Quantitative flow cytometry

Abstract

The present application discloses a method for measuring the cell concentration in a sample, the method comprising the following steps: step (1), using a flow cytometer to measure the cell concentration in a plurality of standard samples of known concentrations; step (2), preparing a calibration curve based on the measured values ​​measured in step (1) and the known concentrations; step (3), using a flow cytometer to measure the cell concentration in a sample of unknown concentration; and step (4), using the calibration curve prepared in step (2) to determine the cell concentration from the measured values ​​measured in step (3).

Description

Technical Field

[0001] The present invention relates to a method for measuring the cell concentration in a sample using a flow cytometer. Background Art

[0002] Flow cytometry is a method for measuring the size, DNA content, and distribution of membrane antigen expression of each cell in a cell population based on the principle of irradiating particles in a water flow with excitation light and measuring the fluorescence emitted from each particle. The measuring device used to perform this flow cytometry is called a flow cytometer.

[0003] A method for measuring the absolute count of cells using such flow cytometry is reported in Patent Document 1. The method described in Patent Document 1 is characterized by comprising the following steps.

[0004] Step (a), in order to label cells in each population present in the sample using a cell marker and a known number of fluorescent microparticles, the sample is added to a tube containing a diluent containing one or more cell markers;

[0005] Step (b), setting a fluorescence trigger to count substantially all tiny particles and cells to be counted that are present in the mother population;

[0006] Step (c), setting one or more fluorescence gates to identify each cell marker and microparticle;

[0007] step (d), counting the number of cells from step (c) that meet a fluorescent trigger or exceed the trigger; and

[0008] Step (e), calculating the number of cells per microparticle from each fluorescent gate in step (d), and multiplying by the concentration of the microparticles to obtain an absolute count of the number of cells per unit volume.

[0009] A known number of fluorescent microparticles are beads used as internal standards. In the method of Patent Document 1, a known number of beads are added to a sample tube, placed in a flow cytometer together with target cells and measured simultaneously, and then the target cell measured value is normalized based on the bead measured value, thereby reducing the measurement deviation between samples (improving precision).

[0010] On the other hand, whether the target cells are lost during the sample processing and whether the flow cytometer can be used to measure without omission are not considered. Therefore, although the method of Patent Document 1 is considered as a "method for measuring the absolute count of cells" as mentioned above, its accuracy cannot be guaranteed.

[0011] Prior art literature

[0012] Patent Literature

[0013] Patent Document 1: Japanese Patent Application Laid-Open No. 4-252957 Summary of the invention

[0014] Problems to be solved by the invention

[0015] As mentioned above, in the method of Patent Document 1, although the management of precision is feasible, the accuracy cannot be evaluated because the recovery rate of target cells is not considered. In order to evaluate the accuracy, the recovery rate of target cells must be obtained, and in order to obtain the recovery rate, a calibration curve must be prepared. However, Patent Document 1 does not disclose the recovery rate of standard substances, calibration curves, and accuracy evaluation at all.

[0016] An object of the present invention is to provide a method for measuring the cell concentration in a sample using a flow cytometer, which can achieve highly accurate quantification of the number of cells.

[0017] Means for solving problems

[0018] The inventors of the present application have conducted intensive research to achieve the above-mentioned purpose, and found that highly accurate quantification of cell number can be achieved as follows: a calibration curve is prepared based on the cell concentration in a standard sample measured using a flow cytometer and a known concentration, and the cell concentration is determined based on the measured value of the cell concentration in the sample measured using the flow cytometer using the calibration curve.

[0019] The present invention has been completed through further studies based on the above findings, and provides the following method for measuring the cell concentration in a sample.

[0020] Item 1. A method for measuring the cell concentration in a sample, the method comprising the following steps:

[0021] Step (1), using a flow cytometer to measure the cell concentration in a plurality of standard samples of known concentration;

[0022] Step (2), preparing a calibration curve based on the measured value measured in step (1) and the known concentration;

[0023] Step (3), using a flow cytometer to measure the cell concentration in the sample of unknown concentration; and

[0024] In step (4), the cell concentration is determined from the measured value obtained in step (3) using the calibration curve prepared in step (2).

[0025] Item 2. The method according to Item 1, wherein two or more standard samples of known concentrations are used.

[0026] Item 3. The method according to Item 1 or 2, wherein one or more standard samples are used for each known concentration.

[0027] Item 4. The method according to any one of items 1 to 3, wherein in step (2), the measured value having an error within ±30% relative to the known concentration is used to prepare the calibration curve.

[0028] Item 5. The method according to any one of items 1 to 4, wherein the accuracy of the measured value obtained in step (1) relative to the known concentration is within ±30%.

[0029] Item 6. The method according to any one of Items 1 to 5, wherein the sample is blood.

[0030] Item 7. The method according to any one of Items 1 to 6, wherein the cell is a T cell.

[0031] Effects of the Invention

[0032] The cell concentration measurement method of the present invention enables highly accurate quantification using a flow cytometer. DETAILED DESCRIPTION

[0033] Hereinafter, embodiments of the present invention will be described in detail.

[0034] It should be noted that, in the present specification, “comprise” includes the meaning of “essentially consist of” and the meaning of “consist of only”.

[0035] The "accuracy" used in this specification refers to the degree of accuracy indicating how close a value is to a true value. That is, the closer the average value is to the true value, the higher the "accuracy".

[0036] The term "precision" used in this specification refers to the degree of variation between values ​​obtained by multiple measurements.

[0037] The method for measuring the cell concentration in a sample of the present invention is characterized by comprising the following steps:

[0038] Step (1), using a flow cytometer to measure the cell concentration in a plurality of standard samples of known concentration;

[0039] Step (2), preparing a calibration curve based on the measured value measured in step (1) and the known concentration;

[0040] Step (3), using a flow cytometer to measure the cell concentration in the sample of unknown concentration; and

[0041] In step (4), the cell concentration is determined from the measured value obtained in step (3) using the calibration curve prepared in step (2).

[0042] In the present invention, the sample for measuring the cell concentration is not particularly limited as long as it can be measured by flow cytometry, and examples thereof include blood, tumor, cerebrospinal fluid, liver, lung, spleen, thymus, bone marrow, and the like.

[0043] Before the measurement using the flow cytometer, the sample can be suitably subjected to various pretreatments. For example, when blood is used as the sample, a pretreatment such as hemolysis can be performed before the measurement. Such hemolysis can be performed by a known method, for example, as a method for dissolving red blood cells, adding a surfactant can be cited. As other pretreatments, cell immobilization can be cited. As a cell fixative used in such cell immobilization, for example, paraformaldehyde, glutaraldehyde, methanol, etc. can be cited.

[0044] In the present invention, the cells whose concentration is measured are not particularly limited as long as they are cells whose cell number can be measured by flow cytometry, and examples thereof include neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp hepatocytes, ES cells, iPS (induced pluripotent stem) cells, tissue precursor cells, blood cells (erythrocytes, leukocytes and platelets), epithelial cells, endothelial cells, muscle cells, fibroblasts, smooth muscle cells, hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells, brain cells, lung cells, kidney cells, adipocytes, cardiomyocytes, osteoblasts, neurons, vascular endothelial cells, osteocytes, osteoclasts, chondrocytes, chondrogenic cells, corneal cells, retinal cells, dendritic cells, primary culture cells, passage cells, and lineage cells. Examples of the above-mentioned leukocytes include monocytes, lymphocytes (T cells, B cells, and NK cells), dendritic cells, macrophages, eosinophils, neutrophils, basophils, etc. T cells include helper T cells, cytotoxic T cells, regulatory T cells, suppressor T cells, CAR-T cells (chimeric antigen receptor T cells), etc.

[0045] There are no particular limitations on the flow cytometer used in the present invention, and various commercially available products can be used. Examples of such commercially available products include Beckman Coulter (e.g., Gallios, Navios, Navios EX, CytoFLEX, CytoFLEX S, CytoFLEX LX, Cytomics FC 500), BD Biosciences (e.g., BD FACSCanto TM II flow cytometer, BD FACSVerse TM Flow cytometer, BD FACSLyric TM Flow cytometer, BDLSRFortessa TM Flow cytometer, BD LSRFortessa TM The determination of cell concentration using a flow cytometer can be carried out according to known methods. For example, it can be carried out according to the manual of the manufacturer of the flow cytometer.

[0046] In flow cytometry, target cells can be detected based on cell size (forward scattered light: FSC), cell density (side scattered light: SSC), cell surface antigens (fluorescence from fluorescently labeled antibodies), etc.

[0047] When measuring using a flow cytometer, a fluorescently labeled antibody capable of detecting target cells can be appropriately selected and used. As such a fluorescently labeled antibody, for example, a fluorescently labeled antibody that specifically binds to various CD antigens can be cited. For fluorescently labeled antibodies, a variety of antibodies are commercially available, so commercially available products can be used, or antibody preparation and fluorescent labeling can be performed to obtain fluorescently labeled antibodies. As a compound for fluorescently labeling an antibody, a known fluorescently labeled compound can be widely used.

[0048] When measuring by flow cytometry, fluorescent beads can also be used as internal standards (see Patent Document 1). The known number of beads mentioned above is placed in a sample tube, placed in a flow cytometer together with target cells and measured simultaneously, and the measured value of the target cells is normalized according to the measured value of the beads, that is, calibrated according to the known number of beads in the sample tube, so that the number of cells that may be present in the sample tube can be calculated.

[0049] Step (1)

[0050] In step (1), in order to prepare a calibration curve, the cell concentrations in a plurality of standard samples of known concentrations are measured using a flow cytometer.

[0051] There is no particular limitation on the number of concentrations of the standard samples used in step (1) as long as a calibration curve can be drawn. For example, it can be 2 or more, preferably 3 or more, more preferably 4 or more, further preferably 5 or more, and particularly preferably 6 or more. The upper limit is, for example, 30.

[0052] As a method for preparing a standard sample, a method that can adjust the concentration of cells can be widely used. For example, a method of visually counting using a hemocytometer with a microscope can be mentioned. As long as one standard sample can be prepared, other standard samples can be prepared by gradient dilution.

[0053] In addition, there is no particular restriction on the number of standard samples of each known concentration used in step (1), for example, it is more than 1, preferably more than 2, more preferably more than 3, further preferably more than 4, particularly preferably more than 5, most preferably more than 6, and the upper limit is, for example, 90.

[0054] The accuracy of the measured values ​​obtained by step (1) relative to the known concentration is preferably within ±30%, more preferably within ±25%, further preferably within ±20%, and particularly preferably within ±15%. The value of "accuracy" herein is the value (relative error) of the difference between the average value of the measured values ​​and the known concentration expressed as a percentage relative to the known concentration. By using the measured values ​​with such accuracy for the preparation of the calibration curve, more accurate quantification of the cell concentration can be achieved.

[0055] In addition, the accuracy of the measured values ​​obtained in step (1) relative to the known concentration is preferably within ±30%, more preferably within ±25%, further preferably within ±20%, and particularly preferably within ±15%. The value of "accuracy" herein refers to the coefficient of variation (CV). By using the measured values ​​with such accuracy for the preparation of the calibration curve, more accurate quantification of the cell concentration can be achieved.

[0056] Step (2)

[0057] In step (2), a calibration curve is prepared based on the measured values ​​obtained in step (1) and the known concentrations.

[0058] The calibration curve can be prepared by a common method such as the least square method using software etc. The closer the correlation coefficient of the prepared calibration curve is to 1, the more ideal it is.

[0059] By creating a calibration curve as described above, the recovery rate of target cells in the sample preparation step can be determined, thereby enabling accuracy evaluation.

[0060] In step (2), (especially when the slope of the calibration curve is approximately 1), it is desirable to use the measured values ​​whose error relative to the known concentration falls within ±30% (preferably within ±25%, more preferably within ±20%, and further preferably within ±15%) for the preparation of the calibration curve. By using the measured values ​​with the error range as described above for the preparation of the calibration curve, more accurate quantification of the cell concentration can be achieved.

[0061] It should be noted that when the slope of the calibration curve is not 1 (for example, when the cell recovery rate is not 100%), it is desirable to use the calibration curve formula to calculate the number of cells from the measured values ​​and verify the accuracy of the calculated value relative to the known concentration.

[0062] Step (3)

[0063] In step (3), the cell concentration in a sample of unknown concentration is measured using a flow cytometer.

[0064] Step (4)

[0065] In step (4), the cell concentration is determined from the measured value obtained in step (3) using the calibration curve prepared in step (2).

[0066] By determining the cell concentration using the calibration curve in this way, highly accurate quantification using a flow cytometer can be performed.

[0067] Example

[0068] Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0069] <Method>

[0070] 50 μL of CB-17 SCID mouse blood was used as a sample, and human T cells (peripheral blood T cells, Cryo-T8: Human CD8 + Negatively Selected (5-8M cells), Precision Bioservices) and samples without human T cell lysate. These samples were placed in commercially available tubes (TruCOUNT Tubes, BD Biosciences) and BD Tritest TM CD3 / CD8 / CD45 reagent (BD Biosciences) was mixed. This reagent contains fluorescein isothiocyanate (FITC)-labeled anti-CD3 antibody (clone SK7), phycoerythrin (PE)-labeled anti-CD8 antibody (clone SK1), and peridinin chlorophyll protein (PerCP)-labeled anti-CD45 antibody (clone 2D1).

[0071] After standing at room temperature for 15 minutes, BD FACS TM Lysis solution (BD Biosciences) was used to hemolyze the blood and immobilize the cells. After standing for another 15 minutes, flow cytometry was performed for quantification. Flow cytometry was performed using BD FACS Lyric TM Flow cytometry was performed by BD Biosciences. FlowJo software (version 10, BD Biosciences) was used for analysis, and human CD45 / CD3 / CD8 positive cells were detected as target cells. Samples were prepared and measured at n=3 for each concentration.

[0072] <Results>

[0073] The results of the calibration curve preparation are shown in Table 1. The linearity of the calibration curve was good, and the recovery rate was about 100%. The intra-day and inter-day accuracy (accuracy, relative error) and precision (precision, coefficient of variation) were also good. The lower limit of quantification was 30 cells / 50 μL.

[0074] Table 1

[0075]

Claims

1. A method for measuring the cell concentration in a sample, the method comprising the following steps: Step (1), using a flow cytometer to measure the cell concentration in a plurality of standard samples of known concentration; Step (2), preparing a calibration curve based on the measured value obtained in step (1) and the known concentration to determine the cell recovery rate and accuracy; Step (3), using a flow cytometer to measure the cell concentration in the sample of unknown concentration; as well as In step (4), the cell concentration is determined from the measured value obtained in step (3) using the calibration curve prepared in step (2).

2. The method of claim 1, wherein: Use two or more standard samples of known concentrations.

3. The method according to claim 1 or 2, wherein: One or more standard samples are used for each known concentration.

4. The method according to any one of claims 1 to 3, wherein: In step (2), the measured values ​​whose errors relative to the known concentrations fall within ±30% are used to prepare the calibration curve.

5. The method according to any one of claims 1 to 4, wherein: The accuracy of the measured values ​​obtained by step (1) relative to the known concentration is within ±30%.

6. The method according to any one of claims 1 to 5, wherein: The sample is blood.

7. The method according to any one of claims 1 to 6, wherein: The cells are T cells.

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