Patent Description:
<CIT> refers to a kit for detecting human regulatory T cell subtypes and a detection method and belongs to the technical field of cell subtype detection. The provided kit comprises components as follows: a blood diluent, a mononuclear cell separation medium, a cell culture fluid, a lymphocyte activation solution, dead cell removal dyes, an FcR blocking agent, a fluorescence labeled antibodyresisting human cell surface labeling molecules, a fluorescence labeled antibody resisting human intracellular molecules, a PBS buffer solution, lipopolysaccharide, a washing buffer solution, a celldyeing buffer solution, a cell fixing solution and a permeable membrane lotion. During detection, firstly, mononuclear cells in whole blood are separated, then, mononuclear cells of human peripheral blood are stimulated, finally, a fluorescent antibody is stained, and the regulatory T cell subtypes can be detected.

<CIT> refers to a method for detecting avian peripheral blood T lymphocyte subsets by flow cytometry; the method comprises the steps of collecting avian peripheral venous blood, preparing anticoagulant blood, and treating with lymphocyte separate to obtain peripheral blood leukocytes; centrifugally washing the peripheral blood leukocytes with PBS phosphate buffer solution to prepare single-cell suspension; sucking the single-cell suspension, adding anti-chicken CD3, CD4 and CD8 monoclonal antibodies, mixing well by cyclone, and staining in the shade at <NUM>; after staining, washing with PBS, resuspending cells, and detecting with a flow cytometer; analyzing detection results, the ratio of avian peripheral blood T lymphocyte subsets.

A flow cytometric sample is mainly used in blood sample detection, while the preparation of a cell sample for use in a conventional flow cytometry detection usually adopts the following methods: (<NUM>) analyzing the influence of different treatment methods of peripheral blood on the results of a flow cytometric analysis; and (<NUM>) detecting T-lymphocyte subsets phenotype in <NUM> lung cancer patients by using flow cytometry, including adding corresponding volume of antibody to a flow tube, then adding <NUM> or <NUM>µl of anticoagulant peripheral blood samples, mixing, incubating without light at room temperature for <NUM> minutes, adding <NUM>×hemolysin (i.e. red blood cell lysis solution), respectively, and mixing before detection with a flow cytometer. The current method for preparing flow cytometric sample mainly includes incubating the peripheral blood with antibody for <NUM>-<NUM> minutes, adding red blood cell lysis solution, performing lysis for <NUM>-<NUM> minutes, and then directly performing detection on a flow cytometer, or detecting on a flow cytometer after washing once. The preparation method suffers from the following problems. The first problem is the incubation time after adding the red blood cell lysis solution. Sufficient amount of hemolysin is needed based on the number of red cells, normally by observing whether the solution is clear after adding hemolysin. If it is clear, it means that the red blood cell is completely lysed, but it is easy to cause damage to lymphocyte. If the time of the lysis is insufficient, the red blood cell cannot be completely lysed, which will influence the results of detection and analysis. The second problem lies in that the number of lymphocyte samples cannot be strictly controlled. If the same volume of antibody is added in spite of the number of lymphocytes in the original blood sample, it is easy to cause oversaturated or insufficient amount of antibody. If the number of cell samples to be detected is small, trace amount of cell subtype is difficult to be detected or inaccurately detected due to small number of samples, which cannot accurately reflect the actual situation of some lymphocyte subgroups. When the number of cell samples is too large, for example when the lymphocytes of some patients are above a standard level, the amount of the antibody will be insufficient, which will lead to a lower detection result. The third problem lies in that, the sample obtained from the lysis has too much debris, and the background is not clean, which will affect the collection and analysis of flow cytometer data, and may cause bias to the adjustment of lymphocyte compensation.

<CIT> discloses a method and kit for lymphocyte immunotyping. The method includes adding mixed antibody into two flow tubes, adding 50µl anticoagulant peripheral blood samples, fully mixing, incubating without light at room temperature for <NUM> minutes, lysing red cells with red cell lysis solution for <NUM> minutes, adding <NUM> PBS to wash once (3500rpm, <NUM>), adding 200µl PBS, and performing detection on a flow cytometer. This invention also suffers from the above technical problem.

Therefore, it is necessary to develop a new method for preparing lymphocyte samples for use in flow cytometry analysis.

In view of the status of the current technology, the present invention provides a method for preparing flow cytometric sample in human immune cells in vitro for flow cytometry, which includes the steps of:.

As an example, the concentration can be <NUM>×<NUM><NUM>/ml, <NUM>×<NUM><NUM>/ml, <NUM>×<NUM><NUM>/ml, <NUM> ×<NUM><NUM>/ml, <NUM>×<NUM><NUM>/ml, or <NUM>×<NUM><NUM>/ml.

In the method of the present application, as one of the embodiments, the PBS solution is <NUM>-<NUM> PBS solution at pH <NUM>-<NUM>. In the method of the present application, as an example, the <NUM>-<NUM> PBS solution can be <NUM> PBS, <NUM> PBS, <NUM> PBS, <NUM> PBS, <NUM> PBS or <NUM> PBS solution.

As one of the embodiments, alternatively, the PBS buffer solution may contain calf serum and/or EDTA, and the concentration of calf serum or EDTA can be a conventional concentration in the art.

In the method of the present application, as one of the embodiments, the PBS-EDTA solution is a mixed solution at pH <NUM>-<NUM> containing <NUM>-<NUM> PBS and a final concentration of <NUM>-<NUM> EDTA.

In the method of the present application, as one of the embodiments, step <NUM> of the present application further includes centrifuging conditions of <NUM>-3500rpm, <NUM>-<NUM>; preferably <NUM>-2900rpm and <NUM>-<NUM>.

In the method of the present application, as one of the embodiments, in step <NUM>), the blood cell precipitate and PBS solution are added in a volume ratio of <NUM>:<NUM>-<NUM>:<NUM> and preferably <NUM>:<NUM>.

In the method of the present application, as one of the embodiments, step <NUM>) of the present application further includes centrifuging conditions of <NUM>-3500rpm, <NUM>-<NUM>, <NUM>; preferably <NUM>-2900rpm, <NUM>, <NUM>.

In the method of the present application, as one of the embodiments, step <NUM>) of the present application further includes centrifuging conditions of <NUM>-3500rpm for <NUM>-<NUM> minutes; preferably <NUM>-2900rpm for <NUM>-<NUM> minutes.

In the method of the present application, as one of the embodiments, step <NUM>) of the present application further includes centrifuging conditions of <NUM>-2900rpm for <NUM>-<NUM> minutes.

In the method of the present application, as one of the embodiments, the amount of PBS solution added in step <NUM>) is an amount equal to the volume of blood cell precipitate.

In the method of the present application, as one of the embodiments, the PBS solution in step <NUM>) is <NUM>-<NUM> PBS solution at pH <NUM>-<NUM>; as one of the embodiments, alternatively, the PBS buffer solution can contain calf serum and/or EDTA, and the concentration of the calf serum or EDTA can be a conventional concentration in the art; as one of the embodiments, the PBS-EDTA solution is a mixed solution at pH <NUM>-<NUM> containing <NUM>-<NUM> PBS and a final concentration of <NUM>-<NUM> EDTA.

In the method of the present application, as one of the embodiments, the lymphocyte includes, but not limited to, lymphocyte subpopulations, which can include memory lymphocyte and other lymphocyte subpopulations that need to be detected, as well as functional analysis, cytokine analysis and dendritic cell maturity detection for lymphocyte subpopulations.

As one of the embodiments, the method of the present application includes.

As an example, the concentration can be <NUM>×<NUM><NUM>/ml, <NUM>×<NUM><NUM>/ml, <NUM>×<NUM><NUM>/ml, <NUM> ×<NUM><NUM>/ml, <NUM>×<NUM><NUM>/ml or <NUM>×<NUM><NUM>/ml.

Each of the flow tubes is added with 100µl cell suspension, and added with flow cytometric antibody for labeling lymphocyte subpopulations, gently shookin a vortex mixer at <NUM>-<NUM>, and incubated without light for <NUM>-<NUM> minutes, preferably <NUM>-<NUM> minutes and most preferably <NUM> minutes.

After staining, the cells are resuspended in <NUM> pre-cooled PBS solution, and centrifuged at <NUM>-<NUM> rpm for <NUM>-<NUM>. The supernatant is discarded, and 400µl PBS-EDTA solution at <NUM>-<NUM> is added under stirring.

The present application solves the technical problems existing in the prior art. In the present application, blood plasma is separated by centrifugation, monocytes are purified by using lymphocyte separation solution, and the antibody is incubated and washed. At present, this method adopts lymphocyte separation solution and auxiliary centrifugation, and performs quantification according to the counting of lymphocytes, that is, adding a specified amount of lymphocytes and adding the antibody in an amount corresponding thereto, thereby solving the problem existing in the prior art, while avoiding a secondary binding caused by oversaturation of the antibody, or insufficient amount of antibody due to excessive lymphocytes.

Compared with traditional samples, the present application enriches and purifies lymphocyte subpopulations, so that part of background cell subpopulations such as granulocytes, red blood cells, platelets, etc. are removed, providing the detection with the advantages of obvious lymphocyte subpopulations, clean background, easy compensation, reduced amount of antibody, and more accurate and convenient flow cytometric analysis and detection. The present application saves time and the use of antibody, reduces the detection cost, makes the background clean, the clustering is clear and obvious, and the trace of the subsets such as DC cells are enriched, and the clustering is obvious, so that the detection results are accurate.

The following examples and test examples are used to further elaborate the application, not intended to limit the effective scope of the present application in any way.

preparing PBMC samples needed for flow cytometric detection (also referred to as "samples" in the context of the present invention).

There are relatively complete antigens or receptors remained on the surface of living cells. Fluorescence labeled antibodies are used to bind to corresponding antigens on the cell surface, and fluorescence intensity and positive percentage are measured to provide the density and distribution of the corresponding antigens.

Super Clear Workbench, Era Beili low speed centrifuge DT5-<NUM>, a blood cell analyzer, a vortex mixer, and pipettes of various ranges.

<NUM> centrifuge tube, <NUM> pipette, <NUM> centrifuge tube, a flow tube, <NUM> pasteurizer pipette, and pipette tips of various ranges.

Routine flow cytometric antibodies: anti-CD3 antibody, lin1[Lineage cocktail <NUM>], anti-CD123 antibody and anti-CD11 antibody.

A flow tube was added with 100µl lymphocyte suspension, added with flow cytometric antibody for labeling lymphocyte subpopulations, mixed evenly to homogenize the antibody and the cells, and incubated at <NUM>-<NUM> without light for <NUM> minutes.

After staining, the cells were resuspended in <NUM> pre-cooled PBS solution, and centrifuged at <NUM>-<NUM> rpm for <NUM>-<NUM> minutes.

The comparative observation results of the sample prepared by the present application and the sample treated by hemolysin are as follow:.

<NUM> the sample treated with hemolysin provides the following observation results: it can be seen that, in the obtained cell populations, the number of cells is relatively small, the cell populations are too close to each other, and some of the granulocytes and lymphocytes adhere seriously, so that the cell populations cannot be identified; and there is a relatively large number of discrete cell debris, as particularly shown in <FIG>.

The sample obtained by the present application provides the following observation results: after separation and enrichment, the number of cells is relatively large, the populations are obviously separated, the boundary is clear, and the part of the cell populations adhered to the granulocytes are removed during treatment, as particularly shown <FIG>.

It can be seen by comparing the cells obtained in <FIG> that, the sample of the present application had successfully achieved separation and enrichment of cells, with a relatively large number of cells, obviously separated populations and clear boundaries, and the part of the cell populations adhered to the granulocytes were removed during treatment.

<NUM> the comparative observation results of enriched cells from the sample prepared by the present application and the sample treated with hemolysin are as follow:
<FIG> and <FIG> show stained antibody lin1[Lineage Cocktail <NUM>], anti-CD123 and anti-CD11; FITC is the first channel, and the stained antibody is antibody lin1[Lineage Cocktail <NUM>]; PerCP is the third channel, and the stained antibody is anti-CD123; APC is the fourth channel, and the stained antibody is anti-CD11. In particular, <FIG> is the cell diagram obtained by hemolysin method; <FIG> is the flow cytometric diagram obtained by lymphocyte separation solution and centrifugation method. It is apparent that the cells in <FIG> are enriched in comparison with those in <FIG>.

In terms the number of cells, the sample of the present application has a better effect of cell enrichment. The particular analysis process is as follows: first, drawing a histogram, circling door <NUM>: FL1 -, then circling FL3-H, FL4-H cell subpopulation-<NUM> inside door <NUM>, that is, cell surface marker lin1-CD123+CD11C- represents plasma cell-derived DC cells (pDC);.

the cell surface marker lin1-CD11C+ represents myeloid cell-derived DC cells (mDC); in particular, in the field of flow cytometry detection, the more cells can be collected, the more accurate the results will be. It is obvious that the number and populations of mDC cells and pDC cells in <FIG> are better than those in <FIG>.

<NUM> From the comparison, it can be seen that, the samples obtained by lysis had too much debris and unclean background, which will influence the collection and analysis of cytometer data, and might cause bias to the compensation adjustment of lymphocyte (see <FIG>).

In particular, from the observation of lymphocyte cells inside the door of the circle in the analysis figure of lymphocyte subpopulations obtained by lysis, it can be seen that the lymphocyte populations and cell debris populations are separated far from ideal (see three figures in the first row of <FIG>).

The population inside the door of the circle in the analysis figure of lymphocyte subpopulations obtained by the present application is lymphocyte population, and it can be seen that the lymphocyte populations and cell debris populations are widely separated, being very ideal (see three figures in the second row of <FIG>).

<NUM> It can be seen from <FIG> that, when using fluorescence antibody, the present application saves <NUM>% or more of the antibodies by adding fluorescent antibody in proportion based on the counted number of lymphocytes. The use amount of the combined antibody during detection in the present application is 15µl, and the use amount of the antibody in the lysis method is 60µl. The method of the present application does not cause excessive or insufficient amount of antibody. From <FIG>, it can be seen that what are labeled in Group <NUM> are CD3 FITC and CD8PerCP; and what are labeled in Group <NUM> are CD3 FITC and CD4PerCP. Due to the different samples obtained by different treatment methods, for the same in vitro peripheral blood sample, the results are that the proportion of double positive groups in the samples obtained by the lysis method is obviously reduced, and the amount of antibody cannot be quantified. The samples obtained by the method of the present application can be added with antibody by an amount corresponding to that of the cells. Besides, it is obvious that the cells obtained by separation method have been enriched to a certain extent.

Claim 1:
A method for preparing flow cytometric sample in human immune cells in vitro for flow cytometry, characterized by comprising the steps of:
<NUM>) separating plasma and blood precipitation by centrifuging in vitro anticoagulant blood samples to obtain blood cell precipitate;
<NUM>) mixing the blood cell precipitate with PBS solution to obtain a blood cell dilution;
<NUM>) adding the blood cell dilution into a centrifuge tube containing the lymphocyte separation solution having a volume equal to that of the blood cell dilution, centrifuging, and discarding plasma to obtain buffy coat cells;
<NUM>) adding the buffy coat cells to a centrifuge tube, adding PBS solution under stirring, centrifuging, discarding supernatant, and then adding PBS solution to adjust the cell concentration to provide lymphocyte suspension having a concentration of <NUM>×<NUM><NUM>/ml - <NUM>×<NUM><NUM>/ml, further preferably <NUM>×<NUM><NUM>/ml - <NUM>×<NUM><NUM>/ml;
<NUM>) adding the lymphocyte suspension into a flow tube, then adding flow cytometric antibody to label lymphocyte subpopulations, mixing evenly, and incubating at <NUM>-<NUM> without light;
<NUM>) adding PBS solution to the flow tube, mixing, and centrifuging to remove unbound antibody;
<NUM>) completion of samples;
① if a detection on a flow cytometer is performed immediately, the supernatant is discarded and the precipitate is mixed with PBS-EDTA solution at <NUM>-<NUM> for flow cytometric detection; or
② if the detection cannot be performed immediately, formalin is added, mixed, and left to stand at <NUM>-<NUM>°Cwithout light; and before detection, each of the tubes is added with PBS solution, centrifuged to discard the supernatant, and evenly mixed with PBS-EDTA solution at <NUM>-<NUM> for flow cytometric detection.