the quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 is also provided.

[0006] To address the above problems, the present invention proposes the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing a quality control sample for flow cytometry detection of leukocyte differentiation antigen CD34, comprising the following steps:

[0008] S1, Construction of the expression vector: The gene encoding the leukocyte differentiation antigen CD34 was amplified using genetic engineering technology, and the gene fragment was inserted into the expression vector to obtain the CD34 expression vector; the amino acid sequence of the leukocyte differentiation antigen CD34 is SEQ NO.1 or SEQ NO.2;

[0009] S2, Packaging of viral vector: The CD34 expression vector and packaging plasmid are transfected into packaging cell lines to obtain a virus containing the gene encoding the leukocyte differentiation antigen CD34;

[0010] S3, Target cell infection: The virus obtained in S2 is used to infect target cells, causing the gene encoding leukocyte differentiation antigen CD34 to be integrated into the genome of the target cells and express leukocyte differentiation antigen CD34.

[0011] S4, Magnetic bead sorting of CD34 positive cells: Infected target cells are sorted out by CD34 antibody magnetic beads to select qualified CD34 positive cell lines.

[0012] S5, CD34-positive cell expansion culture: Qualified CD34-positive cell lines were expanded through culture until the cell number reached 1×10⁻⁶. 9 When the cell number reaches 1 × 10⁶ cells or more, aliquot the cells into cryovials at the first preset concentration to serve as first-generation seed cells, and freeze them in liquid nitrogen. Take one vial of first-generation seed cells for further expansion culture. When the number of cultured cells reaches 1 × 10⁶ cells / cells... 9 When the cell number reaches 1 × 10⁶ cells or more, aliquot the cells into cryovials according to the second preset concentration to serve as second-generation seed cells, and freeze them in liquid nitrogen; take one vial of second-generation seed cells for further expansion culture, and when the number of cultured cells reaches 1 × 10⁶ cells / cells... 9 When cells or more are used, they are dispensed into cryovials according to the third preset concentration and used as working cells, and then frozen in an ultra-low temperature freezer for later use.

[0013] S6, Obtain blood cells: Collect peripheral blood and count the white blood cells in the peripheral blood, ensuring the white blood cell concentration is within 4.0 × 10⁻⁶. 9 ~10.0×10 9 Peripheral blood within the cells / L range was centrifuged at low temperature to separate plasma from blood cells;

[0014] S7. CD34-positive cells are mixed and fixed with blood cells: The working cells obtained in S5 are added to the blood cells obtained in S6 at a predetermined ratio to obtain mixed cells; the mixed cells are fixed to obtain fixed mixed cells; the fixed mixed cells are prepared into quality control samples and stored at 4℃ ~ 8℃; wherein, the predetermined ratio is: in CD34... +In low-value quality control samples, the proportion of CD34-positive cells in peripheral blood leukocytes was 0.1% to 0.3%; CD34 + In high-value quality control products, the proportion of CD34-positive cells in peripheral blood leukocytes is 0.3% to 0.6%.

[0015] A further technical solution is that the target cells are any one of nalm6 cells, KG-1a cells, Jurkat cells, or daudi cells.

[0016] A further technical solution is that, in step S1, the expression vector is selected from pCDH-EF1-MCS-T2A-Puro, pCDH-CMV-MCS-EF1-puro,

[0017] Any one of pCDH-CMV-MCS-EF1-copGFP, pCDH-CMV-MCS-EF1-copGFP-T2A-puro, pCDH-CMV-MCS-EF1-RFP, or CDH-MCS-T2A-puro-MSCV.

[0018] A further technical solution is that, in step S2, the packaging plasmid is selected from any one of the following combinations:

[0019] Combination 1: pMD2.G, pRSV-rev, and pMDLg / pRRE;

[0020] Combination 2: pPACKH1-GAG, pPACKH1-REV, and pVSV-G;

[0021] Combination 3: pMD2.G and psPAX2.

[0022] A further technical solution is that, in step S4, the infected target cells are screened with CD34 antibody magnetic beads to identify the initial CD34 positive cell lines, and then the initial CD34 positive cell lines are screened a second time using flow cytometry. The initial CD34 positive cell lines with a positive rate of 97% or higher are considered as qualified CD34 positive cell lines.

[0023] A further technical solution is that, in step S5, the range of the first preset concentration, the second preset concentration, and / or the third preset concentration is 0.5 × 10⁻⁶. 7 ~1.5×10 7 cells / mL.

[0024] A further technical solution is that, in step S6, the fixing method is as follows:

[0025] Prepare a 0.005–0.75% (w / v) CrCl3 solution and a 0.1–0.5% (w / v) paraformaldehyde solution in advance using solvents;

[0026] Mix the CrCl3 solution with the mixed cells at a volume ratio of 1:1, fix at 0-8℃ for 5 min-18 h, centrifuge to remove the supernatant, wash the mixed cells with the solvent 1-5 times, and discard the supernatant; then mix the mixed cells with paraformaldehyde solution at a volume ratio of 1:1, fix at 0℃-8℃ for 16-22 h, centrifuge to remove the supernatant, wash the mixed cells with the solvent 1-5 times, and discard the supernatant to obtain the fixed mixed cells;

[0027] Add paraformaldehyde solution to the fixed mixed cells and bring the volume up to the original peripheral blood volume to obtain the quality control product; or, add plasma separated from S6 to the fixed mixed cells to obtain the quality control product.

[0028] A further technical solution is that the solvent is DPBS or PBS; the pH value of the solvent is 6.5 to 7.

[0029] A further technical solution is that step S7 further includes setting values ​​for the CD34 positive cell rate and the absolute number of white blood cells in the quality control product: using a flow cytometer to determine the CD34 positive cell rate of the quality control product and determining the target value, and using a blood cell five-part differential counter to determine the absolute number of white blood cells in the quality control product and determining the target value.

[0030] Secondly, the present invention provides a quality control material for flow cytometry detection of leukocyte differentiation antigen CD34, which is prepared by the method described in the first aspect.

[0031] Compared with the prior art, the technical effects achieved by the present invention include:

[0032] The present invention provides a method for preparing quality control material for flow cytometry detection of leukocyte differentiation antigen CD34. Using this method, quality control material for flow cytometry detection of leukocyte differentiation antigen CD34 was successfully prepared.

[0033] The quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 provided by this invention is the first quality control product in China used for CD34 flow cytometry counting, filling a gap in the domestic product market. The quality control product is similar in performance to imported reagents, and the operation method is the same, without adding any additional learning costs. Moreover, the product's shelf life can be stably maintained at 45 to 50 days, and the cost of use is far lower than that of imported quality control products. Attached Figure Description

[0034] Figure 1 This is the result of the low-value quality control group of Comparative Example 1;

[0035] Figure 2The results are for the low-value quality control experimental group of Comparative Example 1;

[0036] Figure 3 This is the result of the high-value quality control group in Comparative Example 1;

[0037] Figure 4 The results are from the high-value quality control experimental group of Comparative Example 1;

[0038] Figure 5 This is the result of the low-value quality control group of Example 1;

[0039] Figure 6 The results are from the low-value quality control experimental group of Example 1;

[0040] Figure 7 The results are for the high-value quality control control group of Example 1;

[0041] Figure 8 The results are from the high-value quality control experimental group of Example 1;

[0042] Figure 9 This is a flowchart of the preparation method for Example 1. Detailed Implementation

[0043] The invention is further illustrated below by specific application examples, but these examples do not limit the invention to the scope of the specific applications described. Experimental methods not specifying particular conditions in the following specific application examples should be performed according to conventional methods and conditions, or as selected in the product instructions. Unless otherwise specified, temperature generally refers to reactions conducted at room temperature; in this invention, room temperature refers to 16°C to 30°C.

[0044] Unless otherwise specified, the experimental methods used in the following application examples are conventional methods.

[0045] Unless otherwise specified, all materials and reagents used in the following application examples are commercially available.

[0046] Example 1

[0047] This invention provides a quality control material for flow cytometry detection of leukocyte differentiation antigen CD34 and its preparation method. The flowchart of the preparation method in this embodiment is shown below. Figure 9 The following is a detailed explanation of each step:

[0048] Step S1: Construction of CD34 lentiviral expression vector

[0049] The human leukocyte differentiation antigen CD34 gene was found in the GenBank database of the U.S. National Library of Medicine. Two variants were identified: Homo sapiens CD34 molecule (CD34), transcript variant 1 (SEQ NO.1) and Homo sapiens CD34 molecule (CD34), transcript variant 2 (SEQ NO.2). Transcript variant 1 (SEQ NO.1) has a longer sequence and more antigenic epitopes to choose from; therefore, this embodiment selected transcript variant 1 (SEQ NO.1) as the target gene for cloning.

[0050] It should be noted that, in other embodiments, those skilled in the art may also choose transcript variant 2 (SEQ NO. 2) as the target gene for cloning to obtain the CD34 gene.

[0051] Umbilical cord vein mRNA was extracted and PCR template was obtained by reverse transcription. Two pairs of specific amplification primers were designed based on the transcript variant 1 (SEQ NO.1) gene sequence of the CD34 gene. Coarse amplification was performed first, followed by fine amplification. The nucleotide sequences of the amplification primers are shown in SEQ NO.3–SEQ NO.6. Typically, cloning amplification only requires one pair of primers for fine amplification. However, in this embodiment of the invention, a pair of coarse amplification primers was designed before fine amplification for coarse amplification, thus improving the success rate of cloning the target gene and successfully obtaining the target gene with restriction enzyme sites (Xba I and EcoRI). Primer information is shown in Table 1 below.

[0052] Table 1. Amplification Primer Information

[0053]

[0054] The obtained target gene fragment was inserted into plasmid pCDH-EF1-MCS-T2A-Puro to obtain a recombinant plasmid, which was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were translated into amino acid sequences and compared with the amino acid sequence of CD34. The topological results of the amino acids were analyzed. Amino acid sequences without mutations, with mutation sites not in the transmembrane region of the CD34 protein, or with mutated amino acid residues similar in properties to the original amino acid residues were selected for lentiviral packaging.

[0055] The vector plasmid CD34-pCDH-EF1-MCS-T2A-Puro, packaging plasmids pMD2.G, pRSV-REV, and pMDLg-pRREE were transformed into competent cells and cultured in large quantities. Plasmids were extracted in large quantities using the alkaline lysis method (Beijing Tiangen Biotech Co., Ltd. Endotoxin-free plasmid extraction kit) for transfection.

[0056] S2, HIV-1 lentiviral packaging

[0057] In this embodiment, HEK293T cells were used as packaging cells. HEK293T cells in the logarithmic growth phase were collected 24 hours before transfection using trypsin digestion. 7 × 10⁻⁶ cells were then packaged into 10⁻⁶ cells. 6 Cells were seeded onto 10 cm culture plates, lentiviral packaging medium was added, and the plates were incubated at 37°C with 5% CO2 for 24 h. Transfection was performed when the cells reached 60-80% conjugation. The lentiviral packaging medium contained 5% FBS and 1 mM sodium pyruvate and MEM I serum-depleted medium.

[0058] The packaging mixture was prepared according to the mass ratio of pMD2.G-Kan: pRSV-REV-Kan: pMDLg-pRREE-Kan = 1~10: 1~10: 1~10. For example, in other embodiments, the packaging mixture is prepared at a mass ratio of pMD2.G-Kan: pRSV-REV-Kan: pMDLg-pRREE-Kan = 1:1:1; or, at a mass ratio of pMD2.G-Kan: pRSV-REV-Kan: pMDLg-pRREE-Kan = 1:2:2; or, at a mass ratio of pMD2.G-Kan: pRSV-REV-Kan: pMDLg-pRREE-Kan = 1:2:3; or, at a mass ratio of pMD2.G-Kan: pRSV-REV-Kan: pMDLg-pRREE-Kan = 1:1:2; or, at a mass ratio of pMD2.G-Kan: pRSV-REV-Kan: pMDLg-pRREE-Kan = 1:1:2. A packaging mixture is prepared at a mass ratio of pMD2.G-Kan:pMDLg-pRREE-Kan = 1:3:2; or, a packaging mixture is prepared at a mass ratio of pMD2.G-Kan:pRSV-REV-Kan:pMDLg-pRREE-Kan = 1:4:2; or, a packaging mixture is prepared at a mass ratio of pMD2.G-Kan:pRSV-REV-Kan:pMDLg-pRREE-Kan = 1:1:8; or, a packaging mixture is prepared at a mass ratio of pMD2.G-Kan:pRSV-REV-Kan:pMDLg-pRREE-Kan = 1:1:10; or, a packaging mixture is prepared at a mass ratio of pMD2.G-Kan:pRSV-REV-Kan:pMDLg-pRREE-Kan = 1:10:1.

[0059] Prepare the packaging plasmid and expression vector ratios according to the Invitrogen Lipofectamine 3000 transfection reagent instructions, and prepare the liposome-DNA complex according to the instructions.

[0060] Before adding the liposome-DNA complex, remove the 10 cm culture plate from the incubator, remove 6 ml of culture medium, leaving a total volume of 6 ml per well. Carefully add 3 ml of the liposome-DNA complex against the plate wall, gently agitate the plate to distribute it evenly, and incubate at 37°C and 5% CO2 for 4 h. Replace the culture medium in the plate afterward. Carefully aspirate the culture medium containing the liposome-DNA complex from each well and replace it with 12 mL of preheated lentiviral packaging medium. Return the plate to the incubator and incubate overnight at 37°C and 5% CO2. 24 h after transfection, collect 12 mL of cell supernatant from each well, transfer it to a 50 mL conical tube, and store at 4°C. Replace the collected medium with 12 mL of preheated lentiviral packaging medium and incubate overnight at 37°C and 5% CO2. 52 h after transfection, 12 ml of cell supernatant was collected from each well and mixed with the first collection of supernatant to make a total volume of 24 ml. The collected 4 ml of supernatant was centrifuged at 2000 rpm for 10 minutes at room temperature. The supernatant was collected and transferred to remove cell pellets. The clarified lentivirus supernatant was filtered through a 0.2 μm filter to remove residual cell debris. After aliquoting, it was stored at -80℃ for later use.

[0061] S3, Target cell infection

[0062] 7.5 × 10⁻⁶ samples were collected 24 hours before infection. 6 One nalm6 cell was seeded in a T75 culture flask and grown in 15 mL of RMMI 1640 complete medium (containing antibiotics) with 10% FBS and 1× penicillin-streptomycin solution. The cells were then cultured at 37°C in a 5% CO2 incubator for 24 h.

[0063] nalm6 cells cultured for 24 hours were then injected with 2×10⁻⁶ cells. 5 1 mL of the virus was seeded per well in a 6-well plate, with a total of 3 wells. The CD34 recombinant lentivirus, stored at -80°C, was thawed at 4°C, and 200 μL, 600 μL, and 1200 μL of viral stock solution were added, respectively. In the well containing 200 μL of viral stock solution, 9.6 μL of polybrene solution with a final concentration of 1–10 μg / mL was added. In the well containing 600 μL of viral stock solution, 12.8 μL of polybrene solution with a final concentration of 1–10 μg / mL was added. In the well containing 1200 μL of viral stock solution, 17.6 μL of polybrene solution with a final concentration of 1–10 μg / mL was added. The mixture was then thoroughly mixed. In other embodiments, the concentration of the polybrene solution could be 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, or 10 μg / mL.

[0064] After inoculation, cells were cultured at 37°C and 5% CO2 for 4 h. Then, 1.3 mL of pre-warmed RPMI 1640 complete medium (containing antibiotics) was added to wells containing 200 μL of viral stock solution; 0.9 mL of pre-warmed RPMI 1640 complete medium (containing antibiotics) was added to wells containing 600 μL of viral stock solution; and 0.3 mL of pre-warmed RPMI 1640 complete medium (containing antibiotics) was added to wells containing 1200 μL of viral stock solution. At this point, the total liquid volume in the plate was 2.5 mL, and the cell density was 0.8 × 10⁻⁶. 5 cells / mL, continue culturing for 20 hours.

[0065] After culturing for 24 h, the cells were shaken well, transferred to centrifuge tubes, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in 0.5 mL of preheated RPMI 1640 complete medium (containing antibiotics). The viable cell density was measured using the AO / PI method as follows: CD34nalm6-200: 6.70 × 10⁻⁶. 5 cells / mL; CD34nalm6-600: 4.97×10 5 cells / mL; CD34nalm6-1200: 6.01×10 5 Cells / mL. Transfer the cell suspension to 24-well plates and continue culturing for 6 days, supplementing with pre-warmed RPMI 1640 complete medium (containing antibiotics) every two days to adjust the cell concentration to approximately 0.5 × 10⁻⁶ cells / mL. 6 cells / mL.

[0066] After 6 days of culture, the viable cell density, measured using the AO / PI method, was: CD34nalm6-200: 9.43 × 10⁻⁶. 5 cells / mL; CD34nalm6-600: 1.05×10 6 cells / mL; CD34nalm6-1200: 1.06×10 6 cells / mL. Continue culturing for 1 day. Take 500 μL of cell suspension and use flow cytometry to measure the CD34 positive cell rate: CD34nalm6-200: 7.35%; CD34nalm6-600: 38.2%; CD34nalm6-1200: 67.13%.

[0067] Continue culturing for 13 days, supplementing with preheated RPMI 1640 complete medium (containing antibiotics) every two days to adjust the cell concentration to approximately 0.5 × 10⁻⁶ cells / day. 6 cells / mL.

[0068] S4, magnetic bead sorting of CD34-positive cells

[0069] The infected CD34nalm6 cells were sorted for CD34-positive cells according to the method in the CD34 MicroBead Kit instructions.

[0070] After sorting, the viable cell density was measured using the AO / PI method as follows: CD34nalm6-200: 1.13 × 10⁻⁶ 6 cells / mL, viability 64.9%; CD34nalm6-600: 5.46 × 10⁻⁶ 6 cells / mL, viability 91.1%; CD34nalm6-1200: 1.24×10⁻⁶ 7 The cell count was [number of cells / mL], with a viability of 92.3%. CD34nalm6-200 cells had too low viability and a small cell count after screening, so they were discarded. Simultaneously, CD34nalm6-600 cells were supplemented with 9 mL of RPMI 1640 complete medium (containing antibiotics) and transferred to T75 culture flasks; CD34nalm6-1200 cells were supplemented with 24 mL of RPMI 1640 complete medium (containing antibiotics) and transferred to T175 culture flasks. Culture was continued for 5 days, with pre-warmed RPMI 1640 complete medium (containing antibiotics) added every two days to adjust the cell concentration to approximately 0.5 × 10⁻⁶ cells / mL. 6 cells / mL.

[0071] Take 500 μL of the above cell suspension and use flow cytometry to measure the CD34 positive cell rate: CD34nalm6-600: 93.52%; CD34nalm6-1200: 94.71%.

[0072] After five days of culture, the cells were sorted again for CD34 positive cells according to the instructions in the CD34 MicroBead Kit manual.

[0073] After sorting, the viable cell density was measured using the AO / PI method as follows: CD34nalm6-600: 2.23 × 10⁻⁶ 6 cells / mL, viability 79.6%; CD34nalm6-1200: 2.92×10⁻⁶ 6The cell density was 89.1% (cells / mL). 8 mL of RPMI 1640 complete medium (with antibiotics) was added to CD34nalm6-600, and the cells were transferred to T75 culture flasks for further culture. 12 mL of RPMI 1640 complete medium (containing antibiotics) was added to CD34nalm6-1200, and the cells were transferred to T75 culture flasks for further culture. Cells were cultured continuously for 3 days. On the second day, the viable cell density was measured using the AO / PI method, and RPMI 1640 complete medium (containing antibiotics), 6 mL of CD34nalm6-600, and 7 mL of CD34nalm6-1200 were added respectively, to achieve a viable cell density of approximately 0.5 × 10⁻⁶ cells / mL. 6 cells / mL.

[0074] Take 500 μL of the above cell suspension and use flow cytometry to measure the CD34 positive cell rate: CD34nalm6-600: 98.97%; CD34nalm6-1200: 99.48%. Use the above two cell lines as qualified CD34 positive cell lines for large-scale culture.

[0075] S5 and CD34 positive cells expanded culture

[0076] Two flasks of qualified CD34-positive cells, namely CD34nalm6-600 and CD34nalm6-1200 cells, were mixed together, and the viable cell density was measured using the AO / PI method to be 1.14 × 10⁻⁶. 6 The cell density was 95.7% viable at 30 mL. 40 mL of RPMI 1640 complete medium (containing antibiotics) was added to adjust the viable cell density to approximately 0.5 × 10⁶ cells / mL. 6 cells / mL. Continue culturing for 3 days.

[0077] After 3 days of culture, the viable cell density, measured using the AO / PI method, was 1.24 × 10⁻⁶. 6 The cells were collected at 70 mL in 147 mL RPMI 1640 complete medium (containing antibiotics and antibiotics), with a viability of 94.5%. The cells were transferred to two 50 mL centrifuge tubes, centrifuged at 300 g for 10 minutes, the supernatant was discarded, and all cells were resuspended in 147 mL RPMI 1640 complete medium (containing antibiotics and antibiotics). The cells were then divided into three equal portions and aliquoted into new T175 culture flasks and cultured for another 2 days.

[0078] After two days of culture, the viable cell density, measured using the AO / PI method, was 2.29 × 10⁻⁶. 6The cells were collected at a concentration of 147 mL, with a viability of 96.6%. The cells were transferred to two 50 mL centrifuge tubes, centrifuged at 300 g for 10 minutes, the supernatant was discarded, and all cell pellets were resuspended in 33.663 mL of Cellbanker cell cryopreservation medium to adjust the cell concentration to 1 × 10⁻⁶ cells / mL. 7 cells / mL. Aliquoted into cryopreservation tubes, 1 mL per tube, as first-generation seed cells, and stored in liquid nitrogen.

[0079] Take 1×10 of the first-generation seed cells. 7 Cells were cultured continuously for 9 days in RPMI 1640 complete medium (containing antibiotics), with RPMI 1640 complete medium (containing antibiotics) added every 2 days to adjust the cell density to 0.5 × 10⁶ cells / day. 6 After 9 days of culture, the viable cell density, measured using the AO / PI method, was 1.11 × 10⁶ cells / mL. 6 Cells / mL, viability 93.6%, volume 200 mL. Transferred to four 50 mL centrifuge tubes, centrifuged at 1000 rpm for 5 min, supernatant discarded. Resuspend all cell pellet in 22.2 mL CELLBANKER cell cryopreservation medium, adjusting cell density to 1×10⁶ cells / mL. 7 cells / mL. Aliquoted into cryopreservation tubes, 1 mL per tube, as second-generation seed cells, and stored in liquid nitrogen.

[0080] Take 1×10 of the second-generation seed cells. 7 Cells were cultured continuously for 12 days in RPMI 1640 complete medium (containing antibiotics), with RPMI 1640 complete medium (containing antibiotics) added every 2 days to adjust the cell density to 0.5 × 10⁶ cells / day. 6 Cells / mL. After 12 days of culture, all cells were collected into a 50 mL centrifuge tube, resuspended in 20 mL of Cellbanker cell cryopreservation medium, and then transferred to a T75 culture flask. 40 mL of Cellbanker was added to bring the final volume to 60 mL. The viable cell density, measured using the AO / PI method, was 1.75 × 10⁻⁶ cells / mL. 7 The cell density was 98% viability, with cells / mL. 44.7 mL of cellbanker was added to adjust the cell density to 1×10⁻⁶ cells / mL. 7 cells / mL. Aliquot 1 mL into cryovials and store at -80°C for use as working cells.

[0081] S6. Obtaining blood cells

[0082] Using EDTA as an anticoagulant, 110 mL of peripheral blood was collected from an adult. 2 mL of this blood was then used to perform 15 consecutive white blood cell counts using a five-part differential hematology analyzer. The average white blood cell density of the 15 counts was calculated to be 5.09 × 10⁻¹². 9 cells / L, CV: 1.29%.

[0083] Transfer peripheral blood to a 250 mL preparation bottle free of mold, sterility, and pyrogens, mix well, and transfer 50 mL of the mixture to two 50 mL centrifuge tubes, labeling them as low and high values. Centrifuge at 4℃ and 2000g for 10 min to separate plasma and blood cells. Aspirate the plasma and label it as low and high values ​​respectively. Store the plasma at 2~8℃ for later use, while retaining the blood cells in the preparation bottle.

[0084] S7 and CD34 positive cells were mixed with blood cells and fixed.

[0085] A single CD34-positive working cell line stored at -80℃ was serially diluted and the mean total cell density was measured using the AO / PI method, which yielded a result of 5.532 × 10⁻⁶ cells / mL. 6 cells / mL.

[0086] The mean peripheral blood white blood cell density measured by S6 was 5.09 × 10⁻⁶. 9 cells / L, mean total cell density of CD34 positive cells 5.532 × 10⁻⁶ 6 The peripheral blood volume was 50 mL. Based on the low-value control (0.170% CD34-positive cells) and high-value control (0.580% CD34-positive cells), the calculated amounts of CD34-positive cells were added to the preparation bottle containing blood cells. A mixed cell line of blood cells and CD34-positive cells was obtained and fixed.

[0087] The fixation method is as follows: 0.1% (w / v) CrCl3 solution and 0.35% (w / v) paraformaldehyde solution are prepared in advance using DPBS.

[0088] Mix the 0.1% CrCl3 solution with the mixed cells at a 1:1 volume ratio and fix at 2–8°C for 1 h. Then centrifuge at 4°C, 2000g for 10 min, discard the supernatant, wash the mixed cells twice with DPBS, and discard the supernatant again. Next, mix the 0.35% paraformaldehyde solution with the mixed cells at a 1:1 volume ratio and fix at 2–8°C for 18 h. Then centrifuge at 4°C, 2000g for 10 min, discard the supernatant, wash the mixed cells twice with DPBS, and discard the supernatant again to obtain the fixed mixed cells.

[0089] The plasma preserved in S6 was added back to the corresponding fixed mixed cells and mixed thoroughly to prepare CD34. + Low-value and high-value quality control products should be stored at 2-8℃.

[0090] In other embodiments, if the fixed mixed cells are not immediately required to be used to prepare quality control samples, a prepared paraformaldehyde solution can be added as a preservation solution for preservation. When the preserved mixed cells are needed to prepare quality control samples, paraformaldehyde solution is added and the volume is adjusted to the original peripheral blood volume (50 ml in this embodiment) to obtain CD34. + Low-value quality control products and high-value quality control products.

[0091] S8 and CD34 positive cell rates and absolute white blood cell count

[0092] Take CD34 respectively + 2.5 mL of low-value and high-value quality control samples were used to perform 20 consecutive white blood cell counts using a five-part differential hematology analyzer. The mean white blood cell density of the 20 counts was calculated as follows: low-value quality control: 5028 cells / μL, CV: 1.29%; high-value quality control: 5005 cells / μL, CV: 1.26%.

[0093] CD34 was analyzed using flow cytometry. + The positive rates of CD34-positive cells in low-value and high-value quality control samples were tested 20 times consecutively, and the average positive rates of CD34-positive cells in the 20 results were calculated as follows: low-value quality control: 0.172% (0.111~0.232%); high-value quality control: 0.437% (0.349~0.525%).

[0094] I. Stability Test Experiment

[0095] The CD34 positivity rate of the low-value and high-value quality control samples prepared in Example 1 was tested for 58 consecutive days (days 0, 1, 2, 5, 9, 16, 23, 30, 37, 44, 51, and 58). The changes in the CD34 positivity rate were observed, and the results showed that the CD34 positivity rate of the low-value and high-value quality control samples remained within the target range from day 0 to day 51 (low-value quality control: 0.111–0.232%, high-value quality control: 0.349–0.525%). However, the test results on day 58 showed off-target effects. Therefore, the quality control samples prepared in Example 1 of this invention can be stably stored for 51 days. This demonstrates that the quality control samples for leukocyte differentiation antigen CD34 flow cytometry detection prepared by the method of this invention have a shelf life of up to 50 days.

[0096] II. Performance Testing

[0097] The quality control sample prepared using Example 1 of this invention was compared with the quality control sample manufactured by BD Company (product name: Stem CellControl CD34). + The performance of Whole Blood Process Control was compared. The experimental group received PE-CD34 fluorescent antibody, while the control group received PE without antibody fluorescent tag.

[0098] Comparative Example 1 is a quality control sample manufactured by BD Company. The test results are shown below. Figure 1-4 The test results of Embodiment 1 of the present invention are shown in [the table below]. Figure 5-8 Results from flow cytometry Figures 1-8 It can be seen that the quality control products provided in the embodiments of the present invention are very close in performance to the imported quality control products of BD Company.

[0099] In summary, the quality control product for flow cytometry detection of leukocyte differentiation antigen CD34 provided by this invention is the first quality control product in China used for CD34 flow cytometry counting, filling a product gap in this field. The quality control product is similar in performance to imported reagents, and the operation method is the same, without adding additional learning costs; moreover, the product shelf life can be stably maintained at 45 to 50 days, and the cost of use is far lower than that of imported quality control products.

[0100] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0101] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a quality control sample for flow cytometry detection of leukocyte differentiation antigen CD34, characterized in that, Includes the following steps: S1, Construction of the expression vector: The gene encoding the leukocyte differentiation antigen CD34 was amplified using genetic engineering technology, and the gene fragment was inserted into the expression vector to obtain the CD34 expression vector; the amino acid sequence of the leukocyte differentiation antigen CD34 is SEQ NO.1 or SEQ NO.2; S2, Packaging of viral vector: The CD34 expression vector and packaging plasmid are transfected into packaging cell lines to obtain a virus containing the gene encoding the leukocyte differentiation antigen CD34; S3, Target cell infection: The virus obtained in S2 is used to infect target cells, causing the gene encoding leukocyte differentiation antigen CD34 to be integrated into the genome of the target cells and express leukocyte differentiation antigen CD34. S4, Magnetic bead sorting of CD34 positive cells: Infected target cells are sorted out by CD34 antibody magnetic beads to select qualified CD34 positive cell lines. S5, CD34-positive cell expansion culture: Qualified CD34-positive cell lines were expanded through culture until the cell number reached 1×10⁻⁶. 9 When the cell number reaches 1 × 10⁶ cells or more, aliquot the cells into cryovials at the first preset concentration to serve as first-generation seed cells, and freeze them in liquid nitrogen. Take one vial of first-generation seed cells for further expansion culture. When the number of cultured cells reaches 1 × 10⁶ cells / cells... 9 When the cell number reaches 1 × 10⁶ cells or more, aliquot the cells into cryovials according to the second preset concentration to serve as second-generation seed cells, and freeze them in liquid nitrogen; take one vial of second-generation seed cells for further expansion culture, and when the number of cultured cells reaches 1 × 10⁶ cells / cells... 9 When cells or more are used, they are dispensed into cryovials according to the third preset concentration and used as working cells, and then frozen in an ultra-low temperature freezer for later use. S6, Obtain blood cells: Collect peripheral blood and count the white blood cells in the peripheral blood, ensuring the white blood cell concentration is within 4.0 × 10⁻⁶. 9 ~ 10.0×10 9 Peripheral blood within the cells / L range was centrifuged at low temperature to separate plasma from blood cells; S7. CD34-positive cells are mixed and fixed with blood cells: The working cells obtained in S5 are added to the blood cells obtained in S6 at a predetermined ratio to obtain mixed cells; the mixed cells are fixed to obtain fixed mixed cells; the fixed mixed cells are prepared into quality control samples and stored at 4℃ ~ 8℃; wherein, the predetermined ratio is: in CD34... + In low-value quality control samples, the proportion of CD34-positive cells in peripheral blood leukocytes was 0.1% to 0.3%; CD34 + In high-value quality control products, the proportion of CD34-positive cells in peripheral blood leukocytes is 0.3% to 0.6%.

2. The method for preparing the quality control material for flow cytometry detection of leukocyte differentiation antigen CD34 as described in claim 1, characterized in that, The target cells are any one of nalm6 cells, KG-1a cells, Jurkat cells, or Daudi cells.

3. The method for preparing the quality control material for flow cytometry detection of leukocyte differentiation antigen CD34 as described in claim 1, characterized in that, In step S1, the expression vector is selected from pCDH-EF1-MCS-T2A-Puro. pCDH-CMV-MCS-EF1-Puro, pCDH-CMV-MCS-EF1-copGFP, pCDH-CMV-MCS-EF1-copGFP-T2A-Puro, pCDH-CMV-MCS-EF1-RFP, Or, any one of CDH-MCS-T2A-Puro-MSCV.

4. The method for preparing the quality control material for flow cytometry detection of leukocyte differentiation antigen CD34 as described in claim 1, characterized in that, In step S2, the packaging plasmid is selected from any of the following combinations: Combination 1: pMD2.G, pRSV-rev, and pMDLg / pRRE; Combination 2: pPACKH1-GAG, pPACKH1-REV, and pVSV-G; Combination 3: pMD2.G and psPAX2.

5. The method for preparing the quality control material for flow cytometry detection of leukocyte differentiation antigen CD34 as described in claim 1, characterized in that, In step S4, the infected target cells are screened using CD34 antibody magnetic beads to identify the initial CD34 positive cell lines. Then, the initial CD34 positive cell lines are screened a second time using flow cytometry. The initial CD34 positive cell lines with a positive rate of 97% or higher are considered as qualified CD34 positive cell lines.

6. The method for preparing the quality control material for flow cytometry detection of leukocyte differentiation antigen CD34 as described in claim 1, characterized in that, In step S5, the range of the first preset concentration, the second preset concentration, and / or the third preset concentration is 0.5 × 10⁻⁶. 7 ~1.5×10 7 cells / mL.

7. The method for preparing the quality control material for flow cytometry detection of leukocyte differentiation antigen CD34 as described in claim 1, characterized in that, In step S6, the fixing method is as follows: Prepare a 0.005–0.75% (w / v) CrCl3 solution and a 0.1–0.5% (w / v) paraformaldehyde solution in advance using solvents; Mix the CrCl3 solution with the mixed cells at a volume ratio of 1:1, fix at 0-8℃ for 5 min-18 h, centrifuge to remove the supernatant, wash the mixed cells with the solvent 1-5 times, and discard the supernatant; then mix the mixed cells with paraformaldehyde solution at a volume ratio of 1:1, fix at 0℃-8℃ for 16-22 h, centrifuge to remove the supernatant, wash the mixed cells with the solvent 1-5 times, and discard the supernatant to obtain the fixed mixed cells; Add paraformaldehyde solution to the fixed mixed cells and bring the volume up to the original peripheral blood volume to obtain the quality control product; or, add plasma separated from S6 to the fixed mixed cells to obtain the quality control product.

8. The method for preparing the quality control material for flow cytometry detection of leukocyte differentiation antigen CD34 as described in claim 7, characterized in that, The solvent is DPBS or PBS; the pH value of the solvent is 6.5 to 7.

9. The method for preparing the quality control material for flow cytometry detection of leukocyte differentiation antigen CD34 as described in claim 1, characterized in that, Step S7 further includes setting values ​​for the CD34 positive cell rate and the absolute number of white blood cells in the quality control product: using a flow cytometer to determine the CD34 positive cell rate of the quality control product and determining the target value, and using a blood cell five-part differential counter to determine the absolute number of white blood cells in the quality control product and determining the target value.

10. A quality control material for flow cytometry detection of leukocyte differentiation antigen CD34, characterized in that, It is prepared by the method described in any one of claims 1-9.

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