阅读说明：本技术 一种用于t淋巴细胞亚群计数标准物质的冻干保护剂及其应用 (Freeze-drying protective agent for T lymphocyte subpopulation counting standard substance and application thereof ) 是由 李妍 郭柏松 朱文婷 单宇 刘刚 闻艳丽 梁文 李兰英 许丽 王乐乐 于 2020-05-13 设计创作，主要内容包括：本发明提供一种用于T淋巴细胞亚群计数标准物质的冻干保护剂及其应用。所述冻干保护剂包括质量分数为2～10％的蔗糖和3～10％的海藻糖,并以细胞固定液为溶剂,使用所述冻干保护剂制备得到的T淋巴细胞亚群计数标准物质稳定性高。同时,所述T淋巴细胞亚群计数标准物质具有明确量值,均匀性良好,在4℃下可以稳定保存15天,在-20℃条件下保存稳定性在1个月以上,能够实现流式细胞术检测结果的质量控制,保证检测结果的准确性,因此对于流式细胞仪的计量校准、T淋巴细胞流式细胞检测过程的方法验证和检测结果质量控制等方面具有重要的意义。(The invention provides a freeze-drying protective agent for a T lymphocyte subpopulation counting standard substance and application thereof. The freeze-drying protective agent comprises 2-10% of sucrose and 3-10% of trehalose by mass, a cell fixing solution is used as a solvent, and the T lymphocyte subpopulation counting standard substance prepared by using the freeze-drying protective agent is high in stability. Meanwhile, the T lymphocyte subpopulation counting standard substance has a definite quantity value and good uniformity, can be stably stored for 15 days at 4 ℃, has the storage stability of more than 1 month at-20 ℃, can realize the quality control of the detection result of the flow cytometry, and ensures the accuracy of the detection result, thereby having important significance for the aspects of metering calibration of the flow cytometer, method verification of the T lymphocyte flow cytometry detection process, quality control of the detection result and the like.)

1. A freeze-drying protective agent for a T lymphocyte subpopulation counting standard substance is characterized by comprising 2-10% of cane sugar and 3-10% of trehalose by mass, and a cell fixing solution is used as a solvent.

2. The lyoprotectant according to claim 1, wherein the sucrose is 5 to 9% by mass, preferably 7.8 to 8.2% by mass;

preferably, the mass fraction of the trehalose is 6-9%, preferably 7.8-8.2%;

preferably, the cell fixing solution is diluted by 5 to 12 times before use, and preferably by 9.5 to 10.5 times.

3. A T lymphocyte subpopulation enumeration standard prepared using a lyoprotectant according to claim 1 or 2.

4. A method for preparing a standard substance for T lymphocyte subpopulation counting according to claim 3, comprising:

sorting peripheral blood mononuclear cells to obtain T lymphocytes, mixing the T lymphocytes with the freeze-drying protective agent according to claim 1 or 2, and freeze-drying to obtain the T lymphocyte subpopulation counting standard substance.

5. The method of claim 4, wherein the method for sorting the peripheral blood mononuclear cells is immunomagnetic bead separation;

preferably, the cell density of the mixed T lymphocytes is (2-3) × 10⁶Per mL;

preferably, the procedure of freeze-drying is: the pre-freezing temperature is-45.5 to-44.5 ℃, the temperature of primary sublimation is-40.5 to-39.5 ℃, the pressure is set to be 0 to 2Pa, and the decompression drying temperature is 24.5 to 25.5 ℃.

6. The production method according to claim 4 or 5, characterized by comprising: sorting peripheral blood mononuclear cells by immunomagnetic bead separation to obtain T lymphocytes, and mixing with the freeze-drying protective agent as described in claim 1 or 2;

and (3) carrying out freeze drying according to the procedures that the pre-freezing temperature is-45.5 to-44.5 ℃, the temperature of primary sublimation is-40.5 to-39.5 ℃, the pressure is set to be 0 to 2Pa, and the decompression drying temperature is 24.5 to 25.5 ℃, so as to obtain the T lymphocyte subpopulation counting standard substance.

7. A method for quantifying a standard substance for a subpopulation of T lymphocytes according to claim 3, comprising the steps of:

re-dissolving the T lymphocyte subpopulation counting standard by using a re-solution to obtain a solution to be detected, wherein the re-solution is a PBS buffer solution containing BSA;

and adding the solution to be detected into a flow tube, mixing with a staining antibody, incubating, cleaning, and performing on-machine detection to obtain the standard value of the T lymphocyte subpopulation counting standard substance.

8. The method according to claim 7, wherein the cell concentration in the test solution after reconstitution with the reconstituted solution is 2000-3000 cells/μ L;

preferably, the mass fraction of BSA in the complex solution is 1.5-2.5%, preferably 2%;

preferably, the staining antibody is a mixed-standard antibody or a single-standard antibody;

preferably, the mixed standard antibody comprises PE-CD4/FITC-CD3/PerCP-CD45 antibody;

preferably, the single-label antibody comprises a FITC-CD4 antibody;

preferably, the number of times of cleaning is 1-2 times, and preferably 1 time.

9. A method of valuing according to claim 7 or 8, wherein the method of valuing includes the steps of:

redissolving the T lymphocyte subpopulation counting standard substance by using a complex solution to obtain a to-be-detected solution, wherein the complex solution is a PBS (phosphate buffer solution) containing 1.5-2.5% of BSA (bovine serum albumin), and the cell concentration in the to-be-detected solution after redissolving by using the complex solution is 2000-3000 cells/mu L;

and adding the solution to be detected into a flow tube, mixing with a staining antibody, incubating, cleaning for 1-2 times, and detecting on a computer to obtain the standard value of the T lymphocyte subpopulation counting standard substance.

10. Use of a T lymphocyte subpopulation counting standard according to claim 3 or a quantification method according to any one of claims 7-9 for calibrating a flow cytometer, flow cytometry method validation or quality control of flow cytometry results.

Technical Field

The invention relates to a single cell quantitative analysis and sorting technology, in particular to a preparation method and a value-fixing method of a T lymphocyte subpopulation counting standard substance, and particularly relates to a freeze-drying protective agent for the T lymphocyte subpopulation counting standard substance and application thereof.

Background

Flow Cytometry (FCM) is a single cell quantitative analysis and sorting technique using a flow cytometer, and is a comprehensive technique involving numerous disciplines, mainly involving computer techniques, cytobiology techniques, physics techniques, laser techniques, immunology techniques, and the like. The development of flow cytometry is based on the rapid flow of cells followed by the development of analytical processes on the cells in flow.

Currently, clinical flow cytometry has become a hot spot in examining the development of medicine. In the clinical examination process, the flow cytometry technology is used as the main detection technology of the immune condition of the organism, and the examination indexes mainly comprise the levels of T lymphocytes, B lymphocytes and natural killer lymphocytes. The detection treatment process needs to be assisted by surface marker substances so as to know and master the in vivo levels of various cells and different cytokines in the cells, realize effective detection treatment on the immune condition of a patient by measuring the number of lymphocyte subsets of the patient, and provide reliable and effective guidance for clinical subsequent diagnosis and related treatment. Compared with the traditional immunological detection treatment measures, the flow cytometry technology is combined with the monoclonal antibody technology, so that accurate quantitative detection can be realized.

The greatest advantage of flow cytometry is the counting of the sub-population cells in the mixed cell population, for example, lymphocytes can be divided into T lymphocytes (CD3+), B lymphocytes (CD19+), NK cells (CD16+56+/CD3-), T lymphocytes can be further divided into helper/inducer T lymphocytes (CD3+ CD4+ CD8-), suppressor/cytotoxic T lymphocytes (CD3+ CD4-CD8+) and the like according to the difference of surface markers of the lymphocytes. For example, by measuring the expression levels of subpopulations of T lymphocytes in peripheral blood of a naive aged Multiple Myeloma (MM) patient, the prognostic value of subpopulations of T lymphocytes in a naive aged MM patient can be assessed. The ratio of CD4/CD8 and the reduction of CD4+ T cell count in the initial diagnosis are poor prognostic factors of the MM of the old, the level of T lymphocyte subpopulation can be used as a potential index influencing prognosis judgment, and the reduction of CD4+ T cells is commonly seen in malignant tumors, hereditary immunodeficiency, AIDS and the like.

At present, few researches are carried out on standard substances (CRM) for flow cytometry detection at home and abroad, and the standard substances play an important role in detecting and calibrating indication errors and repeatability of a flow cytometry analysis instrument. For example, the T lymphocyte subpopulation counting standard substance can efficiently measure and calibrate the operation condition of the flow cytometer, ensure the uniformity of the quantity value and ensure the validity of the detection result. However, only lymphocyte CD4+ cells, developed by the Chinese institute of metrology science, were retrieved as a percentage of total lymphocyte counts for the standard substance (GBW (E)090938), with a standard value of 49.4% and an uncertainty of 4.8%.

Therefore, the provision of the T lymphocyte subpopulation counting standard substance with definite quantity value, good uniformity and high stability has great significance in the aspects of metering calibration of a flow cytometer, method verification of a T lymphocyte flow cytometry detection process, detection result quality control and the like.

Disclosure of Invention

In view of the problems in the prior art, the invention provides a lyoprotectant for a T lymphocyte subpopulation counting standard substance and application thereof. The T lymphocyte subpopulation counting standard substance prepared by using the freeze-drying protective agent has good appearance and high stability, can be used for a detected cell sample, and ensures the accuracy of a detection result.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a freeze-drying protective agent for a T lymphocyte subpopulation counting standard substance, which comprises 2-10% of sucrose and 3-10% of trehalose by mass, and takes a cell fixing solution as a solvent.

The survival rate of cells in the freeze drying process can be improved by adding the freeze-drying protective agent into the standard substance, and the types of the protective agent are more and mainly divided into four types: osmotic antifreeze, non-osmotic antifreeze, antioxidants, and colloids. The cell fixing solution, the sucrose and the trehalose are cooperatively matched and interact, so that the survival rate of the T lymphocytes in the freeze drying process can be obviously improved, the stability of the standard substance is ensured, and the reliability of the standard substance is higher when the indication error and the repeatability of a flow cytometry analyzer are detected and calibrated.

In the invention, the cell fixing solution is IC hybridization Buffer, the component is phosphate buffered saline (Parafamaldehyde in phosphate buffered saline) containing 4% Paraformaldehyde, and the pH value is 7.3; the IC fire Buffer selected for use in the present invention is available from Thermo. The cell fixing solution can fix the cell morphology, can be compatible with an antibody coupled with a fluorescent dye for surface dyeing, can be used as a short-term storage buffer solution for dyeing cells by the fluorescent dye (including a tandem dye), and can be stored for 3 days at the temperature of 2-8 ℃ at most (being stored in a dark place). It can be used without dilution.

The sucrose may be 2 to 10% by mass, for example, 2%, 3%, 4%, 5%, 5.2%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%. Preferably, the mass fraction of the sucrose is 5-9%, and more preferably 7.8-8.2%.

The trehalose may be, for example, 3%, 4%, 5%, 5.2%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, or the like, in a mass fraction of 3 to 10%. Preferably, the mass fraction of the trehalose is 6-9%, and more preferably 7.8-8.2%.

Preferably, the cell fixing solution is diluted 5 to 12 times before use, for example, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, or the like; preferably 9.5 to 10.5 times.

In the invention, the freeze-drying protective agent preferably comprises 8% of sucrose and 8% of trehalose by mass, and when 10-time diluted cell fixing solution is used as a solvent, the appearance and the stability of the freeze-dried product of the obtained T lymphocyte subpopulation counting standard substance are better.

In a second aspect, the T lymphocyte subpopulation enumeration standard substance produced by the production method according to the first aspect. The standard substance has a definite quantity value, is good in uniformity and can stably exist for a long time; the method is a cell sample which can be practically used for detection, can realize the quality control of the detection result of the flow cytometry, and ensures the accuracy of the detection result.

In a third aspect, the present invention provides a method for preparing a standard substance for counting subpopulations of T lymphocytes, the method comprising:

sorting peripheral blood mononuclear cells to obtain T lymphocytes, mixing the T lymphocytes with the freeze-drying protective agent according to the first aspect, and freeze-drying to obtain the T lymphocyte subpopulation counting standard substance.

In a preferred embodiment of the present invention, the method for sorting peripheral blood mononuclear cells is an immunomagnetic bead separation method.

Illustratively, the method of sorting selected T lymphocytes of the invention comprises the steps of:

1) 2.5 × 10⁸Individual PBMC cells were resuspended in 1mL MACS buffer, and evenly divided into 5 EP tubes, 200 μ L each; adding 50 μ L of PanT Biotin-Antibody in human total T cell sorting kit into each branch, mixing uniformly, standing at 4 deg.C for 5 min; each was added with 150. mu.L of MACS buffer, and then 100. mu.L of Pan TMicrobead in the human total T cell sorting kit was added; after vortex mixing, standing for 10min at 4 ℃, and preparing 5 samples of 500 microlitres on a separation column;

2) LS Columns were wetted by adding 3mL of MACS buffer; add 500. mu.L of sample, take care not to touch the column wall, place a new 15mL centrifuge tube below to collect the filtered liquid; before the sample is filtered, adding 3mL of buffer solution to wash the sorting column, repeatedly washing for three times, and collecting 9.5mL of target cells in a centrifugal tube; the same procedure was repeated 4 times to complete the remaining cell sorting collection.

Preferably, the cell density of the mixed T lymphocytes is (2-3) × 10⁶cell/mL, at experimental work, cell pellet after centrifugation was about 1 × 10⁸Adding protective agent 40mL lyophilized protective agent directly into each cell, mixing, subpackaging 200 tubes with 200 μ L of each tube and average cell density of about 5 × 10⁵One tube per tube.

Preferably, the procedure of freeze-drying is: the pre-freezing temperature is-45.5 to-44.5 ℃, the temperature of primary sublimation is-40.5 to-39.5 ℃, the pressure is set to be 0 to 2Pa, and the decompression drying temperature is 24.5 to 25.5 ℃.

Since the cell freezing medium or cell fixing medium has complicated composition and contains Na in large amount⁺、K⁺Plasma with low freeze-drying parameters and high freeze-drying difficulty, the invention adopts a conservative process, namely the pre-freezing temperature is-45 ℃, the temperature is-40 ℃ for one-time sublimation and 0Pa, and decompression drying is carried out at 25 ℃ after one-time sublimation is finished.

As a preferred technical scheme of the invention, the preparation method of the T lymphocyte subpopulation counting standard substance comprises the following steps: sorting peripheral blood mononuclear cells by immunomagnetic bead separation to obtain T lymphocytes, and mixing the T lymphocytes with the freeze-drying protective agent in the first aspect; and (3) carrying out freeze drying according to the procedures that the pre-freezing temperature is-45.5 to-44.5 ℃, the temperature of primary sublimation is-40.5 to-39.5 ℃, the pressure is set to be 0 to 2Pa, and the decompression drying temperature is 24.5 to 25.5 ℃, so as to obtain the T lymphocyte subpopulation counting standard substance.

In a fourth aspect, the present invention provides a method of valuing a T lymphocyte subpopulation count standard substance according to the third aspect, said method comprising the steps of:

re-dissolving the T lymphocyte subpopulation counting standard by using a re-solution to obtain a solution to be detected, wherein the re-solution is a PBS buffer solution containing BSA; and adding the solution to be detected into a flow tube, mixing with a staining antibody, incubating, cleaning, and performing on-machine detection to obtain the standard value of the T lymphocyte subpopulation counting standard substance.

As a preferable embodiment of the present invention, the cell concentration in the test solution after reconstitution with the reconstituted solution is 2000 to 3000 cells/μ L, and may be 2000 cells/μ L, 2100 cells/μ L, 2200 cells/μ L, 2300 cells/μ L, 2400 cells/μ L, 2500 cells/μ L, 2600 cells/μ L, 2700 cells/μ L, 2800 cells/μ L, 2900 cells/μ L, 3000 cells/μ L, or the like.

Preferably, the mass fraction of BSA in the double solution is 1.5 to 2.5%, and may be, for example, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, or the like, and preferably 2%.

Preferably, the staining antibody is a mixed-standard antibody or a single-standard antibody.

Preferably, the mixed standard antibody comprises PE-CD4/FITC-CD3/PerCP-CD45 antibody. The PE-CD4/FITC-CD3/PerCP-CD45 antibody refers to a mixed antibody which can simultaneously mark CD4, CD3 and CD45 and can emit different fluorescence.

Preferably, the single-label antibody comprises a FITC-CD4 antibody.

Preferably, the number of times of cleaning is 1-2 times, and preferably 1 time.

As a preferred technical solution of the present invention, the method for setting the value includes the steps of: redissolving the T lymphocyte subpopulation counting standard substance by using a complex solution to obtain a to-be-detected solution, wherein the complex solution is a PBS (phosphate buffer solution) containing 1.5-2.5% of BSA (bovine serum albumin), and the cell concentration in the to-be-detected solution after redissolving by using the complex solution is 2000-3000 cells/mu L; and adding the solution to be detected into a flow tube, mixing with a staining antibody, incubating, cleaning for 1-2 times, and detecting on a computer to obtain the standard value of the T lymphocyte subpopulation counting standard substance.

In a fifth aspect, the present invention also provides a use of the T lymphocyte subpopulation count standard according to the second aspect or the valued method according to the fourth aspect for calibrating a flow cytometer, validating a flow cytometry method or controlling the quality of flow cytometry results.

The T lymphocyte subpopulation counting standard substance which is fixed by the fixing method is widely applied to the calibration of flow cytometry, the verification of the flow cytometry detection method or the quality control of the flow cytometry detection result.

The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) the freeze-drying protective agent provided by the invention comprises 10 times of diluted cell fixing liquid and high-concentration sugar (8% of sucrose and 8% of trehalose), and after the freeze-drying protective agent is used and subjected to the freeze-drying process, the obtained cell freeze-dried substance has good appearance and high stability, and is suitable for freeze-drying protection of a T lymphocyte subset counting standard substance;

(2) the T lymphocyte subpopulation counting standard substance provided by the invention has definite magnitude and good uniformity, can be stably stored for 15 days at 4 ℃, and has the storage stability of more than 1 month at-20 ℃; meanwhile, according to the result of common measurement in multiple laboratories, the total average value of the proportion of CD4 positive cells in the standard substance is 74%, and the standard substance is a cell sample which can be actually used for detection, so that the quality control of the detection result of the flow cytometry can be realized, and the accuracy of the detection result is ensured;

(3) the method for fixing the value of the T lymphocyte subpopulation counting standard substance provided by the invention selects a proper redissolution type and a redissolution volume, ensures that the cell density is proper when a sample is measured, has the characteristics of higher precision, monochromatic dyeing without adjusting fluorescence channel compensation, meeting the fixed value requirement of the T lymphocyte subpopulation counting standard substance and the like, and has good repeatability, the repeatability RSD is 0.82%, the reproducibility is good, and the reproducibility RSD is 0.39%.

Drawings

FIG. 1(a) is a graph showing the result of CD4 lymphocyte positive proportion detection after reconstitution of a sample lyophilized using the lyoprotectant provided in example 1; FIG. 1(b) is a graph showing the results of detection after example 2 is used; FIG. 1(c) is a graph showing the results of measurement after comparative example 1 was used; fig. 1(d) is a graph showing the results of the test using comparative example 2.

FIG. 2(a) is a graph showing the results of the detection of the percentage of PE-CD4+ in the multi-colored stained antibody after adjustment compensation after using the mixed standard antibody in application example 2; FIG. 2(b) shows the results of detection of FITC-CD3 antibody, PE-CD4 antibody and PerCP-CD45 antibody after adjustment compensation in peak diagrams A, B and C, respectively, after the mixed standard antibody is used in application example 2; scatter plots i, ii, and iii are plots of results after double staining with different antibodies; FIG. 2(c) is a graph showing the results of percentage detection of TIFC-CD4+ after using a single-standard antibody in application example 2.

FIG. 3(a) is a graph showing the results of flow cytometry at day0 of a sample reconstituted with PBS containing 2% BSA as a reconstitution solution in application example 2; FIG. 3(b) is a graph showing the results of flow cytometry at day 1;

FIG. 3(c) is a graph showing the results of flow cytometry at day 2; FIG. 3(d) is a graph showing the flow cytometry results of the sample on day0 after being reconstituted with PBS as the reconstitution liquid in application example 2; FIG. 3(e) is a graph showing the results of flow cytometry at day 1; FIG. 3(f) is a graph showing the results of flow cytometry at day 2.

FIG. 4 is a scattergram of flow cytometry detection of a reconstituted sample (1000. mu.L) using the method (1) in application example 2.

FIG. 5 is a diagram showing the peak fluorescence signal of FITC-CD4 detected by flow cytometry in three replicates of a reconstituted sample (100. mu.L) by the method (2) in application example 2.

FIG. 6 is a diagram showing a peak of a fluorescence signal of FITC-CD4 detected by flow cytometry on a reconstituted sample (100. mu.L, 200. mu.L, 300. mu.L) by the method (3) in application example 2.

FIG. 7(a) is a flow cytometric scattergram obtained by storing the standard substance of application example 3 at 20 ℃ for 3 days; FIG. 7(b) is a flow cytometric scattergram obtained after 7 days of storage; FIG. 7(c) is a flow cytometric scattergram obtained after 10 days of storage; fig. 7(d) is a flow cytometric scattergram obtained after 15 days of storage.

Detailed Description

The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.

In the following examples, the flow cytometer is purchased from BD, usa; low temperature centrifuges were purchased from Eppendorf, germany; centrifuges were purchased from ruxiang instruments, china; ultra-low temperature refrigerators were purchased from Thermo Fisher Scientific, usa; the freeze dryer was purchased from Tofflon, china.

In the following examples, PBMC CELLS used were purchased from ALL CELLS; human total T cell sorting kit, MACSbuffer, LS Columns and Pre-Separation Filters were purchased from Miltenyibitotec; fetal bovine serum BSA was purchased from MP; cell cryopreservation solution and cell fixative solution (IC lysis Buffer) were purchased from Thermo corporation; 1 XPBS (pH7.2) from Gibco; CD4-FITC antibody was purchased from Beckman Coulter; PE-CD4/FITC-CD3/PerCP-CD45 mixed antibody was purchased from BD corporation; d-trehalose was purchased from Runjie chemical; sucrose was purchased from national drug companies.