Quality control substance for lymphocyte subpopulation analysis and preparation method thereof

Abstract

The invention relates to the technical field of medical quality control products, in particular to a quality control substance for lymphocyte subpopulation analysis and a preparation method thereof, and the quality control substance comprises human monocyte simulation particles, human granulocyte simulation particles, human lymphocyte simulation particles, human erythrocyte simulation particles, human platelet simulation particles and a preserving fluid, adding the human monocyte simulation particles, the human granulocyte simulation particles, the human lymphocyte simulation particles, the human erythrocyte simulation particles and the human platelet simulation particles into a preserving fluid, and uniformly mixing according to the concentration of the human body particles, so as to obtain the quality control substance for lymphocyte subpopulation analysis. Compared with the prior art, the quality control substance for lymphocyte subpopulation analysis and the preparation method thereof have the advantages that the preparation method is simple to operate, small in batch difference and good in stability, and can be applied to quality control in the field of detection of quality control substances for flow cytometry lymphocyte subpopulation analysis.

Description

[Technical field]

[0001] The present invention relates to the technical field of medical quality control products, and in particular to a quality control material for lymphocyte subpopulation analysis and a preparation method thereof. [Background technology]

[0002] Lymphocyte subsets refer to a type of lymphocytes in the blood, including T cells, B cells and NK lymphocyte subsets. They play different roles in the immune system, such as T cells are responsible for cellular immune responses, B cells are responsible for humoral immune responses, and NK cells can directly kill virus-infected target cells or tumor cells.

[0003] Lymphocytes are the main cell type that constitutes the immune system, accounting for 20%-45% of the total number of peripheral blood leukocytes. There are about 1012 lymphocytes in an adult. T lymphocytes (Tlymphocyte) are referred to as T cells. T cells originate from bone marrow hematopoietic stem cells and differentiate and mature into T cells under the influence of thymosin and the thymus microenvironment, so T cells are also called thymus-dependent lymphocytes. T cells are mainly involved in cellular immunity and express CD3 antigens. Among them, T cells include helper T cells (Th) and cytotoxic T cells (Tc), which express CD4 and CD8 respectively. B cells are mainly involved in humoral immunity and express CD19 antigens; NK cells express CD16 and / or CD56, and spontaneously exert cytotoxic effects in the body without relying on antigen stimulation. Lymphocytes play a core role in immune response.

[0004] Immune function is a double-edged sword. Too strong or too weak will endanger human health. Therefore, it is necessary to monitor and evaluate the immune function of patients clinically. The proportion, number and function of different lymphocyte subsets will directly affect the body's immune status, and lymphocyte subset analysis is an important indicator for detecting immune function. It can reflect the body's current immune function, status and balance level, and can assist in the diagnosis of certain diseases. It is of great significance for analyzing pathogenesis, observing efficacy and detecting prognosis. In the peripheral blood of tumor patients, the values ​​of T lymphocyte subsets are abnormal, characterized by a significant decrease in CD3+ cells and CD4+ cells in the patient's body, a significant increase in CD8+ cells, and a significant decrease in the CD4+ / CD8+ ratio, indicating that the cellular immune function of tumor patients is in an immunosuppressive state, and the patient's ability to identify and kill mutant cells is reduced, forming tumor growth and metastasis. Proliferation and differentiation disorders of bone marrow hematopoietic cells are also related to abnormal T cell subsets. For example, in aplastic anemia and granulocytopenia, the number of CD4+ cells in the patient's peripheral blood decreases, the number of CD8+ cells increases, and the CD4+ / CD8+ ratio decreases significantly.

[0005] An increase in B lymphocytes is seen in bacterial infections, allergies, autoimmune diseases, etc.; a decrease in B lymphocytes indicates a low immune function, which is seen in recurrent infections, severe viral infections, etc.

[0006] NK cells (CD3-CD16+ and / or CD56+) can mediate cytotoxic effects on certain tumor cells and virus-infected cells. Increased NK cells are mainly seen in the early stages of infection, early stages of tumors, long-term use of interferon and other drugs, after bone marrow transplantation, recurrent miscarriage, etc. Decreased NK cells indicate low immune function, which is seen in viral infections, malignant tumors, immunodeficiency, HIV, etc.

[0007] At present, the quality control materials used in flow cytometry for lymphocyte subset analysis include whole blood quality control and frozen cell quality control, but there are the following problems: 1. The storage period is short. The shelf life of human whole blood is 3 months, and the raw materials are not easy to obtain; 2. The use process is complicated, and frozen cells need to be revived first. 3. The batch-to-batch difference is large, and it is impossible to accurately control the quality of the detection system, which seriously affects clinical application. [Summary of the invention]

[0008] In order to overcome the above problems, the present invention provides a quality control substance for lymphocyte subpopulation analysis and a preparation method thereof which can effectively solve the above problems.

[0009] The present invention provides a technical solution to solve the above technical problems: providing a quality control material for lymphocyte subpopulation analysis and a preparation method thereof, including human monocyte simulation particles, human granulocyte simulation particles, human lymphocyte simulation particles, human red blood cell simulation particles, human platelet simulation particles and preservation fluid.

[0010] Preferably, the human monocyte simulation 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 simulating particles include second microspheres, the surface of which is coated with CD45 antigen by physical adsorption or covalent binding.

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

[0013] Preferably, the human red blood cell simulation particles are prepared by centrifuging and separating fresh mammalian anticoagulated whole blood to obtain red blood cells, washing them three times with 10 mM PBS buffer, adding 0.05%-5% formaldehyde to fix the purified red blood cells, and then washing and concentrating the fixed red blood cells to obtain the human red blood cell simulation particles;

[0014] The human platelet simulation particles are prepared by centrifuging and separating fresh mammalian anticoagulated whole blood to obtain platelets, which are then washed three times with 10 mM PBS buffer. The purified platelets are fixed by adding 0.05%-5% formaldehyde, and then the fixed platelets are washed and concentrated to obtain the platelet simulation particles.

[0015] Preferably, the preservation solution comprises a buffer, a dispersant, an antibacterial agent and a matrix, the concentration of the buffer 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 microspheres are microspheres with a particle size of 7-8 μm, carboxyl groups or amino groups on the surface, and are magnetic or non-magnetic microspheres; the second microspheres are microspheres with a particle size of 6-7 μm, carboxyl groups or amino groups on the surface, and are magnetic or non-magnetic microspheres; the third microspheres are microspheres with a particle size of 5-6 μm, carboxyl groups or amino groups on the surface, and are magnetic or non-magnetic microspheres.

[0017] A method for preparing a quality control material for lymphocyte subpopulation analysis comprises the following steps:

[0018] Step S1, material preparation;

[0019] Step S2, preparation of human monocyte simulated particles;

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

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

[0022] Step S5, preparation of human red blood cell simulating particles and human platelet simulating particles;

[0023] Step S6, preparation of quality control material for lymphocyte subset analysis.

[0024] Preferably, in step S2, the preparation of human monocyte simulated particles comprises the following steps:

[0025] Step S21, selecting microspheres and antigens, and diluting the antigens with MES buffer to a concentration of 1 mg / mL;

[0026] Step S22, taking the microsphere suspension, adding EDC solution and Sulfo-NHS solution in sequence, mixing, and reacting at 25° C. for 30 minutes;

[0027] Step S23, magnetically separating the microspheres and discarding the supernatant, resuspending with MES buffer, mixing, and magnetically separating again and discarding the supernatant;

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

[0029] Step S25, magnetically separate the microspheres in step S24, discard the supernatant, add 0.4 mL of blocking solution, mix well and perform blocking reaction at 25° C. for 4-18 hours;

[0030] Step S26, microsphere magnetic separation, discard the supernatant, add 10-50mL preservation solution to resuspend, and store at 4°C.

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

[0032] Step S41, antigen preparation;

[0033] Step S42, taking the microspheres, magnetically separating them, discarding the supernatant, and adding MES buffer to resuspend them;

[0034] Step S43, taking EDC and Sulfo-NHS, and preparing a 50 mg / mL solution with MES buffer;

[0035] Step S44, adding EDC solution and Sulfo-NHS solution to the microsphere suspension in sequence, mixing, and reacting at 25° C. for 30 minutes;

[0036] Step S45, magnetically separate the microspheres and discard the supernatant, resuspend them in MES buffer, mix them and evenly divide them into multiple tubes and mark them, with 0.2 mL in each tube, and magnetically separate them again and discard the supernatant;

[0037] Step S46, respectively add corresponding antigens to the multi-tube microspheres of step S45, add MES buffer to 0.2 mL, mix well, and react at 25° C. for 3 hours;

[0038] Step S47, magnetic separation of microspheres, discarding the supernatant, adding 0.4 mL of blocking solution to each tube of microspheres, mixing and blocking reaction at 25° C. for 4-18 hours;

[0039] Step S48, magnetic separation of microspheres, discarding the supernatant, adding 10-100 mL of preservation solution to each tube of microspheres for resuspending, and storing at 4°C.

[0040] Compared with the prior art, the quality control material for lymphocyte subpopulation analysis and the preparation method thereof of the present invention have stable performance, small batch difference, low cost, and are very important for the quality control material for lymphocyte subpopulation analysis with wide clinical application; the preparation method is simple to operate and has good stability, and can be applied to quality control in the field of quality control material detection for lymphocyte subpopulation analysis using flow cytometry methodology.

Brief Description of the Drawings

[0041] Figure 1 The flowchart of the method for preparing the quality control material for lymphocyte subpopulation analysis of the present invention;

[0042] Figure 2 A lymphocyte (Lym) distribution diagram of the quality control material for lymphocyte subpopulation analysis and the preparation method thereof of the present invention;

[0043] Figure 3 A T lymphocyte distribution diagram of the quality control material for lymphocyte subset analysis and the preparation method thereof of the present invention;

[0044] Figure 4 CD4 and T lymphocyte distribution diagram of the quality control material for lymphocyte subpopulation analysis and its preparation method of the present invention;

[0045] Figure 5 CD8 and T lymphocyte distribution diagram of the quality control material for lymphocyte subpopulation analysis and its preparation method of the present invention;

[0046] Figure 6 A B lymphocyte distribution diagram of the quality control material for lymphocyte subpopulation analysis and the preparation method thereof of the present invention;

[0047] Figure 7 The NK lymphocyte distribution diagram of the quality control material for lymphocyte subpopulation analysis and the preparation method thereof of the present invention. [Specific implementation method]

[0048] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and implementation examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present 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] In addition, in the present invention, descriptions such as "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0051] See also Figures 1 to 7The quality control material for lymphocyte subpopulation analysis of the present invention is based on flow cytometry technology and includes human monocyte simulation particles, human granulocyte simulation particles, human lymphocyte simulation particles, human red blood cell simulation particles, human platelet simulation particles and preservation fluid. The quality control material for lymphocyte subpopulation analysis of the present invention can be used for quality control of T cells, B cells, NK cells, BNK cells and TBNK lymphocyte subpopulations.

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

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

[0054] The human granulocyte simulation particles include second microspheres, and the surface of the second microspheres is coated with CD45 antigens by physical adsorption or covalent bonding.

[0055] The second microspheres are microspheres with a particle size of 6-7 μm, carboxyl groups or amino groups on the surface, and magnetic or non-magnetic microspheres.

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

[0057] The third microspheres are microspheres with a particle size of 5-6 μm, carboxyl groups or amino groups on the surface, and magnetic or non-magnetic microspheres.

[0058] The human red blood cell simulation particles are made of fresh mammalian anticoagulated whole blood, which can be bovine whole blood or porcine whole blood. Red blood cells are obtained by centrifugation and separation, and washed three times with 10mM PBS buffer. The purified red blood cells are fixed by adding 0.05%-5% formaldehyde, and then the fixed red blood cells are washed and concentrated to obtain the human red blood cell simulation particles of the present invention.

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

[0060] The preservation solution comprises a buffer, a dispersant, an antibacterial agent and a matrix. The concentration of the buffer 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 is one of phosphate buffer, HEPES buffer, Tris buffer or glycine buffer.

[0062] The dispersant is a surfactant, and the surfactant can 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 P rclin-950.

[0064] The matrix is ​​preferably: BSA (0.1-0.5% w / v) and / or animal serum (20-50% v / v) and the like.

[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 a quality control material for lymphocyte subpopulation analysis of the present invention comprises the following steps:

[0067] Step S1, material preparation.

[0068] Step S2, preparation of human monocyte simulated particles.

[0069] Step S3, preparation of human granulocyte simulated particles.

[0070] Step S4, preparation of human lymphocyte simulation particles.

[0071] Step S5, preparation of human red blood cell simulating particles and human platelet simulating particles.

[0072] Step S6, preparation of quality control material for lymphocyte subset analysis.

[0073] In step S2, the preparation of human monocyte simulated particles comprises the following steps:

[0074] Step S21, select microspheres and antigens, and dilute the antigen to a concentration of 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, the microspheres are magnetically separated and the supernatant is discarded, and the microspheres are resuspended with MES buffer, mixed, and magnetically separated again and the supernatant is discarded.

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

[0078] Step S25, magnetically separate the microspheres in step S24, discard the supernatant, add 0.4 mL of blocking solution, mix well and perform blocking reaction at 25°C for 4-18 hours.

[0079] Step S26, microsphere magnetic separation, discard the supernatant, add 10-50mL preservation solution to resuspend, and store at 4°C. 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 simulation 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 simulation particles includes the following steps:

[0083] Step S41, antigen preparation.

[0084] Step S42, taking the microspheres, magnetically separating them, discarding the supernatant, and adding MES buffer to resuspend them.

[0085] Step S43, take EDC and Sulfo-NHS, and prepare a 50 mg / mL solution with 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, the microspheres are magnetically separated and the supernatant is discarded, and the microspheres are resuspended with MES buffer. After mixing, they are evenly divided into multiple tubes and labeled, with 0.2 mL in each tube, and magnetically separated again and the supernatant is discarded.

[0088] Step S46, respectively add corresponding antigens to the multi-tube microspheres of step S45, supplement with MES buffer to 0.2 mL, mix well, and react at 25° C. for 3 hours.

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

[0090] Step S48, magnetic separation of microspheres, discarding the supernatant, adding 10-100 mL of preservation solution to each tube of microspheres for resuspending, and storing at 4°C.

[0091] In step S5, the preparation of human red blood cell simulating particles and human platelet simulating particles includes:

[0092] Preparation of human red blood cell simulating particles and preparation of human platelet simulating particles.

[0093] The preparation of the human red blood cell simulation particles preferably selects fresh mammalian anticoagulated whole blood, which can be bovine whole blood, and obtains red blood cells by centrifugation and separation, and washes them three times with PBS buffer. The purified red blood cells are fixed by adding 0.05%-5% formaldehyde, and then the fixed red blood cells are washed and concentrated to obtain the human red blood cell simulation particles.

[0094] The preparation of the human platelet simulation particles preferably selects fresh mammalian anticoagulated whole blood, which can be bovine whole blood, and obtains platelets by centrifugation and separation, which are washed three times with PBS buffer, and the purified platelets are fixed by adding 0.05%-5% formaldehyde, and then the fixed platelets are washed and concentrated to obtain the human platelet simulation particles.

[0095] In step S6, the quality control material for lymphocyte subpopulation analysis is prepared by adding human monocyte simulation particles, human granulocyte simulation particles, human lymphocyte simulation particles, human red blood cell simulation particles, and human platelet simulation particles into the preservation solution, and mixing them according to the human particle concentration to obtain the quality control material for lymphocyte subpopulation analysis.

[0096] The preparation method of the quality control material for lymphocyte subpopulation analysis of the present invention is described below through specific examples.

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

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

[0099]

[0100] Step A1, material preparation:

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

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

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

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

[0105] EDC;

[0106] Sulfo-NHS;

[0107] PBS buffer (10 mM, pH 7.4), the osmotic concentration can be adjusted with sodium chloride in the range of 240-280 m mol / L;

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

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

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

[0111] Preservative solution: BSA (0.1% w / v), PBS (10mmol / L), animal serum (50% v / v), proclin300 (0.1% v / v), Tween 20 (0.2‰ v / v), adjusting the osmotic concentration within the range of 240-280m mol / L, pH within the range of 7.0-7.4; magnet, etc.

[0112] Step A2, preparation of human monocyte-simulating particles:

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

[0114] Step A22, antigen: 10-100 μg of CD45 antigen was taken and diluted to a concentration of 1 mg / mL with MES buffer;

[0115] Step A23, first take 1 mL of the microsphere suspension of step A21, add 0.08 mL of EDC solution and 0.08 mL of Sulfo-NHS solution in sequence, mix well, and react at 25°C for 30 minutes;

[0116] Step A24, magnetically separate the microspheres and discard the supernatant, resuspend with 0.8 mL of MES buffer, mix, and magnetically separate again and discard the supernatant;

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

[0118] Step A26, magnetically separate the microspheres in step A25, discard the supernatant, add 0.4 mL of blocking solution, mix well and perform blocking reaction at 25°C for 4-18 hours;

[0119] Step A27, microsphere magnetic separation, discard the supernatant, add 10-50mL preservation solution to resuspend, and store at 4°C. The concentration is not easy to be diluted too much, which is convenient for the subsequent addition of other components.

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

[0121] Step A31, microspheres: select magnetic microspheres with a diameter of 7 μm (concentration: 8×10 6 / mL, surface carboxyl activated).

[0122] Step A32, antigen: Take 10-100 μg of CD45 antigen and dilute to a concentration of 1 mg / mL with MES buffer.

[0123] Preparation: Same as the preparation method of "human monocyte simulated particles" above, add 10-50mL preservation solution to resuspend, store at 4℃, the concentration is not easy to be diluted too much, and it is convenient to add other components later.

[0124] Step A4, preparation of human lymphocyte-simulating particles.

[0125] Step A41, Antigen Preparation:

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

[0127] Antigen B: Take 10-100 μg of CD45 and CD3 antigens respectively, mix until uniform, and dilute to a concentration of 1 mg / mL for each antigen with MES buffer;

[0128] C antigen: Take 10-100 μg of CD45 antigen and dilute to 1 mg / mL with MES buffer;

[0129] Step A42, take 3.75 mL of microspheres, separate magnetically, discard the supernatant, and add 0.6 mL of MES buffer to resuspend;

[0130] Step A43, weigh 5 and 50 mg of EDC and Sulfo-NHS respectively, and prepare a 50 mg / mL solution with 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, magnetically separate the microspheres and discard the supernatant, resuspend with 0.6 mL MES buffer, mix and evenly divide into 3 tubes marked A, B, and C, each with 0.2 mL, and magnetically separate again and discard the supernatant;

[0133] Step A46, add corresponding antigens to the three tubes of microspheres in step A45, namely, the above-mentioned antigen A, antigen B, and antigen C, add 10-50 μL of each, add MES buffer to 0.2 mL, mix well, and react at 25° C. for 3 hours;

[0134] Step A47, magnetic separation of microspheres, discarding the supernatant, adding 0.4 mL of blocking solution to each tube of microspheres, mixing and blocking at 25°C for 4-18 hours;

[0135] Step A48, magnetic separation of microspheres, discarding the supernatant, adding 10-100 mL of the preservation solution in this protocol to each tube of microspheres to resuspend, and store at 4°C.

[0136] In step A5, the preparation of human red blood cell simulating particles and human platelet simulating particles comprises:

[0137] Preparation of human red blood cell simulating particles and preparation of human platelet simulating particles.

[0138] The preparation of the human red blood cell simulation particles preferably selects fresh mammalian anticoagulated whole blood, which can be bovine whole blood, and obtains red blood cells by centrifugation and separation, and washes them three times with PBS buffer. The purified red blood cells are fixed by adding 0.05%-5% formaldehyde, and then the fixed red blood cells are washed and concentrated to obtain the human red blood cell simulation particles.

[0139] The preparation of the human platelet simulation particles preferably selects fresh mammalian anticoagulated whole blood, which can be bovine whole blood, and obtains platelets by centrifugation and separation, which are washed three times with PBS buffer, and the purified platelets are fixed by adding 0.05%-5% formaldehyde, and then the fixed platelets are washed and concentrated to obtain the human platelet simulation particles.

[0140] Step A6, preparation of quality control material for T cell analysis: adding the above-mentioned simulated particles into the preservation solution of the present invention, mixing them according to the human particle concentration, and obtaining the preparation of quality control material for T cell analysis.

[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] The rest is the same as in Example 1.

[0147] The preparation of human monocyte-simulating particles, human granulocyte-simulating particles, human erythrocyte-simulating particles and human platelet-simulating particles is the same as in Example 1.

[0148] Step B3, preparation of human lymphocyte-simulating particles:

[0149] Step B31, Antigen Preparation:

[0150] Antigen A: Take 10-100 μg of CD45 and CD3 antigens respectively, mix until uniform, and dilute to a concentration of 1 mg / mL for each antigen with MES buffer;

[0151] Antigen B: Take 10-100 μg of CD45 and CD19 antigens respectively, mix until uniform, and dilute to a concentration of 1 mg / mL for each antigen with MES buffer;

[0152] C antigen: Take 10-100 μg of CD45 antigen and dilute to 1 mg / mL with MES buffer;

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

[0154] Step B4, Preparation of Quality Control for B Cell Analysis:

[0155] The simulated particles are added to the preservation solution of the present invention and mixed according to the human particle concentration to obtain the preparation of B cell analysis quality control material.

[0156] Example 3 Preparation of NK cell analysis quality control material

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

[0158]

[0159]

[0160] Step C1, material preparation:

[0161] Antigens (CD45, CD3, CD16)

[0162] The rest is the same as in Example 1.

[0163] The preparation of human monocyte-simulating particles, human granulocyte-simulating particles, human erythrocyte-simulating particles and human platelet-simulating particles is the same as in Example 1.

[0164] Step C3, preparation of human lymphocyte-simulating particles:

[0165] Step C31, Antigen Preparation:

[0166] Antigen A: Take 10-100 μg of CD45 and CD3 antigens respectively, mix until uniform, and dilute to a concentration of 1 mg / mL for each antigen with MES buffer;

[0167] Antigen B: Take 10-100 μg of CD45 and CD16 antigens respectively, mix until uniform, and dilute to a concentration of 1 mg / mL for each antigen with MES buffer;

[0168] C antigen: Take 10-100 μg of CD45 antigen and dilute to 1 mg / mL with MES buffer;

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

[0170] Step C34, preparation of quality control material for NK cell analysis:

[0171] The simulated particles are added to the preservation solution of the present invention and mixed according to the human particle concentration to obtain the preparation of 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] The rest is the same as in Example 1.

[0178] The preparation of human monocyte-simulating particles, human granulocyte-simulating particles, human erythrocyte-simulating particles and human platelet-simulating particles is the same as in Example 1.

[0179] Step D3, preparation of human lymphocyte-simulating particles:

[0180] Step D31, Antigen Preparation:

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

[0182] Antigen B: Take 10-100 μg of CD45 and CD3 antigens respectively, mix until uniform, and dilute to a concentration of 1 mg / mL for each antigen with MES buffer;

[0183] Antigen C: Take 10-100 μg of CD45 and CD19 antigens respectively, mix until uniform, and dilute to a concentration of 1 mg / mL for each antigen with MES buffer;

[0184] D antigen: Take 10-100 μg of CD45 and CD16 antigens respectively, mix until uniform, and dilute to a concentration of 1 mg / mL for each antigen with MES buffer;

[0185] E antigen: Take 10-100 μg of CD45 antigen and dilute to 1 mg / mL with MES buffer;

[0186] Step D32, take 6.25 mL of microspheres, separate magnetically, discard the supernatant, and add 1 mL of MES buffer to resuspend;

[0187] Step D33, weigh 5 and 50 mg of EDC and Sulfo-NHS respectively, 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, magnetically separate the microspheres and discard the supernatant, resuspend with 1 mL of MES buffer, mix and evenly divide into 5 tubes marked A, B, C, D, and E, 0.2 mL per tube, and magnetically separate again and discard the supernatant;

[0190] Step D36, add corresponding antigens to the 5 microspheres in step D35, which are the above-mentioned antigen A, antigen B, antigen C, antigen D and antigen E, add 10-50 μL of each, add MES buffer to 0.2 mL, mix well, and react at 25° C. for 3 hours;

[0191] Step D37, magnetic separation of microspheres, discarding the supernatant, adding 0.4 mL of blocking solution to each tube of microspheres, mixing and blocking at 25°C for 4-18 hours;

[0192] Step D38, magnetic separation of microspheres, discarding the supernatant, adding 10-100 mL of the preservation solution in this protocol to each tube of microspheres to resuspend, and store at 4°C.

[0193] Step D 4, Preparation of quality control material for TBNK lymphocyte subset analysis:

[0194] The simulated particles are added to the preservation solution of the present invention and mixed according to the human particle concentration to obtain the preparation of the TBNK lymphocyte subset analysis quality control material.

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

[0196] The quality control material obtained in this example was tested experimentally and can be stored at 2-8°C in a sealed container for 6 months. The grouping is obvious and the attenuation rate is ≤10%, which meets clinical requirements.

[0197] The following experimental data illustrate the stability and grouping of the quality control materials described in this application, and the data are as follows:

[0198] project Zero Time 6 months Decay rate 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 subpopulation analysis and the preparation method thereof of the present invention have the following beneficial effects:

[0200] 1. It has the advantages of stable performance, low production cost, mass production, small batch difference, wider clinical application, etc., which can better control the quality of lymphocyte subset analysis system.

[0201] 2. It can be flexibly combined. Microspheres coated with different antigen components can be added separately to perform quality control on T cell, B cell, NK cell or BNK cell analysis respectively, or they can be mixed and added for quality control of TBNK lymphocyte subset analysis.

[0202] 4. It can be widely used in inter-laboratory quality assessment and 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 in the patent protection scope of the present invention.

Claims

1. A quality control substance for lymphocyte subpopulation analysis, characterized in that: The invention comprises human monocyte simulation particles, human granulocyte simulation particles, human lymphocyte simulation particles, human red blood cell simulation particles, human platelet simulation particles and preservation solution.

2. The quality control material for lymphocyte subpopulation analysis according to claim 1, characterized in that: The human monocyte simulation particles include a first microsphere, the surface of which is coated with CD45 antigen by physical adsorption or covalent binding.

3. The quality control material for lymphocyte subpopulation analysis according to claim 2, characterized in that: The human granulocyte simulation particles include second microspheres, and the surface of the second microspheres is coated with CD45 antigens by physical adsorption or covalent bonding.

4. The quality control material for lymphocyte subpopulation analysis according to claim 3, characterized in that: The human lymphocyte simulation particles include a third microsphere, the surface of which is coated with one or several antigens among CD45, CD3, CD4, CD8, CD19 and CD16 by physical adsorption or covalent binding.

5. The quality control material for lymphocyte subpopulation analysis according to claim 1, characterized in that: The human red blood cell simulation particles are prepared by centrifuging and separating fresh mammalian anticoagulated whole blood to obtain red blood cells, washing them three times with 10 mM PBS buffer, adding 0.05%-5% formaldehyde to fix the purified red blood cells, and then washing and concentrating the fixed red blood cells to obtain the human red blood cell simulation particles; The human platelet simulation particles are prepared by centrifuging and separating fresh mammalian anticoagulated whole blood to obtain platelets, which are then washed three times with 10 mM PBS buffer. The purified platelets are fixed by adding 0.05%-5% formaldehyde, and then the fixed platelets are washed and concentrated to obtain the platelet simulation particles.

6. The quality control material for lymphocyte subpopulation analysis according to claim 1, characterized in that: The preservation solution comprises a buffer, a dispersant, an antibacterial agent and a matrix. The concentration of the buffer 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%.

7. The quality control material for lymphocyte subpopulation analysis according to claim 4, characterized in that: The first microspheres are microspheres with a particle size of 7-8 μm, carboxyl groups or amino groups on the surface, and are magnetic or non-magnetic microspheres; the second microspheres are microspheres with a particle size of 6-7 μm, carboxyl groups or amino groups on the surface, and are magnetic or non-magnetic microspheres; the third microspheres are microspheres with a particle size of 5-6 μm, carboxyl groups or amino groups on the surface, and are magnetic or non-magnetic microspheres.

8. A method for preparing a quality control material for lymphocyte subpopulation analysis, characterized in that: The steps include: Step S1, material preparation; Step S2, preparation of human monocyte simulated particles; Step S3, preparation of human granulocyte simulated particles; Step S4, preparation of human lymphocyte simulation particles; Step S5, preparation of human red blood cell simulating particles and human platelet simulating particles; Step S6, preparation of quality control material for lymphocyte subset analysis.

9. The method for preparing a quality control material for lymphocyte subpopulation analysis according to claim 8, characterized in that: In step S2, the preparation of human monocyte simulated particles comprises the following steps: Step S21, selecting microspheres and antigens, and diluting the antigens with MES buffer to a concentration of 1 mg / mL; Step S22, taking the microsphere suspension, adding EDC solution and Sulfo-NHS solution in sequence, mixing, and reacting at 25° C. for 30 minutes; Step S23, magnetically separating the microspheres and discarding the supernatant, resuspending with MES buffer, mixing, and magnetically separating again and discarding the supernatant; Step S24, add 10-50 μL of the antigen in step S21 to the microspheres in step S23, add MES buffer to 0.2 mL, mix well, and react at 25° C. for 3-12 hours; Step S25, magnetically separate the microspheres in step S24, discard the supernatant, add 0.4 mL of blocking solution, mix well and perform blocking reaction at 25° C. for 4-18 hours; Step S26, microsphere magnetic separation, discard the supernatant, add 10-50mL preservation solution to resuspend, and store at 4°C.

10. The method for preparing a quality control material for lymphocyte subpopulation analysis according to claim 8, characterized in that: In step S4, the preparation of human lymphocyte simulation particles includes the following steps: Step S41, antigen preparation; Step S42, taking the microspheres, magnetically separating them, discarding the supernatant, and adding MES buffer to resuspend them; Step S43, taking EDC and Sulfo-NHS, and preparing a 50 mg / mL solution with MES buffer; Step S44, adding EDC solution and Sulfo-NHS solution to the microsphere suspension in sequence, mixing, and reacting at 25° C. for 30 minutes; Step S45, magnetically separate the microspheres and discard the supernatant, resuspend them in MES buffer, mix them and evenly divide them into multiple tubes and mark them, with 0.2 mL in each tube, and magnetically separate them again and discard the supernatant; Step S46, respectively add corresponding antigens to the multi-tube microspheres of step S45, add MES buffer to 0.2 mL, mix well, and react at 25° C. for 3 hours; Step S47, magnetic separation of microspheres, discarding the supernatant, adding 0.4 mL of blocking solution to each tube of microspheres, mixing and blocking reaction at 25° C. for 4-18 hours; Step S48, magnetic separation of microspheres, discarding the supernatant, adding 10-100 mL of preservation solution to each tube of microspheres for resuspending, and storing at 4°C.

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