A METHOD FOR PREPARING LYMPHOCYTE SAMPLE FOR FLOW CYTOMETRY ANALYSIS

Abstract

The invention provides methods for preparing lymphocyte samples for flow cytometric analysis. The method includes: centrifuging anticoagulant blood samples to obtain blood cell precipitate, diluting the blood cell precipitate with PBS solution, then adding into a centrifuge tube containing lymphocyte separation solution, centrifuging, discarding the plasma to obtain buffy coat cells; then adding the buffy coat cells into a centrifuge tube, adding PBS solution, centrifuging, discarding the supernatant, adding PBS solution to adjust the concentration of cells, adding the cell solution into a flow tube; then adding antibody, mixing, incubating without light and centrifuging, discarding the supernatant and resuspending the cells in pre-cooled PBS-EDTA solution. The samples obtained enriches and purifies lymphocytes, fully preserves the activity of lymphocytes, removes granulocytes, red blood cells, platelets or other blood components, and the subpopulations to be detected are purified, the analysis and detection are more accurate, and costs for analyzing antibodies and reagents are saved.

Claims

1. A method for preparing flow cytometric sample in immune cells for flow cytometry, comprising the steps of: 1) separating plasma and blood precipitation by centrifuging in vitro anticoagulant blood samples to obtain blood cell precipitate; 2) mixing the blood cell precipitate with PBS solution to obtain a blood cell dilution; 3) 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; 4) 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 1×10.sup.5/ml-1×10.sup.8/ml; 5) adding the lymphocyte suspension into a flow tube, then adding flow cytometric antibody to label lymphocyte subpopulations, mixing evenly, and incubating at 2-8° C. without light; 6) adding PBS solution to the flow tube, mixing, and centrifuging to remove unbound antibody; 7) completion of samples; {circle around (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 2-8° C. for flow cytometric detection; or {circle around (2)} If the detection cannot be performed immediately, formalin is added, mixed, and left to stand at 2-8° C. without 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 2-8° C. for flow cytometric detection.

2. The method according to claim 1, wherein, the PBS solution is 0.005-0.05M PBS solution at pH 7.2-7.4; and the PBS-EDTA solution is a mixed solution at pH 7.2-7.4 containing 0.005-0.05M PBS and a final concentration of 2-3 mM EDTA.

3. The method according to claim 1, wherein, step 1 further comprises centrifuging conditions of 1500-3500 rpm, 5-30 min.

4. The method according to claim 1, wherein, in step 2), the blood cell precipitate and PBS solution are added in a volume ratio of 1:0.5-1:2.

5. The method according to claim 1, wherein, step 3) further comprises centrifuging conditions of 1500-3500 rpm, 10-20 min, 4° C.

6. The method according to claim 1, wherein, step 4) further comprises centrifuging conditions of 1500-3500 rpm for 5-20 minutes.

7. The method according to claim 1, wherein, step 6) further comprises centrifuging conditions of 1500-2900 rpm for 5-10 minutes.

8. The method according to claim 1, wherein, the amount of PBS solution added in step 6) is an amount equal to the volume of blood cell precipitate.

9. The method according to claim 1, wherein, the sample obtained in step 7) is treated by: {circle around (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 2-8° C. for flow cytometric detection; or {circle around (2)} If the detection is not be performed immediately, 0.05-5% formalin, preferably 1% formalin, is added, mixed, and left to stand at 2-8° C. without 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 2-8° C. for flow cytometric detection.

10. The method according to claim 9, wherein, the PBS solution is 0.005-0.05M PBS solution at pH 7.2-7.4; and the PBS-EDTA solution is a mixed solution at pH 7.2-7.4 containing 0.005-0.05M PBS and a final concentration of 2-3 mM EDTA.

11. The method according to claim 1, comprising the steps of: (I) separation of human peripheral blood mononuclear cells (1) obtaining an in vitro anticoagulant peripheral blood sample and counting; (2) centrifuging the blood sample in a centrifuge tube and discarding the upper plasma to provide lower blood cell precipitate; (3) diluting the blood cell precipitate with PBS solution by a volume ratio of 1:1 to provide blood cell dilution; (4) adding lymphocyte separation solution having a volume equal to the volume of the blood cell dilution into a centrifuge tube; (5) adding the blood cell dilution into the centrifuge tube in step (4) slowly for keeping the layer of the separation solution clear; (6) Slowly placing the centrifuge tube into a low-speed centrifuge, balancing, and centrifuging; (7) discarding the supernatant after centrifuging to provide buffy coat cells; (8) placing the buffy coat cells into a centrifuge tube, adding PBS solution, counting, and centrifuging; and (9) removing the supernatant after centrifuging, and adding PBS solution at 2-8° C. according to the result of counting, to obtain a cell suspension having a cell density of 1.5-3.0×10.sup.6/ml; (II) Preparation of flow cytometric samples (10) sample staining added 100 μl lymphocyte suspension into a flow tube, adding corresponding antibody, mixed evenly, and incubating at 2-8° C. without light for 10-30 minutes, preferably 15-20 minutes, and most preferably 20 minutes; (11) sample washing after dyeing, adding PBS solution pre-cooled at 2-8° C. to resuspend the cells, centrifuging for 5-10 minutes, and removing unbound antibody.

12. The method of claim 1, wherein lymphocyte suspension has a concentration of 1.5×10.sup.6/ml-3×10.sup.6/ml.

13. The method according to claim 3, wherein step 1 further comprises centrifuging conditions of 1500-2900 rpm and 15-20 min.

14. The method according to claim 4, wherein in step 2), the blood cell precipitate and PBS solution are added in a volume ratio of 1:1.

15. The method according to claim 5, wherein step 3) further comprises centrifuging conditions of 1500-2900 rpm, 20 min, 4° C.

16. The method according to claim 6, wherein step 4) further comprises centrifuging conditions of 1500-2900 rpm for 5-10 minutes.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0063] FIG. 1: flow cytometric diagram of a sample obtained with method of literature using hemolysin, i.e., lysis method, in example 1;

[0064] FIG. 2: flow cytometric diagram of a sample obtained with the present application, in example 1;

[0065] FIG. 3: flow cytometric diagram of a sample obtained with the present application, in example 1;

[0066] FIG. 4: flow cytometric diagram of a sample obtained with method of literature in example 1;

[0067] FIG. 5: flow cytometric diagram of a sample obtained with the present application, in example 1;

[0068] FIG. 6: a comparison diagram of the cell populations of the sample obtained according to the method of example 1 in Test example 2 and the sample obtained with lysis method of literature, (the upper 3 pictures are the flow cytometry diagrams obtained by the lysis method, and the lower 3 pictures are graphs correspond to the flow cytometry obtained by the sample separation method).;

[0069] FIG. 7: comparative diagram of cell enrichment in the sample obtained by the method of example 1 and the sample obtained by the lysis method of literature in test example 2;

[0070] FIG. 8: comparative diagram of enrichment of different types of cells in the sample obtained by the method of example 1 and the sample obtained with lysis method of literature in test example 2.

DETAILED DESCRIPTION

[0071] 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.

EXAMPLE 1

Preparation of PBMC Samples Needed for Flow Cytometry

[0072] 1. Purpose

[0073] preparing PBMC samples needed for flow cytometric detection (also referred to as “samples” in the context of the present invention)

[0074] 2. Principle

[0075] 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.

[0076] 3. Preparation of Instruments and Consumable Materials

[0077] 3.1 Instruments

[0078] Super Clear Workbench, Era Beili low speed centrifuge DT5-4, a blood cell analyzer, a vortex mixer, and pipettes of various ranges

[0079] 3.2 Consumable Materials

[0080] 15 ml centrifuge tube, 10 ml pipette, 1.5 ml centrifuge tube, a flow tube, 3 ml pasteurizer pipette, and pipette tips of various ranges.

[0081] 3.3 Formulations of Reagents

TABLE-US-00001 Stock Name solution Formulation 1 × PBS 10 × PBS stock solution: purified water = 1:9 0.05-5% 10% stock solution: 1 × PBS = 1:9 formalin formalin PBS solution 1 × PBS 1 × PBS (0.01M PBS, pH = 7.2~7.4) PBS-EDTA 1 × PBS adding 0.5M EDTA to 1 × PBS (0.01M solution PBS; pH = 7.2~7.4) until the final concentration of EDTA is 2.5 mM

[0082] Routine flow cytometric antibodies: anti-CD3 antibody, lin1[Lineage cocktail 1], anti-CD123 antibody and anti-CD11 antibody.

[0083] 4. Operation Procedure (Illustrated by Taking 5 ml Anticoagulant Blood Sample as an Example)

[0084] 4.1. Separation of PBMC from Peripheral Blood

[0085] (1) obtaining 0.2 ml original blood and counting;

[0086] (2) adding the rest of the original blood to a centrifuge tube, centrifuging at 1500-2900 rpm for 15-20 min, and removing upper plasma;

[0087] (3) diluting cell precipitate of the rest of the original blood with PBS solution by volume;

[0088] (4) providing a centrifuge tube and adding lymphocyte separation solution having a volume equal to the volume of the blood cell dilution to the centrifuge tube by using a pipette;

[0089] (5) adding blood cell dilution to the centrifuge tube in step (4) slowly for keeping the layer of separation solution clear;

[0090] (6) slowly placing the centrifuge tube into a centrifuge, balancing, and centrifuging at 1500-2900 rpm for 15-20 min;

[0091] (7) slowly suctioning and discarding the supernatant with a pipette after centrifuging;

[0092] (8) pipetting middle buffy coat cells into a new centrifuge tube, adding PBS solution to a volume of 10 ml, counting 100 μl of the solution with a whole blood cell counter for subsequent dilution, and centrifuging the rest of the cell suspension at 1500-2900 rpm for 5-10 min;

[0093] (9) discarding the supernatant after centrifuging, and removing residual liquid by using a 1 ml pipette. According to the result of counting, appropriate amount of pre-cooled PBS solution at 4° C. was added to adjust the density of cells to 1.5-3.0×10.sup.6/ml, for example, 2×10.sup.6/ml.

[0094] 4.2 Preparation of Flow Cytometric Samples (The Temperature was Controlled in this Operation, and the Samples and Antibodies Were Operated in an Ice Box Throughout the Operation)

[0095] (1) Sample Staining

[0096] A flow tube was added with 100 μl lymphocyte suspension, added with flow cytometric antibody for labeling lymphocyte subpopulations, mixed evenly to homogenize the antibody and the cells, and incubated at 2-8° C. without light for 20 minutes.

[0097] (3) Sample Washing

[0098] After staining, the cells were resuspended in 2 ml pre-cooled PBS solution, and centrifuged at 1500-2900 rpm for 5-10 minutes.

[0099] (4) Completion of Samples

[0100] {circle around (1)} If the detection on a flow cytometer is performed immediately, the supernatant is discarded and the cells are mixed in 400 μl pre-cooled PBS-EDTA solution for flow detection;

[0101] {circle around (2)} If the detection cannot be performed immediately, 500 μl 0.05-5% formalin is added, fully mixed, and left to stand at 2-8° C. without light. Before detection, each of the tubes is added with 2 ml PBS solution, and centrifuged at 1500-2900 rpm for 5-10 minutes to discard the supernatant, and the cells are evenly mixed with 400 μl pre-cooled PBS-EDTA solution for detection on a flow cytometer.

TEST EXAMPLE 1

Comparative Test Between a Sample Prepared by the Present Application and a Sample Obtained by the Method of Treating with Hemolysin

[0102] 1.1 Samples and Reagents:

[0103] Lymphocyte sample of the present application: the sample was prepared from in vitro blood sample by using the method according to the above example 1;

[0104] Comparative lymphocyte samples: the sample was prepared from in vitro blood sample by hemolysin treatment of in vitro blood sample specified in section 1.2.1

[0105] 5 ml anticoagulant blood sample;

[0106] PBS solution: prepared by 10× PBS solution: purified water=1:9 by volume

[0107] PBS-EDTA solution: prepared by adding 0.5M EDTA to 1× PBS until the final concentration of EDTA is 2.5 mM;

[0108] Routine flow cytometric antibodies: anti-CD3 antibody, lin1[Lineage Cocktail 1], anti-CD123 antibody, and anti-CD11 antibody.

[0109] 1.2 Test Method:

[0110] 1.2.1 Hemolysin Treatment Method:

[0111] (1) sample staining: adding 100 μl anticoagulant blood sample into each of the flow tubes, adding flow cytometric antibody into the tube to label lymphocyte subpopulations, slowly shaking the tube in a vortex mixer to mix the antibody and the cells evenly, and incubating the tube without light at 2-8° C. for 20 minutes;

[0112] (2) lysing: adding 2 ml 1× BD hemolysin (i.e. erythrocyte lysis solution) to the flow tube for 5 minutes;

[0113] (3) sample washing: after 5 minutes, centrifuging the tube at 1500-2900 rpm for 10 minutes, discarding the supernatant after centrifuging, adding 2 ml pre-cooled PBS solution to resuspend the cells, centrifuging the tube at 1500-2900 rpm for 5-20 minutes, discarding the supernatant, adding 400 μL PBS-EDTA solution at 2-8° C. into the tube, and vortex mixing for 3 seconds to obtain the comparative lymphocyte sample.

[0114] 1.2.2 Detection Method on Flow Cytometer:

[0115] a) checking the status of the cytometer before starting up;

[0116] b) warming up the flow cytometer system for 5-10 minutes and performing perfusion;

[0117] c) performing quality control procedure and proceeding to the next step after qualification;

[0118] d) setting the parameters of the flow cytometer, adjusting the voltage of each of detection channels, and setting the liquid flow speed to 35-40 μL/min;

[0119] e) vortex vibrating the prepared lymphocyte samples of the present application and the comparative lymphocyte samples for 3 seconds, and respectively subjecting the samples to detection under the conditions of: 15000 cells in the door; and collecting samples.

[0120] 1.3 Results of Experiments:

[0121] The comparative observation results of the sample prepared by the present application and the sample treated by hemolysin are as follow:

[0122] 1.3.1 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. 1.

[0123] 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. 2.

[0124] It can be seen by comparing the cells obtained in FIG. 1 and FIG. 2 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.

[0125] 1.3.2 the comparative observation results of enriched cells from the sample prepared by the present application and the sample treated with hemolysin are as follow:

[0126] FIGS. 3, 4 and 5 show stained antibody lin1[Lineage Cocktail 1], anti-CD123 and anti-CD11; FITC is the first channel, and the stained antibody is antibody lin1[Lineage Cocktail 1]; 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. 4 is the cell diagram obtained by hemolysin method; FIG. 5 is the flow cytometric diagram obtained by lymphocyte separation solution and centrifugation method. It is apparent that the cells in FIG. 5 are enriched in comparison with those in FIG. 4.

[0127] 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 1: FL1-, then circling FL3-H, FL4-H cell subpopulation-1 inside door 1, that is, cell surface marker lin1.sup.−CD123.sup.+CD11C.sup.− represents plasma cell-derived DC cells (pDC);

[0128] the cell surface marker lin1.sup.−CD11C.sup.+ 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. 5 are better than those in FIG. 4.

TEST EXAMPLE 2

Comparison Between a Sample Prepared by the Present Application and a Sample Obtained by Hemolysin Lysis

[0129] 2.1 Samples and Reagents:

[0130] Lymphocyte sample of the present application: the sample was prepared from in vitro blood sample by using the method according to the above example 1;

[0131] Comparative lymphocyte samples: the sample was prepared from in vitro blood sample by a method specified in the following section 2.2.1

[0132] 5 ml anticoagulant blood sample;

[0133] PBS solution: prepared by 10× PBS solution: purified water=1:9 by volume

[0134] PBS-EDTA solution: prepared by adding 0.5M EDTA to 1× PBS until the final concentration of EDTA is 2.5 mM;

[0135] Routine flow cytometric antibodies: anti-CD3 antibody, anti-CD123 antibody, anti-CD11 antibody, CD3 FITC, CD8PerCP, and CD4PerCP.

[0136] 2.2 Test Method:

[0137] 2.2.1 Hemolysin Lysis Treatment Method:

[0138] (1) sample staining: adding 100 μl anticoagulant blood sample into each of the flow tubes, adding flow cytometric antibody to the tube to label lymphocyte subpopulations, fully mixing the antibody and the cells, and incubating the tube at 2-8° C. without light for 20 minutes;

[0139] (2) lysing: adding 2 ml 1× BD hemolysin (i.e. erythrocyte lysis solution) to the flow tube for 5 minutes;

[0140] (3) sample washing: after 5 minutes, centrifuging the tube at 1500 rpm for 10 minutes, discarding the supernatant after centrifuging, adding 2 ml pre-cooled PBS solution to resuspend the cells, centrifuging the tube at 1500 rpm for 5 minutes, discarding the supernatant, adding 400 μL PBS-EDTA solution at 2-8° C. into the tube, and mixing evenly.

[0141] 2.2.2 Detection Method on Flow Cytometer:

[0142] a) checking the status of the cytometer before starting up;

[0143] b) warming up the flow cytometer system for 5-10 minutes and performing perfusion;

[0144] c) performing quality control procedure and proceeding to the next step after qualification;

[0145] d) setting the parameters of the flow cytometer, adjusting the voltage of each of detection channels, and setting the liquid flow speed to 35-40 μL/min;

[0146] e) vortex vibrating the prepared lymphocyte samples of the present application and the comparative lymphocyte samples for 3 seconds, and respectively subjecting the samples to detection under the conditions of: 15000 cells in the door; and collecting samples.

[0147] 2.3 Results of Experiments:

[0148] 2.3.1 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. 6).

[0149] 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. 6).

[0150] 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. 6).

[0151] 2.3.2 It can be seen from FIG. 7 that, when using fluorescence antibody, the present application saves 70% 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 15 μl, and the use amount of the antibody in the lysis method is 60 μl. The method of the present application does not cause excessive or insufficient amount of antibody. From FIG. 7, it can be seen that what are labeled in Group 1 are CD3 FITC and CD8PerCP; and what are labeled in Group 2 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.

[0152] 2.3.3 it can be seen from FIG. 8 that, the sample prepared by the present application has enrichment effect for the sample with small proportion of cell subpopulations. The proportion of cell subpopulation MDC and PDC in the same in vitro peripheral blood samples is relatively small. The observation results of the samples obtained by lysis method and the present method are different, and the cell subpopulations in the samples obtained by the present application are enriched, which can be shown from the number and percentage of cells.