A quality control substance for lymphocyte subset analysis and a method for preparing the same

Abstract

The present application relates to the technical field of medical quality control products, in particular to a quality control substance for lymphocyte subset analysis and a preparation method thereof, comprising human monocyte simulation particles, human granulocyte simulation particles, human lymphocyte simulation particles, human red blood cell simulation particles, human platelet simulation particles and a preservative, wherein the human monocyte simulation particles, the human granulocyte simulation particles, the human lymphocyte simulation particles, the human red blood cell simulation particles and the human platelet simulation particles are added into the preservative, and mixed according to the concentration of human particles to obtain the quality control substance for lymphocyte subset analysis. Compared with the prior art, the quality control substance for lymphocyte subset analysis and the preparation method thereof have the advantages of simple operation, small batch difference and good stability, and can be applied to the field of quality control of flow cytometry methodological lymphocyte subset analysis.

Description

[Technical Field]

[0001] This invention relates to the field of medical quality control technology, and in particular to a quality control substance for lymphocyte subset analysis and its preparation method. [Background Technology]

[0002] Lymphocyte subsets refer to a class of lymphocytes in the blood, including T cells, B cells, and NK lymphocyte subsets. They play different roles in the immune system; for example, T cells are responsible for cell-mediated immunity, B cells are responsible for humoral immunity, and NK cells can directly kill virus-infected target cells or tumor cells.

[0003] Lymphocytes are the main cell class constituting the immune system, accounting for 20%-45% of the total number of leukocytes in peripheral blood. An adult body contains approximately 10¹² lymphocytes. T lymphocytes (T cells) originate from hematopoietic stem cells in the bone marrow and differentiate into T cells under the influence of thymosin and the thymic microenvironment; therefore, T cells are also called thymus-dependent lymphocytes. T cells mainly participate in cell-mediated immunity, expressing the CD3 antigen. T cells include helper T cells (Th cells) and cytotoxic T cells (Tc cells), which express CD4 and CD8, respectively. B cells mainly participate in humoral immunity, expressing the CD19 antigen; NK cells express CD16 and / or CD56, spontaneously exerting cytotoxic effects in the body without antigen stimulation. Lymphocytes play a central role in the immune response.

[0004] The immune system is a double-edged sword; both excessive and insufficient immune function can harm human health. Therefore, clinical monitoring and assessment of patients' immune function are essential. The proportion, quantity, and function of different lymphocyte subsets directly affect the body's immune status, and lymphocyte subset analysis is a crucial indicator for detecting immune function. It reflects the body's current immune function, status, and balance, and can assist in the diagnosis of certain diseases. It is also significant for analyzing pathogenesis, observing treatment efficacy, and predicting prognosis. Abnormal T lymphocyte subset values ​​are observed in the peripheral blood of cancer patients. A characteristic feature is a significant decrease in CD3+ and CD4+ cells, while CD8+ cells are significantly increased, and the CD4+ / CD8+ ratio is significantly reduced. This indicates that cancer patients are in a state of immunosuppression, with a decreased ability to recognize and kill mutated cells, leading to tumor growth and metastasis. Impaired proliferation and differentiation of bone marrow hematopoietic cells are also related to abnormal T cell subsets. For example, in aplastic anemia and granulocytopenia, patients have a decreased number of CD4+ cells and an increased number of CD8+ cells in their peripheral blood, and the CD4+ / CD8+ ratio is significantly reduced.

[0005] Elevated B lymphocyte counts are seen in bacterial infections, allergic reactions, and autoimmune diseases; decreased B lymphocyte counts indicate weakened immune function and are seen in recurrent infections and severe viral infections.

[0006] NK cells (CD3-CD16+ and / or CD56+) can mediate cytotoxic effects against certain tumor cells and virus-infected cells. Elevated NK cell counts are mainly seen in the early stages of infection, early stages of tumors, long-term use of drugs such as interferon, after bone marrow transplantation, and recurrent miscarriage; decreased NK cell counts indicate weakened immune function and are seen in viral infections, malignant tumors, immunodeficiency, HIV, etc.

[0007] Currently, flow cytometry uses whole blood and cryopreserved cell quality control materials for lymphocyte subset analysis. However, these methods have the following drawbacks: 1. Short storage period: human whole blood has a shelf life of only 3 months, and the raw materials are not readily available; 2. Complex usage process: cryopreserved cells require prior cell thawing; 3. Significant batch-to-batch variation, making accurate quality control of the detection system impossible and severely impacting clinical applications. [Summary of the Invention]

[0008] To overcome the above problems, this invention proposes a quality control material for lymphocyte subset analysis and its preparation method that can effectively solve the above problems.

[0009] The present invention provides a technical solution to solve the above-mentioned technical problems by providing a quality control material for lymphocyte subset analysis and its preparation method, including human monocyte mimic particles, human granulocyte mimic particles, human lymphocyte mimic particles, human erythrocyte mimic particles, human platelet mimic particles and preservation solution.

[0010] Preferably, the human mononuclear cell mimic particles include a first microsphere, the surface of which is coated with CD45 antigen by physical adsorption or covalent binding.

[0011] Preferably, the human granulocyte mimic particles include a second microsphere, the surface of which is coated with the CD45 antigen by physical adsorption or covalent binding.

[0012] Preferably, the human lymphocyte mimic particles include a third microsphere, the surface of which is coated with one or more of the antigens of CD45, CD3, CD4, CD8, CD19, and CD16, by physical adsorption or covalent binding.

[0013] Preferably, the human erythrocyte mimic particles are obtained by centrifuging and separating fresh mammalian anticoagulated whole blood, washing the red blood cells three times with 10mM PBS buffer, fixing the purified red blood cells with 0.05%-5% formaldehyde, and then washing and concentrating the fixed red blood cells to obtain the human erythrocyte mimic particles.

[0014] The human platelet mimic particles were obtained by centrifuging and separating fresh mammalian anticoagulated whole blood, washing the platelets three times with 10mM PBS buffer, fixing the purified platelets with 0.05%-5% formaldehyde, and then washing and concentrating the fixed platelets to obtain the platelet mimic particles.

[0015] Preferably, the preservation solution comprises a buffer solution, a dispersant, an antibacterial agent, and a matrix, wherein the concentration of the buffer solution is 10-100 mmol / L, the concentration of the dispersant is 0.005%-5%, and the concentration of the antibacterial agent is 0.005%-5%.

[0016] Preferably, the first microsphere is a microsphere with a particle size of 7-8 μm, with carboxyl or amino groups on the surface, and is magnetic or non-magnetic; the second microsphere is a microsphere with a particle size of 6-7 μm, with carboxyl or amino groups on the surface, and is magnetic or non-magnetic; the third microsphere is a microsphere with a particle size of 5-6 μm, with carboxyl or amino groups on the surface, and is magnetic or non-magnetic.

[0017] A method for preparing quality control materials for lymphocyte subset analysis includes the following steps:

[0018] Step S1, Material Preparation;

[0019] Step S2, preparation of human mononuclear cell mimic particles;

[0020] Step S3, preparation of human granulocyte mimic particles;

[0021] Step S4, preparation of human lymphocyte mimic particles;

[0022] Step S5: Preparation of human erythrocyte mimic particles and human platelet mimic particles;

[0023] Step S6: Preparation of quality control materials for lymphocyte subset analysis.

[0024] Preferably, in step S2, the preparation of human mononuclear cell mimic particles includes the following steps:

[0025] Step S21: Select microspheres and antigen, and adjust the antigen concentration to 1 mg / mL using MES buffer.

[0026] Step S22: Take the microsphere suspension, add EDC solution and Sulfo-NHS solution in sequence, mix well, and react at 25°C for 30 minutes.

[0027] Step S23: After magnetic separation of the microspheres, discard the supernatant, resuspend in MES buffer, mix well, and then magnetically separate again to discard the supernatant.

[0028] Step S24: Add 10-50 μL of the antigen from step S21 to the microspheres from step S23, add MES buffer to make up to 0.2 mL, mix well, and react at 25°C for 3-12 hours.

[0029] Step S25: Magnetically separate the microspheres from step S24, discard the supernatant, add 0.4 mL of blocking solution, mix and block the reaction at 25 °C for 4-18 hours.

[0030] Step S26: Microsphere magnetic separation, discard the supernatant, add 10-50 mL of preservation solution to resuspend, and store at 4℃.

[0031] Preferably, in step S4, the preparation of human lymphocyte mimic particles includes the following steps:

[0032] Step S41, Antigen preparation;

[0033] Step S42: Take the microspheres, magnetically separate them, discard the supernatant, and resuspend them in MES buffer.

[0034] Step S43: Take EDC and Sulfo-NHS and prepare a 50 mg / mL solution using MES buffer.

[0035] Step S44: Add EDC solution and Sulfo-NHS solution to the microsphere suspension in sequence, mix well, and react at 25°C for 30 minutes.

[0036] Step S45: Magnetic separation of microspheres, discarding the supernatant, resuspending in MES buffer, mixing well, dividing evenly into multiple tubes and labeling each tube (0.2 mL), magnetic separation again, discarding the supernatant.

[0037] Step S46: Add the corresponding antigen to the multi-tube microspheres from step S45, add MES buffer to make up to 0.2 mL, mix well, and react at 25°C for 3 hours.

[0038] Step S47: Magnetic separation of microspheres, discard supernatant, add 0.4 mL blocking solution to each tube of microspheres, mix and block at 25 °C for 4-18 hours;

[0039] Step S48: Magnetic separation of microspheres, discard the supernatant, add 10-100 mL of preservation solution to each tube of microspheres for resuspending, and store at 4°C.

[0040] Compared with existing technologies, the quality control material for lymphocyte subset analysis and its preparation method of the present invention are stable, have small batch-to-batch variation, low cost, and are widely used in clinical applications. Therefore, the quality control material for lymphocyte subset analysis is very important. The preparation method is simple to operate and has good stability, and can be applied to the quality control of lymphocyte subset analysis in flow cytometry. [Attached Image Description]

[0041] Figure 1 This is a flowchart of the method for preparing quality control materials for lymphocyte subset analysis according to the present invention;

[0042] Figure 2 This is a distribution map of lymphocytes (Lym) using the quality control material for lymphocyte subset analysis and its preparation method of the present invention.

[0043] Figure 3 This is a T lymphocyte distribution map of the quality control material and its preparation method used for lymphocyte subset analysis in this invention.

[0044] Figure 4 This is a distribution map of CD4 and T lymphocytes of the quality control material and its preparation method used for lymphocyte subset analysis in this invention;

[0045] Figure 5 This is a distribution map of CD8 and T lymphocytes of the quality control material and its preparation method used for lymphocyte subset analysis in this invention.

[0046] Figure 6 This is a B lymphocyte distribution map of the quality control material and its preparation method used for lymphocyte subset analysis in this invention.

[0047] Figure 7 This is a distribution map of NK lymphocytes for the quality control material and its preparation method used in the lymphocyte subset analysis of this invention.

Detailed Implementation Methods

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0049] It should be noted that in the embodiments of the present invention, all directional indications (such as up, down, left, right, front, back, etc.) are limited to relative positions on the specified view, rather than absolute positions.

[0050] Furthermore, in this invention, descriptions involving "first," "second," etc., are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0051] Please see Figures 1 to 7The quality control material for lymphocyte subset analysis of the present invention, based on flow cytometry technology, includes human monocyte mimic particles, human granulocyte mimic particles, human lymphocyte mimic particles, human erythrocyte mimic particles, human platelet mimic particles, and a preservation solution. The quality control material for lymphocyte subset analysis of the present invention can be used for quality control of T cells, B cells, NK cells, BNK cells, and TBNK lymphocyte subsets.

[0052] The human mononuclear cell mimic particles include a first microsphere, the surface of which is coated with the CD45 antigen by physical adsorption or covalent binding.

[0053] The first microsphere is a microsphere with a particle size of 7-8 μm, with carboxyl or amino groups on the surface, and may be magnetic or non-magnetic.

[0054] The human granulocyte mimic particles include a second microsphere coated with CD45 antigen via physical adsorption or covalent binding.

[0055] The second microsphere is a microsphere with a particle size of 6-7 μm, and has carboxyl or amino groups on its surface. It may be a magnetic or non-magnetic microsphere.

[0056] The human lymphocyte mimic particles include a third microsphere, the surface of which is coated with one or more of the antigens of CD45, CD3, CD4, CD8, CD19, and CD16, by physical adsorption or covalent binding.

[0057] The third microsphere is a microsphere with a particle size of 5-6 μm, and has carboxyl or amino groups on its surface. It may be a magnetic or non-magnetic microsphere.

[0058] The human erythrocyte mimic particles are obtained from fresh mammalian anticoagulated whole blood, such as bovine or porcine whole blood. The erythrocytes are obtained by centrifugation and separation, washed three times with 10mM PBS buffer, and then fixed with 0.05%-5% formaldehyde. The fixed erythrocytes are then washed and concentrated to obtain the human erythrocyte mimic particles of the present invention.

[0059] The human platelet mimic particles are obtained from fresh mammalian anticoagulated whole blood, such as bovine or porcine whole blood. Platelets are obtained by centrifugation and separation, washed three times with 10mM PBS buffer, and the purified platelets are fixed in 0.05%-5% formaldehyde. The fixed platelets are then washed and concentrated to obtain the platelet mimic particles of the present invention.

[0060] The preservation solution comprises a buffer solution, a dispersant, an antibacterial agent, and a matrix. The concentration of the buffer solution is 10-100 mmol / L, the concentration of the dispersant is 0.005%-5%, and the concentration of the antibacterial agent is 0.005%-5%.

[0061] The buffer solution is one of phosphate buffer, HEPES buffer, Tris buffer, or glycine buffer.

[0062] The dispersant is a surfactant, which may be one of Tween-20, Tween-40, Tween-80, Triton-100, or poloxamer.

[0063] The antibacterial agent is one of sodium azide, Prclin-300, BND-10, or Prclin-950.

[0064] The preferred matrix is ​​BSA (0.1-0.5% w / v) and / or animal serum (20-50% v / v), etc.

[0065] The pH value of the preservation solution is controlled within the range of 6.0-8.0, and the osmotic concentration is controlled within the range of 240-280 mmol / L.

[0066] The method for preparing quality control materials for lymphocyte subset analysis of the present invention includes the following steps:

[0067] Step S1: Material preparation.

[0068] Step S2, preparation of human mononuclear cell mimic particles.

[0069] Step S3: Preparation of human granulocyte mimic particles.

[0070] Step S4: Preparation of human lymphocyte mimic particles.

[0071] Step S5: Preparation of human erythrocyte mimic particles and human platelet mimic particles.

[0072] Step S6: Preparation of quality control materials for lymphocyte subset analysis.

[0073] In step S2, the preparation of human mononuclear cell mimic particles includes the following steps:

[0074] Step S21: Select microspheres and antigen, and adjust the antigen concentration to 1 mg / mL using MES buffer.

[0075] Step S22: Take the microsphere suspension, add EDC solution and Sulfo-NHS solution in sequence, mix well, and react at 25°C for 30 minutes.

[0076] Step S23: Magnetic separation of microspheres, discarding the supernatant, resuspending in MES buffer, mixing, and magnetic separation again, discarding the supernatant.

[0077] Step S24: Add 10-50 μL of the antigen from step S21 to the microspheres from step S23, add MES buffer to make up to 0.2 mL, mix well, and react at 25°C for 3-12 hours.

[0078] In step S25, the microspheres from step S24 are magnetically separated, the supernatant is discarded, 0.4 mL of blocking solution is added, and the mixture is mixed and the blocking reaction is carried out at 25 °C for 4-18 hours.

[0079] Step S26: Microsphere magnetic separation, discard the supernatant, add 10-50 mL of preservation solution to resuspend, store at 4℃, the concentration should not be diluted too much to facilitate the subsequent addition of other components.

[0080] In step S3, the preparation of human granulocyte mimic particles includes the following steps:

[0081] Steps S31 to S36 are the same as steps S21 to S26.

[0082] In step S4, the preparation of human lymphocyte mimic particles includes the following steps:

[0083] Step S41, antigen preparation.

[0084] Step S42: Take the microspheres, perform magnetic separation, discard the supernatant, and resuspend them in MES buffer.

[0085] Step S43: Take EDC and Sulfo-NHS and prepare a 50 mg / mL solution using MES buffer.

[0086] Step S44: Add EDC solution and Sulfo-NHS solution to the microsphere suspension in sequence, mix well, and react at 25°C for 30 minutes.

[0087] Step S45: Magnetic separation of microspheres, discarding the supernatant, resuspending in MES buffer, mixing well, dividing evenly into multiple tubes and labeling each tube (0.2 mL), magnetic separation again, discarding the supernatant.

[0088] In step S46, add the corresponding antigen to the multi-tube microspheres from step S45, replenish with MES buffer to 0.2 mL, mix well, and react at 25°C for 3 hours.

[0089] Step S47: Magnetic separation of microspheres, discard the supernatant, add 0.4 mL of blocking solution to each tube of microspheres, mix and block the reaction at 25 °C for 4-18 hours.

[0090] Step S48: Magnetic separation of microspheres, discard the supernatant, add 10-100 mL of preservation solution to each tube of microspheres for resuspending, and store at 4°C.

[0091] In step S5, the preparation of human erythrocyte mimic particles and human platelet mimic particles includes:

[0092] Preparation of human erythrocyte mimic particles and human platelet mimic particles.

[0093] The preparation of the human erythrocyte mimic particles preferably involves selecting fresh mammalian anticoagulated whole blood, such as bovine whole blood, centrifuging and separating to obtain erythrocytes, washing them three times with PBS buffer, adding 0.05%-5% formaldehyde to the purified erythrocytes for fixation, and then washing and concentrating the fixed erythrocytes to obtain the human erythrocyte mimic particles.

[0094] The preparation of the human platelet mimic particles preferably involves selecting fresh mammalian anticoagulated whole blood, such as bovine whole blood, centrifuging and separating platelets, washing them three times with PBS buffer, adding 0.05%-5% formaldehyde to the purified platelets for fixation, and then washing and concentrating the fixed platelets to obtain the human platelet mimic particles.

[0095] In step S6, the preparation of quality control material for lymphocyte subset analysis involves adding human monocyte simulated particles, human granulocyte simulated particles, human lymphocyte simulated particles, human erythrocyte simulated particles, and human platelet simulated particles to the preservation solution and mixing them according to the concentration of human particles to obtain the quality control material for lymphocyte subset analysis.

[0096] The preparation method of the quality control material for lymphocyte subset analysis of the present invention will be described below through specific embodiments.

[0097] Example 1: Preparation of quality control materials for T cell analysis

[0098] Table 1. Composition of quality control materials for T cell analysis

[0099]

[0100] Step A1, Material Preparation:

[0101] 8μm magnetic microspheres (concentration: 8×106 particles / mL, surface carboxyl group activated);

[0102] 7μm magnetic microspheres (concentration: 8×106 particles / mL, surface carboxyl group activated);

[0103] 6μm magnetic microspheres (concentration: 8×106 particles / mL, surface carboxyl group activated);

[0104] Antigens (CD45, CD3, CD4, CD8, CD16, CD19);

[0105] EDC;

[0106] Sulfo-NHS;

[0107] PBS buffer (10mM, pH 7.4) can be adjusted with sodium chloride to achieve an osmotic concentration in the range of 240-280 mmol / L.

[0108] Tris buffer (50 mM, pH 8.2);

[0109] MES buffer (50mM, pH 6.0);

[0110] Blocking solution: Tris buffer + 1% BSA;

[0111] Preservative solutions: BSA (0.1% w / v), PBS (10 mmol / L), animal serum (50% v / v), proclin 300 (0.1% v / v), Tween 20 (0.2‰ v / v), with osmotic concentration adjusted to 240-280 mmol / L and pH 7.0-7.4; magnets, etc.

[0112] Step A2, Preparation of human mononuclear cell mimic particles:

[0113] Step A21, Microspheres: Select magnetic microspheres with a diameter of 8 μm (concentration: 8 × 10⁶ microspheres / mL, surface carboxyl group activated);

[0114] Step A22, Antigen: Take 10-100 μg of CD45 antigen and adjust the volume with MES buffer to an antigen concentration of 1 mg / mL;

[0115] Step A23: Take 1 mL of the microsphere suspension from step A21, add 0.08 mL of EDC solution and 0.08 mL of Lulfo-NHS solution in sequence, mix well, and react at 25°C for 30 minutes.

[0116] Step A24: After magnetic separation of the microspheres, discard the supernatant, resuspend in 0.8 mL of MES buffer, mix well, and then perform magnetic separation again to discard the supernatant.

[0117] Step A25: Add 10-50 μL of the antigen from step A22 to the microspheres from step A24, add MES buffer to make up to 0.2 mL, mix well, and react at 25°C for 3-12 hours.

[0118] Step A26: Magnetically separate the microspheres from Step A25, discard the supernatant, add 0.4 mL of blocking solution, mix and block the reaction at 25 °C for 4-18 hours;

[0119] Step A27: Microsphere magnetic separation, discard the supernatant, add 10-50 mL of preservation solution to resuspend, store at 4℃, the concentration should not be diluted too much, so as to facilitate the subsequent addition of other components.

[0120] Step A3, preparation of human granulocyte mimic particles.

[0121] Step A31, Microspheres: Select magnetic microspheres with a diameter of 7 μm (concentration: 8 × 10⁶ microspheres / mL, surface carboxyl group activated).

[0122] Step A32, Antigen: Take 10-100 μg of CD45 antigen and adjust the volume with MES buffer to a concentration of 1 mg / mL for each antigen.

[0123] Preparation: Same as the preparation method of "human mononuclear cell mimic particles" above, add 10-50 mL of preservation solution to resuspend, store at 4℃, the concentration should not be diluted too much, so as to facilitate the subsequent addition of other components.

[0124] Step A4, Preparation of human lymphocyte mimic particles.

[0125] Step A41, Antigen Preparation:

[0126] A antigen: Take 10-100 μg of each of CD45, CD3, CD4 and CD8 antigens, mix until homogeneous, and adjust the volume with MES buffer to a concentration of 1 mg / mL for each antigen;

[0127] B antigen: Take 10-100 μg of CD45 and CD3 antigens, mix them until homogeneous, and adjust the volume with MES buffer to a concentration of 1 mg / mL for each antigen.

[0128] C antigen: Take 10-100 μg of CD45 antigen and adjust the volume with MES buffer to a concentration of 1 mg / mL for each antigen.

[0129] Step A42: Take 3.75 mL of microspheres, magnetically separate them, discard the supernatant, and resuspend them in 0.6 mL of MES buffer.

[0130] Step A43: Weigh 5 mg of EDC and 50 mg of Sulfo-NHS, and prepare a 50 mg / mL solution using MES buffer.

[0131] Step A44: Add 0.06 mL of EDC solution and 0.06 mL of Sulfo-NHS solution to the microsphere suspension in sequence, mix well, and react at 25°C for 30 minutes.

[0132] Step A45: Magnetic separation of microspheres, discarding the supernatant, resuspending in 0.6 mL MES buffer, mixing well, and dividing evenly into 3 tubes labeled A, B, and C, 0.2 mL per tube, followed by magnetic separation and discarding the supernatant again;

[0133] Step A46: Add the corresponding antigens to the three tubes of microspheres from step A45, namely antigen A, antigen B, and antigen C, 10-50 μL of each, and replenish with MES buffer to 0.2 mL. Mix well and react at 25°C for 3 hours.

[0134] Step A47: Magnetic separation of microspheres, discard the supernatant, add 0.4 mL of blocking solution to each tube of microspheres, mix and block at 25 °C for 4-18 hours;

[0135] Step A48: Magnetic separation of microspheres, discard the supernatant, add 10-100 mL of the preservation solution in this protocol to each tube of microspheres for resuspending, and store at 4°C.

[0136] Step A5, the preparation of human erythrocyte mimic particles and human platelet mimic particles, includes:

[0137] Preparation of human erythrocyte mimic particles and human platelet mimic particles.

[0138] The preparation of the human erythrocyte mimic particles preferably involves selecting fresh mammalian anticoagulated whole blood, such as bovine whole blood, centrifuging and separating to obtain erythrocytes, washing them three times with PBS buffer, adding 0.05%-5% formaldehyde to the purified erythrocytes for fixation, and then washing and concentrating the fixed erythrocytes to obtain the human erythrocyte mimic particles.

[0139] The preparation of the human platelet mimic particles preferably involves selecting fresh mammalian anticoagulated whole blood, such as bovine whole blood, centrifuging and separating platelets, washing them three times with PBS buffer, adding 0.05%-5% formaldehyde to the purified platelets for fixation, and then washing and concentrating the fixed platelets to obtain the human platelet mimic particles.

[0140] Step A6, Preparation of T cell analysis quality control material: The above-mentioned simulated particles are added to the preservation solution of the present invention and mixed according to the concentration of human particles to obtain the preparation of T cell analysis quality control material.

[0141] Example 2B: Preparation of quality control materials for cell analysis

[0142] Table 2. Composition of quality control materials for B cell analysis

[0143]

[0144] Step B1, Material Preparation:

[0145] Antigens (CD45, CD3, CD19)

[0146] Everything else is the same as in Example 1.

[0147] The preparation of human mononuclear cell mimic particles, human granulocyte mimic particles, human erythrocyte mimic particles, and human platelet mimic particles is the same as in Example 1.

[0148] Step B3, Preparation of human lymphocyte mimic particles:

[0149] Step B31, Antigen Preparation:

[0150] A antigen: Take 10-100 μg of CD45 and CD3 antigens, mix them until homogeneous, and adjust the volume with MES buffer to a concentration of 1 mg / mL for each antigen.

[0151] B antigen: Take 10-100 μg of CD45 and CD19 antigens, mix them until homogeneous, and adjust the volume with MES buffer to a concentration of 1 mg / mL for each antigen.

[0152] C antigen: Take 10-100 μg of CD45 antigen and adjust the volume with MES buffer to a concentration of 1 mg / mL for each antigen.

[0153] Other operations are the same as in Example 1.

[0154] Step B4, Preparation of quality control materials for B cell analysis:

[0155] The simulated particles described above were added to the preservation solution of this invention and mixed according to the concentration of human particles to obtain the preparation of the B cell analysis quality control material.

[0156] Example 3: Preparation of quality control materials for NK cell analysis

[0157] Table 3. Composition of quality control materials for NK cell analysis

[0158]

[0159]

[0160] Step C1, Material Preparation:

[0161] Antigens (CD45, CD3, CD16)

[0162] Everything else is the same as in Example 1.

[0163] The preparation of human mononuclear cell mimic particles, human granulocyte mimic particles, human erythrocyte mimic particles, and human platelet mimic particles is the same as in Example 1.

[0164] Step C3, Preparation of human lymphocyte mimic particles:

[0165] Step C31, Antigen Preparation:

[0166] A antigen: Take 10-100 μg of CD45 and CD3 antigens, mix them until homogeneous, and adjust the volume with MES buffer to a concentration of 1 mg / mL for each antigen.

[0167] B antigen: Take 10-100 μg of CD45 and CD16 antigens, mix them until homogeneous, and adjust the volume with MES buffer to a concentration of 1 mg / mL for each antigen.

[0168] C antigen: Take 10-100 μg of CD45 antigen and adjust the volume with MES buffer to a concentration of 1 mg / mL for each antigen.

[0169] Other operations are the same as in Example 1.

[0170] Step C34, Preparation of quality control materials for NK cell analysis:

[0171] The simulated particles described above were added to the preservation solution of this invention and mixed according to the concentration of human particles to obtain the preparation of the NK cell analysis quality control material.

[0172] Example 4: Preparation of quality control materials for TBNK lymphocyte subset analysis

[0173] Table 4. Composition of quality control materials for TBNK lymphocyte subset analysis

[0174]

[0175] Step D1, Material Preparation:

[0176] Antigens (CD45, CD3, CD4, CD8, CD16, CD19)

[0177] Everything else is the same as in Example 1.

[0178] The preparation of human mononuclear cell mimic particles, human granulocyte mimic particles, human erythrocyte mimic particles, and human platelet mimic particles is the same as in Example 1.

[0179] Step D3, Preparation of human lymphocyte mimic particles:

[0180] Step D31, Antigen Preparation:

[0181] A antigen: Take 10-100 μg each of CD45, CD3, CD4, CD8 antigen, mix until homogeneous, and adjust the volume with MES buffer to a concentration of 1 mg / mL for each antigen;

[0182] B antigen: Take 10-100 μg of CD45 and CD3 antigens, mix them until homogeneous, and adjust the volume with MES buffer to a concentration of 1 mg / mL for each antigen.

[0183] C antigen: Take 10-100 μg of CD45 and CD19 antigens, mix them until homogeneous, and adjust the volume with MES buffer to a concentration of 1 mg / mL for each antigen.

[0184] D antigen: Take 10-100 μg of CD45 and CD16 antigens, mix them until homogeneous, and adjust the volume with MES buffer to 1 mg / mL for each antigen.

[0185] E antigen: Take 10-100 μg of CD45 antigen and adjust the volume with MES buffer to a concentration of 1 mg / mL for each antigen.

[0186] Step D32: Take 6.25 mL of microspheres, perform magnetic separation, discard the supernatant, and resuspend in 1 mL of MES buffer.

[0187] Step D33: Weigh 5 mg of EDC and 50 mg of Sulfo-NHS, and prepare a 50 mg / mL solution with MES buffer.

[0188] Step D34: Add 0.1 mL of EDC solution and 0.1 mL of Sulfo-NHS solution to the microsphere suspension in sequence, mix well, and react at 25°C for 30 minutes.

[0189] Step D35: Magnetic separation of microspheres, discarding the supernatant, resuspending with 1 mL of MES buffer, mixing well, and dividing evenly into 5 tubes labeled A, B, C, D, and E, with 0.2 mL in each tube, followed by magnetic separation and discarding the supernatant again;

[0190] Step D36: Add the corresponding antigens to the 5 tubes of microspheres from step D35, namely antigen A, antigen B, antigen C, antigen D and antigen E, 10-50 μL of each, and make up to 0.2 mL with MES buffer. Mix well and react at 25°C for 3 hours.

[0191] Step D37: Magnetic separation of microspheres, discard supernatant, add 0.4 mL of blocking solution to each tube of microspheres, mix and block at 25 °C for 4-18 hours;

[0192] Step D38: Magnetic separation of microspheres, discard the supernatant, add 10-100 mL of the preservation solution in this protocol to each tube of microspheres for resuspending, and store at 4°C.

[0193] Step D4, Preparation of quality control materials for TBNK lymphocyte subset analysis:

[0194] The simulated particles described above were added to the preservation solution of this invention and mixed according to the concentration of human particles to obtain the quality control material for TBNK lymphocyte subset analysis.

[0195] Evaluation of quality control materials for TBNK lymphocyte subset analysis

[0196] The quality control material obtained in this embodiment was tested experimentally. When stored at 2-8℃ in a sealed container, it can be stored for 6 months. The material showed obvious clustering and a decay rate of ≤10%, which meets clinical requirements.

[0197] The stability and clustering of the quality control materials described in this application are illustrated below using experimental data:

[0198] CD3+ 75.69％ 72.47％ -4.25％ CD4+ 52.58％ 49.37％ -6.10％ CD3+ / CD4+ 55.69％ 53.17％ -4.53％ CD8+ 41.23％ 39.76％ -3.57％ CD3+ / CD8+ 25.43％ 23.56％ -7.35％ CD19+ 22.19％ 21.45％ -3.33％ CD3- / CD16+ 35.17％ 33.89％ -3.64％

[0199] Compared with the prior art, the quality control material for lymphocyte subset analysis and its preparation method of the present invention have the following beneficial effects:

[0200] 1. It has advantages such as stable performance, low production cost, mass production capability, small batch-to-batch variation, and wider clinical application, which enable better quality control of lymphocyte subset analysis systems.

[0201] 2. It can be flexibly combined. Microspheres coated with different antigen components can be added individually for quality control analysis of T cells, B cells, NK cells or BNK cells, or they can be mixed together for quality control analysis of TBNK lymphocyte subsets.

[0202] 4. It can be widely used in interlaboratory quality assessment and to simulate whole blood for quality control.

[0203] 5. Calibrators that can be used for lymphocyte subset analysis.

[0204] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any modifications, equivalent substitutions and improvements made within the concept of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A quality control substance for lymphocyte subset analysis, characterized in that, Includes human mononuclear cell mimic particles, human granulocyte mimic particles, human lymphocyte mimic particles, human erythrocyte mimic particles, human platelet mimic particles, and preservation solution; The human mononuclear cell mimic particles include a first microsphere and a second microsphere, the surfaces of which are coated with CD45 antigen by physical adsorption or covalent binding. The human lymphocyte mimic particles include a third microsphere, the surface of which is coated with one or more of the antigens of CD45, CD3, CD4, CD8, CD19, and CD16, by physical adsorption or covalent binding. The human erythrocyte mimic particles were obtained by centrifuging and separating fresh mammalian anticoagulated whole blood, washing the red blood cells three times with 10mM PBS buffer, fixing the purified red blood cells with 0.05%-5% formaldehyde, and then washing and concentrating the fixed red blood cells to obtain the human erythrocyte mimic particles. The human platelet mimic particles were obtained by centrifuging and separating fresh mammalian anticoagulated whole blood, washing the platelets three times with 10mM PBS buffer, fixing the purified platelets with 0.05%-5% formaldehyde, and then washing and concentrating the fixed platelets to obtain the platelet mimic particles. The preservation solution comprises a buffer solution, a dispersant, an antibacterial agent, and a matrix. The concentration of the buffer solution is 10-100 mmol / L, the concentration of the dispersant is 0.005%-5%, and the concentration of the antibacterial agent is 0.005%-5%. The first microsphere has a particle size of 7-8 μm, with carboxyl or amino groups on the surface, and may be magnetic or non-magnetic; the second microsphere has a particle size of 6-7 μm, with carboxyl or amino groups on the surface, and may be magnetic or non-magnetic; the third microsphere has a particle size of 5-6 μm, with carboxyl or amino groups on the surface, and may be magnetic or non-magnetic.

2. A method for preparing a quality control material for lymphocyte subset analysis as described in claim 1, characterized in that, Includes the following steps: Step S1, Material Preparation; Step S2, preparation of human mononuclear cell mimic particles; Step S3, preparation of human granulocyte mimic particles; Step S4, preparation of human lymphocyte mimic particles; Step S5: Preparation of human erythrocyte mimic particles and human platelet mimic particles; Step S6: Preparation of quality control materials for lymphocyte subset analysis.

3. The method for preparing quality control materials for lymphocyte subset analysis as described in claim 2, characterized in that, In step S2, the preparation of human mononuclear cell mimic particles includes the following steps: Step S21: Select microspheres and antigen, and adjust the antigen concentration to 1 mg / mL using MES buffer. Step S22: Take the microsphere suspension, add EDC solution and Sulfo-NHS solution in sequence, mix well, and react at 25°C for 30 minutes. Step S23: After magnetic separation of the microspheres, discard the supernatant, resuspend in MES buffer, mix well, and then magnetically separate again to discard the supernatant. Step S24: Add 10-50 μL of the antigen from step S21 to the microspheres from step S23, add MES buffer to make up to 0.2 mL, mix well, and react at 25°C for 3-12 hours. Step S25: Magnetically separate the microspheres from step S24, discard the supernatant, add 0.4 mL of blocking solution, mix and block the reaction at 25°C for 4-18 hours; Step S26: Microsphere magnetic separation, discard the supernatant, add 10-50 mL of preservation solution to resuspend, and store at 4℃.

4. The method for preparing quality control materials for lymphocyte subset analysis as described in claim 2, characterized in that, In step S4, the preparation of human lymphocyte mimic particles includes the following steps: Step S41, Antigen preparation; Step S42: Take the microspheres, magnetically separate them, discard the supernatant, and resuspend them in MES buffer. Step S43: Take EDC and Sulfo-NHS and prepare a 50 mg / mL solution using MES buffer. Step S44: Add EDC solution and Sulfo-NHS solution to the microsphere suspension in sequence, mix well, and react at 25°C for 30 minutes. Step S45: Magnetic separation of microspheres, discarding the supernatant, resuspending in MES buffer, mixing well, dividing evenly into multiple tubes and labeling each tube (0.2 mL), magnetic separation again, discarding the supernatant. Step S46: Add the corresponding antigen to the multi-tube microspheres from step S45, add MES buffer to make up to 0.2 mL, mix well, and react at 25°C for 3 hours. Step S47: Magnetic separation of microspheres, discard the supernatant, add 0.4 mL of blocking solution to each tube of microspheres, mix and block at 25°C for 4-18 hours; Step S48: Magnetic separation of microspheres, discard the supernatant, add 10-100 mL of preservation solution to each tube of microspheres for resuspending, and store at 4°C.

Images