Measuring device, measuring device adjustment method and computer storage medium

Abstract

In order to facilitate adjustment of an apparatus for detecting fluorescence from a sample, the present invention provides a measuring apparatus (100), which comprises: a flow chamber (10) for allowing a sample containing particles to flow; a light source (20) for irradiating light to the sample flowing in the flow chamber (10); a fluorescence detector (30) for detecting fluorescence generated from the sample irradiated with light by the light source (20); and a control unit (40) for allowing a positive control sample containing a fluorescent dye to flow into the flow chamber (10), measuring the fluorescence generated from the positive control sample irradiated with light by the light source (20) through the fluorescence detector (30), comparing the obtained measured value with a standard value, and adjusting the detection sensitivity of the fluorescence detector (30) according to the comparison result.

Description

Technical Field

[0001] The present invention relates to a measuring device, a method for adjusting the measuring device, and a program. Background Art

[0002] Patent Document 1 discloses a flow cytometer that irradiates a fluorescently labeled sample with laser light and measures fluorescence generated from each cell. In order to detect fluorescence from a plurality of fluorescent dyes having different spectra, the flow cytometer is provided with fluorescence detectors corresponding to the respective fluorescent dyes to be detected.

[0003] In this flow cytometer, fluorescence detectors such as photomultiplier tubes (PMTs) and avalanche diodes that can amplify detected signals are used to detect low-level fluorescence. In order to make the measured value of fluorescence from each cell fall within the dynamic range, the detection sensitivity, that is, the signal amplification, of each fluorescence detector is adjusted according to the fluorescent dye to be detected.

[0004] The signal amplification of the PMT is changed by adjusting the voltage applied to the PMT. When setting the flow cytometer, a plurality of different applied voltages are set for the PMT, and the fluorescence from the fluorescently labeled microspheres is measured by each applied voltage. The voltage is determined and stored by experiments. Fig.11 The linear functional relationship between the applied voltage and the output fluorescence value is shown in FIG. 1. Subsequently, after the target fluorescence value of each PMT is set, the applied voltage that outputs the target fluorescence value from the fluorescence detector is calculated using the determined functional relationship.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent document 1: Japanese Patent Application Laid-Open No. 2004-205508. Summary of the invention

[0008] Technical problem to be solved by the invention

[0009] In the flow cytometer of Patent Document 1, the above functional relationship for adjusting the PMT detection sensitivity needs to be prepared in advance according to each PMT and fluorescent dye. Therefore, when a new PMT or a new fluorescent dye is used, it is necessary to perform many new measurements and obtain data in order to obtain the above functional relationship corresponding to mechanical differences (errors caused by individual differences in the device).

[0010] Technical means to solve technical problems

[0011] The present invention relates to a measuring device, which comprises: a flow chamber for a sample containing particles to flow; a light source for irradiating light to the sample flowing in the flow chamber; a fluorescence detector for detecting the fluorescence generated by the sample irradiated by the light source; and a control unit for causing a positive control sample containing a fluorescent dye to flow into the flow chamber, measuring the fluorescence generated by the positive control sample irradiated by the light source through the fluorescence detector, comparing the obtained measured value with a standard value, and adjusting the detection sensitivity of the fluorescence detector according to the comparison result.

[0012] The present invention relates to an adjustment method for a measuring device: allowing a positive control sample containing a fluorescent dye to flow into a flow chamber, irradiating light to the positive control sample flowing in the flow chamber, measuring the fluorescence generated from the positive control sample irradiated with light by a fluorescence detector, comparing the measured value obtained by the fluorescence detector with a standard value, and adjusting the detection sensitivity of the fluorescence detector based on the comparison result.

[0013] The present invention relates to a computer storage medium, which controls a computer to execute the following processing steps: a step of supplying a positive control sample containing a fluorescent dye to a flow chamber, a step of irradiating light to the positive control sample flowing in the flow chamber, a step of measuring the fluorescence generated by the positive control sample irradiated with light by a fluorescence detector, and a step of comparing the measured value obtained by the fluorescence detector with a standard value and adjusting the detection sensitivity of the fluorescence detector according to the comparison result.

[0014] Effects of the Invention

[0015] The measuring device, the measuring device adjustment method, and the program of the present invention can easily adjust the detection sensitivity for detecting light from a sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A schematic diagram for explaining an outline of a method for adjusting the sensitivity of a fluorescence detector of a measuring device;

[0017] Figure 2 is a block diagram for explaining a measuring device;

[0018] Figure 3 An illustration of the optical system of the measuring device;

[0019] Figure 4 is a flowchart for explaining the adjustment process of the measuring device;

[0020] Figure 5 For illustration Figure 5 A flowchart of the sensitivity adjustment process of step S3;

[0021] Figure 6 An explanatory diagram of an example of sensitivity adjustment of a measuring device;

[0022] Figure 7 is a graph showing the relationship between voltage and fluorescence intensity in the sensitivity adjustment of the measuring device;

[0023] Figure 8 A schematic diagram illustrating fluorescence correction of the measuring device;

[0024] Fig. 9 For illustration Figure 5 A flowchart of the fluorescence correction process of step S6;

[0025] Fig.10 An explanatory diagram of an example of fluorescence correction of a measuring device;

[0026] Fig.11 is a block diagram showing a conventional fluorescence analysis device. DETAILED DESCRIPTION

[0027] The following describes the embodiments based on the drawings.

[0028] Figure 1 (A)~ Figure 1 (B) is a schematic diagram for explaining an outline of a method for adjusting the sensitivity of a fluorescence detector of a measuring device. As an example, a flow cytometer equipped with a FITC detector for detecting fluorescein isothiocyanate (hereinafter referred to as FITC) as a fluorescent dye and a PE detector for detecting phycoerythrin (hereinafter referred to as PE) as a fluorescent dye is described in the following description, in which a positive control sample containing a microsphere group labeled with a certain amount of FITC (hereinafter referred to as FITC microsphere group) is measured. Figure 1 (A) and Figure 1 In (B), the horizontal axis is set to the fluorescence intensity (FL1) based on the output of the FITC detector, and the vertical axis is set to the fluorescence intensity (FL2) based on the output of the PE detector, and the fluorescence intensity obtained from each microsphere in the positive control sample containing the FITC microsphere group is shown in a dot graph. Figure 1 As shown in (A), the measurement result of the FITC microsphere group of the positive control sample is represented as a measurement value group 61. The measurement device compares the statistical value A (for example, the median of the fluorescence intensity) of the fluorescence intensity (FL1) generated from the FITC microsphere group with the standard value, and based on the comparison result, Figure 1 As shown in (B), the detection sensitivity of the detector for FITC is adjusted so that the statistical value A is substantially equal to the standard value.

[0029] By implementing the above-described adjustment method, it is possible to easily adjust the detection sensitivity for detecting light from a sample.

[0030] (Details of this embodiment)

[0031] use Figure 2The measurement device 100 will be described. The measurement device 100 is a multicolor flow cytometer. The flow cytometer performs optical measurement of a sample by flow cytometry. The measurement device 100 includes an optical system unit 200 , a sample preparation unit 400 , a voltage application unit 50 , and a control unit 40 .

[0032] The control unit 40 controls the operations of the optical system unit 200 , the sample preparation unit 400 , and the voltage application unit 50 . The control unit 40 is a computer, and includes a CPU (Central Processing Unit) 41 , a memory 42 , an input unit 43 , a display unit 44 , and a storage unit 45 .

[0033] The CPU 41 performs control processing related to the control unit 40. The memory 42 is used as an operating area for performing control processing related to the CPU 41. The memory 42 includes a RAM (random access memory). The input unit 43 accepts instruction input from the user. In addition, the input unit 43 reads information attached to the sample container. The input unit 43 includes a reader / writer such as a keyboard, a mouse, a barcode, and an RFID. The display unit 44 displays the measurement results of the optical system unit 200. In addition, the display unit 44 displays an interface for operation. The storage unit 45 stores programs for processing, measurement results, and information for measurement. The storage unit 45 includes a ROM (read-only memory).

[0034] The sample preparation unit 400 mixes a fluorescent reagent with a sample to be measured and labels the sample with a fluorescent dye, thereby preparing a sample suitable for measurement in the optical system unit 200 .

[0035] The optical system unit 200 includes a flow cell 10, a light source 20, and a plurality of detectors 30 (30a to 30p). The detector 30 includes a photodetector 34 that outputs an electrical signal corresponding to the intensity of the detected light, and an amplifier 33 that amplifies the electrical signal output from the photodetector 34. The detector 30 for fluorescence detection is a photomultiplier tube (PMT), the photodetector 34 includes a photocathode, and the amplifier 33 includes a dynode electrode.

[0036] use Figure 3The optical system unit 200 will be described in further detail. The optical system unit 200 includes a flow cell 10, light sources 20a, 20b, 20c, and detectors 30a to 30p. The flow cell 10 receives a sample prepared by the sample preparation unit 400. The light sources 20a to 20c irradiate light to the sample passing through the flow cell 10. The detectors 30a to 30p detect light from the sample and output detection signals converted into electrical signals. The detector 30a is a detector for detecting forward scattered light, and the detector 30b is a detector for detecting side scattered light. The detectors 30a and 30b are photodiodes. The detectors 30c, 30d, 30e, 30f, 30g, 30h, 30i, 30j, 30k, 30l, 30m, 30n, 30o, and 30p are detectors for detecting fluorescence. The detectors 30c to 30p are photomultiplier tubes (PMTs). The fluorescent dyes used as detection targets in detectors 30c to 30p are different. The PMT includes a photocathode, a dynode, and an anode. The photoelectrons generated by the photocathode are amplified by the dynode and output by the anode. The detection sensitivity of each detector 30c to 30p is adjusted by changing the voltage applied to the dynode. That is, Figure 2 The photodetector 34 shown includes a photocathode, and the amplifier 33 includes a dynode. The voltage application unit 50 applies a voltage of a specified magnitude to the dynode electrodes of the detectors 30c to 30p in accordance with the instruction of the control unit 40.

[0037] The light sources 20a to 20c emit light of different wavelengths. The light source 20a emits light of a wavelength of about 488 nm. The light source 20b emits light of a wavelength of about 642 nm. The light source 20c emits light of a wavelength of about 405 nm. The light emitted by the light source 20a passes through the collimating lens 301a, the dichroic mirrors 303a and 303b, and the condensing lens 302, and is irradiated onto the flow cell 10. The light emitted by the light source 20b passes through the collimating lens 301b, is reflected by the dichroic mirror 303a, passes through the dichroic mirror 303b, passes through the condensing lens 302, and is irradiated onto the flow cell 10. The light emitted by the light source 20c passes through the collimating lens 301c, is reflected by the dichroic mirror 303b, passes through the condensing lens 302, and is irradiated onto the flow cell 10.

[0038] Forward scattered light from the sample passing through the flow cell 10 is transmitted through the condenser 304a, the beam stopper 305a, the aperture plate 305b, and the bandpass filter 307a, and enters the detector 30a.

[0039] The side scattered light and fluorescence from the particles contained in the sample passing through the flow cell 10 are condensed by the condenser 304b. The side scattered light to be incident on the detector 30b is transmitted from the condenser 304b through the dichroic mirrors 308a and 308b, reflected by the dichroic mirror 308c, and transmitted through the dichroic mirrors 308d and 308e and the band pass filter 307b, and is incident on the detector 30b.

[0040] The fluorescence incident on the detectors 30c to 30p is divided into different wavelength bands by the dichroic mirrors 308a to 308n. The fluorescence incident on the detector 30c is transmitted from the condenser 304b through the dichroic mirrors 308a and 308b, reflected by the dichroic mirror 308c, transmitted through the dichroic mirror 308d, reflected by the dichroic mirror 308e, transmitted through the bandpass filter 307c, and incident on the detector 30c. The fluorescence incident on the detector 30d is transmitted from the condenser 304b through the dichroic mirrors 308a and 308b, reflected by the dichroic mirror 308c, reflected by the dichroic mirror 308d, transmitted through the bandpass filter 307d, and incident on the detector 30d.

[0041] The fluorescence that is incident on the detector 30e passes through the dichroic mirrors 308a, 308b, 308c, and 308f from the condenser 304b, passes through the bandpass filter 307e, and enters the detector 30e. The fluorescence that is incident on the detector 30f passes through the dichroic mirrors 308a, 308b, and 308c from the condenser 304b, is reflected by the dichroic mirror 308f, is reflected by the dichroic mirror 308g, passes through the bandpass filter 307f, and enters the detector 30f.

[0042] The fluorescence to be incident on the detector 30g is transmitted from the condenser 304b through the dichroic mirrors 308a, 308b, and 308c, reflected by the dichroic mirror 308f, transmitted through the dichroic mirror 308g and the bandpass filter 307g, and incident on the detector 30g. The fluorescence to be incident on the detector 30h is reflected from the condenser 304b through the dichroic mirror 308a, transmitted through the dichroic mirror 308b, reflected by the dichroic mirror 308h, transmitted through the bandpass filter 307h, and incident on the detector 30h.

[0043] The fluorescence that is incident on the detector 30i is reflected by the dichroic mirror 308a from the condenser 304b, passes through the dichroic mirrors 308b and 308h, is reflected by the dichroic mirror 308i, passes through the bandpass filter 307i, and is incident on the detector 30i. The fluorescence that is incident on the detector 30j is reflected by the dichroic mirror 308a from the condenser 304b, passes through the dichroic mirrors 308b, 308h, and 308i, passes through the bandpass filter 307j, and is incident on the detector 30j.

[0044] The fluorescence to be incident on the detector 30k is reflected by the dichroic mirror 308b from the condenser 304b, transmitted by the dichroic mirror 308a, reflected by the dichroic mirror 308j, transmitted by the dichroic mirrors 308k and 308l and the bandpass filter 307k, and incident on the detector 30k. The fluorescence to be incident on the detector 30l is reflected by the dichroic mirror 308b from the condenser 304b, transmitted by the dichroic mirror 308a, reflected by the dichroic mirror 308j, transmitted by the dichroic mirror 308k, reflected by the dichroic mirror 308l, transmitted by the bandpass filter 307l, and incident on the detector 30l.

[0045] The fluorescence that is incident on the detector 30m is reflected by the dichroic mirror 308b from the condenser 304b, transmitted by the dichroic mirror 308a, reflected by the dichroic mirror 308j, reflected by the dichroic mirror 308k, transmitted by the bandpass filter 307m, and incident on the detector 30m. The fluorescence that is incident on the detector 30n is reflected by the dichroic mirror 308b from the condenser 304b, transmitted by the dichroic mirror 308a, transmitted by the dichroic mirrors 308j and 308m and the bandpass filter 307n, and incident on the detector 30n.

[0046] The fluorescence to be incident on the detector 30o is reflected by the dichroic mirror 308b from the condenser 304b, transmitted by the dichroic mirror 308a, transmitted by the dichroic mirror 308j, reflected by the dichroic mirror 308m, reflected by the dichroic mirror 308n, transmitted by the bandpass filter 307o, and incident on the detector 30o. The fluorescence to be incident on the detector 30p is reflected by the dichroic mirror 308b from the condenser 304b, transmitted by the dichroic mirror 308a, transmitted by the dichroic mirror 308j, reflected by the dichroic mirror 308m, transmitted by the dichroic mirror 308n, transmitted by the bandpass filter 307p, and incident on the detector 30p.

[0047] The detection signals outputted by the detectors 30 a to 30 p are sent to the control unit 40 , and the control unit 40 acquires parameters such as light intensity from the detection signals, and performs sample analysis based on the acquired parameters.

[0048] There may be one, two, or four or more light sources 20. Preferably, the flow cytometer is equipped with at least three light sources 20. The light source 20 is selected according to the wavelength region of the light from the sample. When there are two or more light sources 20, it is preferred that these light sources 20 emit light with different peak wavelengths.

[0049] The number of photodiodes, dichroic mirrors and bandpass filters can be changed according to the peak wavelength of light from the sample. In addition, the types of photodiodes, dichroic mirrors and bandpass filters can be selected according to the peak wavelength or wavelength range of light from the sample and the fluorescence intensity.

[0050] The control unit 40 stores information related to the detection sensitivity of the detector 30 when detecting scattered light and fluorescence, information related to the fluorescence correction corresponding to the combination of detected fluorescence, and information related to the gating for selecting the distribution area of ​​the sample to be detected, and controls the optical system unit 200 to obtain appropriate optical information according to the antigen based on the above information.

[0051] (Adjustment Process of Measuring Device 100)

[0052] Reference Figure 4 , the adjustment process of the measuring device 100 is described.

[0053] First, the process for adjusting the detection sensitivity of the fluorescence detectors (30c, 30d, 30e, 30f, 30g, 30h, 30i, 30j, 30k, 30l, 30m, 30n, 30o, and 30p) performed by the control unit 40 of the measuring device 100 is described. In the adjustment of the detection sensitivity, the detection sensitivity of each fluorescence detector is adjusted in the fluorescence measurement of a specific wavelength, so as to effectively separate the particles labeled with the fluorescent dye and the unlabeled particles. Here, the sensitivity adjustment in the case of multicolor analysis in combination with FITC and PE is described as an example. The fluorescence of FITC is detected by the fluorescence detector 30c, and the fluorescence of PE is detected by the fluorescence detector 30d.

[0054] exist Figure 4 In step S1, the control unit 40 sets a standard value of the fluorescence intensity as a sensitivity adjustment target for the FITC fluorescence detector 30c, and an upper limit and a lower limit of the applied voltage (hereinafter referred to as PMT voltage) for the fluorescence detector 30c. The value of the fluorescence intensity obtained from the positive control sample containing a microsphere group labeled with a certain amount of FITC is sufficiently greater than the fluorescence intensity from the negative control sample containing a non-labeled microsphere group as the standard value. However, if the standard value is large, the amplification factor of the noise signal is also large, so the standard value is set within a range where the influence of the amplified noise signal is small. The user inputs the standard value via the input unit 43.

[0055] The control unit 40 sets the PMT voltage that causes the fluorescence detector 30c to output a measurement value greater than the input standard value as the initial value of the upper limit value. In addition, the control unit 40 sets the PMT voltage that causes the fluorescence detector 30c to output a measurement value less than the input standard value as the initial value of the lower limit value. In step S2, the control unit 40 controls the positive control sample to start supplying to the flow cell 10.

[0056] In step S3 , the control unit 40 performs a process of adjusting the voltage (PMT voltage) applied to the FITC fluorescence detector 30 c to adjust the detection sensitivity of the fluorescence detector 30 c .

[0057] Reference Figure 5 ,right Figure 4 The processing of step S3 will be described.

[0058] exist Figure 5 In step S11, the control unit 40 sets the median of the upper limit value and the lower limit value as the PMT voltage of the fluorescence detector 30c, and performs measurement of the positive control sample. Thus, a statistical value A of the fluorescence intensity obtained from the FITC microsphere group contained in the positive control sample is obtained. The median of the fluorescence intensity obtained from each microsphere of the microsphere group is used as the statistical value A. In step S12, the control unit 40 compares the statistical value A with the standard value to determine whether the statistical value A is approximately equal to the standard value. That is, the control unit 40 determines whether the difference between the statistical value A and the standard value is within a certain range. If the difference between the statistical value A and the standard value is within a certain range, the process proceeds to step S13. If the difference between the statistical value A and the standard value is not within a certain range, the process proceeds to step S14.

[0059] In step S13, the control unit 40 sets the PMT voltage when the statistical value A is obtained as the PMT voltage after the sensitivity adjustment. Figure 4 In step S14, the control unit 40 determines whether the statistical value A is less than the standard value. If the statistical value A is less than the standard value, the process proceeds to step S15. If the statistical value A is greater than the standard value, the process proceeds to step S16.

[0060] In step S15, the control unit 40 sets the current PMT voltage (in Figure 4 In step S16, the control unit 40 changes the current PMT voltage (the median of the upper limit value and the lower limit value set in step S1) to the lower limit value of the PMT voltage. Then, the process returns to step S11. In step S16, the control unit 40 changes the current PMT voltage (the median of the upper limit value and the lower limit value) to the upper limit value of the PMT voltage. Then, the process returns to step S11.

[0061] Reference Figure 6 and Figure 7 The sensitivity adjustment is described in more detail. The horizontal axis is set to the fluorescence intensity (FL1) based on the output of the FITC fluorescence detector 30c, and the vertical axis is set to the fluorescence intensity (FL2) based on the output of the PE fluorescence detector 30d. The measurement results of the FITC microspheres as positive control microspheres are shown in the measurement value group 61. Figure 4 The case where the initial value of the lower limit value of the PMT voltage set in step S1 is 100 V and the initial value of the upper limit value is 1000 V is described as an example.

[0062] First, if Figure 7 As shown, the fluorescence intensity (FL1) of the positive control sample is measured using the median VC1 (550V) of the lower limit value VL1 (100V) and the upper limit value VH1 (1000V) of the PMT voltage. Figure 6 As shown in (A), when the measurement is performed using a PMT voltage of 550 V, the statistical value A1 of the measurement value group 61 is smaller than the standard value, so the PMT voltage (550 V) used for the measurement is set as the next lower limit value.

[0063] Next, if Figure 7 As shown, the fluorescence intensity (FL1) of the positive control sample is measured using the median VC2 (775V) of the updated PMT voltage lower limit value VL2 (550V) and upper limit value VH2 (1000V). Figure 6 As shown in (B), when the measurement is performed using a PMT voltage of 775 V, the statistic value A2 of the measurement value group 61 is smaller than the standard value, so the PMT voltage (775 V) used for the measurement is set as the next lower limit value.

[0064] Next, if Figure 7 As shown, the fluorescence intensity (FL1) of the positive control sample is measured using the median VC3 (887V) of the updated PMT voltage lower limit VL3 (775V) and upper limit VH3 (1000V). Figure 6 As shown in (C), when the measurement is performed using a PMT voltage of 887 V, the statistic A3 of the measurement value group 61 is larger than the standard value, so the PMT voltage (887 V) used for the measurement is set as the next upper limit value.

[0065] Next, if Figure 7 As shown, the fluorescence intensity (FL1) of the positive control sample is measured using the median VC4 (831V) of the updated PMT voltage lower limit value VL4 (775V) and upper limit value VH4 (887V). Figure 6 As shown in (D), when the measurement is performed using a PMT voltage of 831 V, the statistic A4 of the measurement value group 61 is larger than the standard value, so the PMT voltage (831 V) used for the measurement is set as the next upper limit value.

[0066] Next, if Figure 7 As shown, the fluorescence intensity (FL1) of the positive control sample is measured using the median VC5 (803V) of the updated PMT voltage lower limit VL5 (775V) and upper limit VH5 (831V). Figure 6 As shown in (E), when the measurement is performed using the PMT voltage of 803 V, the statistical value A5 of the measured value group 61 is substantially equal to the standard value. Thus, the sensitivity adjustment is completed and the control is turned on. Figure 4 The flow chart is transferred to step S4. In the present embodiment, the condition for the statistical value A to be substantially equal to the standard value is that the difference between the two is less than 5% of the standard value.

[0067] In addition, in the present embodiment, a binary search is used to search for a PMT voltage that matches a standard value. By using the binary search method, the search range can be narrowed and the applied voltage that meets the conditions can be found, and the search can be quickly ended. Finally, it can prevent time from being spent on adjusting the detection sensitivity of the fluorescence detector. Other search methods can also be used. For example, the search method can be a linear search and a bidirectional search.

[0068] In addition, although the median is used as the statistical value A, the mode or the average value may also be used as the statistical value A.

[0069] Next, in Figure 4 In step S4 of the flowchart of FIG. 1 , the control unit 40 performs control to end the supply of the positive control sample to the flow cell.

[0070] Next, in step S5, the control unit 40 measures the statistical value A of the fluorescence intensity of the FITC microsphere group in the positive control sample by the FITC fluorescence detector 30c after the detection sensitivity is adjusted, measures the statistical value B of the fluorescence intensity of the FITC microsphere group in the same positive control sample by the PE fluorescence detector 30d after the detection sensitivity is adjusted, and measures the statistical value C of the fluorescence intensity of the microsphere group in the negative control sample by the PE fluorescence detector 30d after the detection sensitivity is adjusted, and stores them in the storage unit 45. These values ​​are used for the fluorescence correction process in step S6. In this measurement, a mixed sample of the positive control sample and the negative control sample is supplied to the flow cell 10.

[0071] Next, in step S6 , the control unit 40 performs fluorescence correction processing.

[0072] In multicolor flow cytometry, samples are multi-stained and fluorescence from multiple fluorescent dyes is detected simultaneously. In addition, since each fluorescent dye has a spectrum, when the fluorescence is separated by wavelength domain by an optical system, fluorescence from fluorescent dyes other than the fluorescent dye bound to the target molecule may also leak in.

[0073] Figure 8 In the example, the fluorescence of the fluorescent dye F1 and the fluorescent dye F2 leak into each other. The fluorescent dye F1 is measured by detecting the light of the wavelength domain FL1. In addition, the fluorescent dye F2 is measured by detecting the light of the wavelength domain FL2. A part of the fluorescence of the fluorescent dye F1 includes the wavelength domain FL2. In addition, a part of the fluorescence of the fluorescent dye F2 includes the wavelength domain FL1. Here, in the detection of the light of the wavelength domain FL1, the light of the fluorescent dye F2 is removed, thereby detecting the light of the fluorescent dye F1. In addition, in the detection of the light of the wavelength domain FL2, the light of the fluorescent dye F1 is removed, thereby detecting the light of the fluorescent dye F2.

[0074] Reference Fig. 9 ,right Figure 4 Here, a case where a fluorescence correction value for subtracting the fluorescence from FITC leaking into the fluorescence detector for PE is calculated will be described.

[0075] exist Fig. 9 In step S21, the control unit 40 reads the data in the storage unit 45. Figure 4 The statistical values ​​A, B, and C obtained in step S5, that is, the statistical value A of the fluorescence intensity of the FITC microsphere group in the positive control sample measured by the FITC fluorescence detector 30c after the detection sensitivity is adjusted, the statistical value B of the fluorescence intensity of the FITC microsphere group in the same positive control sample measured by the PE fluorescence detector 30d after the detection sensitivity is adjusted, and the statistical value C of the fluorescence intensity of the microsphere group in the negative control sample measured by the PE fluorescence detector 30d after the detection sensitivity is adjusted are read from the storage unit 45.

[0076] Here, the correlation matrix of fluorescence correction can be expressed as shown in formula (1).

[0077]

Number 1

[0078]

[0079] tS 21 : Correction value for correcting the leakage of FITC into the fluorescence detector for PE (correction value set last time or correction value initially set by the user)

[0080] tS 12 : Correction value for correcting the leakage of PE into the fluorescence detector for FITC (correction value set last time or correction value initially set by the user)

[0081] tFL1: The value regarded as the true fluorescence intensity from FITC

[0082] tFL2: The value regarded as the true fluorescence intensity from PE

[0083] PMT1: Measured value of fluorescence detector for FITC

[0084] PMT2: Measured value of fluorescence detector for PE

[0085] Considered as passing correction value tS 21 After removing the fluorescence leakage, the statistical value A is substituted into tFL1, and the statistical value B is substituted into tFL2. According to formula (1), the measured value PMT2 is shown in formula (2).

[0086] (tS 21 ×A) + (1×B) = PMT2・・・(2)

[0087] When the fluorescence correction value is most appropriate, the correlation matrix can be expressed as formula (3).

[0088]

Number 2

[0089]

[0090] S 21 : Correction value for correcting the leakage of FITC into the fluorescence detector for PE

[0091] S 12 : Correction value for correcting the leakage of PE into the fluorescence detector for FITC

[0092] FL1: True fluorescence intensity from FITC

[0093] FL2: True fluorescence intensity from PE

[0094] PMT1: Measured value of fluorescence detector for FITC

[0095] PMT2: Measured value of fluorescence detector for PE

[0096] At this time, the fluorescence intensity of the FITC microspheres in the positive control sample and the fluorescence intensity of the microspheres in the negative control sample measured by the PE fluorescence detector are the same, so B = C. Then, according to formula (3), the measured value PMT2 is as shown in formula (4).

[0097] (S 21 ×A) + (1 × C) = PMT2・・・(4)

[0098] In formula (2) and formula (4), the measured value PMT2 is equal, so formula (5) is derived.

[0099] (S 21 ×A)+(1×C)=(tS 21 ×A)+(1×B)…(5)

[0100] Then, formula (6) is derived from formula (5).

[0101] S 21 = (tS 21 ×A) + (B-C)) / A・・・(6)

[0102] In addition, when setting the fluorescence correction value for the first time, tS 21 = 0. Therefore, S 21 = (B-C) / A. In the next calculation, this S 21 The value of tS 21 use.

[0103] In step S22, the control unit 40 checks the correction value tS calculated using the same type of positive control sample and negative control sample. 21 The historical record or the correction value tS set by the user initially 21 No correction value tS 21 The historical records and the correction value tS set by the user initially 21 If , the process proceeds to step S23. On the other hand, there is a correction value tS 21 The historical record or the correction value tS set by the user initially 21 In this case, proceed to step S24.

[0104] In step S23, the control unit 40 calculates a correction value S for subtracting the fluorescence from FITC leaking into the PE fluorescence detector based on the statistical value A, the statistical value B, and the statistical value C. 21 In step S24, the control unit 40 calculates the correction value tS based on the correction value tS stored in the storage unit 45. 21 Calculate the new correction value S with the statistical values ​​A, B and C 21 .

[0105] As shown above, in the fluorescence correction for subtracting the fluorescence from FITC relative to the fluorescence detector 30d for PE, the control unit 40 calculates the correction value S based on the value (B-C) related to the difference between the statistical value B of the fluorescence intensity of the FITC microsphere group in the positive control sample and the statistical value C of the fluorescence intensity of the microsphere group in the negative control sample measured by the fluorescence detector 30d for PE. 21 .

[0106] In the above-mentioned fluorescence correction, the control unit 40 can calculate the correction value S based on the fluorescence intensity of the FITC microsphere group in the positive control sample measured by the FITC fluorescence detector. 21 .

[0107] In addition, in the present embodiment, a negative control is used to obtain the statistical value C, but the correction value S may be calculated by setting C=0 without using a negative control. 21 .

[0108] The control unit 40 causes the storage unit 45 to store the calculated correction value S 21 . Thus, the calculated correction value S can be easily 21 Used in further correction value S 21 Calculation or measurement of samples.

[0109] In step S25, if Fig.10 As shown in (A) to (B), the control unit 40 uses the calculated correction value S 21 Correction B. After that, Figure 4 The flow chart is transferred to step S7.

[0110] In step S7, the control unit 40 stores the detection sensitivity of each fluorescence detector whose detection sensitivity has been adjusted in the storage unit 45. The control unit 40 sets the detection sensitivity of the fluorescence detector when measuring a sample extracted from the subject to the detection sensitivity stored in the storage unit 45.

[0111] In step S8 , the control unit 40 causes the display unit 44 to display the adjustment of the detection sensitivity and a distribution graph (for example, Figure 6 (E)). Thus, the user can easily determine whether the detection sensitivity adjustment and correction value S of the fluorescence detector are performed correctly by checking the distribution graph. 21 Calculated.

[0112] In addition, the measuring device 100 can receive the detection sensitivity and the correction value S 21 For example, the user can check the distribution graph and adjust the correction value S 21 Thus, the user can check the distribution diagram and adjust the detection sensitivity and correction value S 21 By making minor changes, the detection sensitivity and correction value S can be set with higher precision. 21 .

[0113] The embodiments disclosed this time are all examples and are not restrictive. The scope of the present invention is indicated by the claims, not the description of the above embodiments, and includes all modifications (variations) within the meaning and scope equivalent to the claims.

[0114] For example, in the above embodiment, Figure 4 In the flowchart shown in FIG. 1 , the supply of the positive control is started in step S2, the supply of the positive control is ended in step S4 after the sensitivity adjustment is completed in step S3, and the mixed solution of the positive control and the negative control is supplied in step S5. The present invention is not limited thereto, and the supply of the mixed solution of the positive control and the negative control may be started in step S2, and the supply of the mixed solution may be ended after the sensitivity adjustment process in step S3 and the fluorescence correction process in step S6 are completed. In this way, the number of times the user supplies the control sample to the measuring device can be reduced, and the adjustment operation can be further simplified.

[0115] Furthermore, in the above-described embodiment, PMTs are used as the detectors 30 c to 30 p for fluorescence detection, but avalanche photodiodes may also be used.

[0116] In addition, the detection sensitivity is adjusted by adjusting the voltage applied to the multiplier electrode of the PMT, and the electrical signal output by the detector can be amplified by an amplifier circuit, or the electrical signal output by the detector can be digitized and amplified by the control unit 40.

[0117] The sample to be measured by the measuring device 100 is not limited to a sample containing cells extracted from a test object, and may be a sample containing particles such as microorganisms, animal cells, plant cells, pollen, and plankton.

[0118] In addition, in the above-mentioned embodiment, a sample containing fluorescently labeled microspheres is used as a positive control sample for adjusting the detection sensitivity, but as long as it is a sample that emits fluorescence, for example, a sample containing fluorescently labeled cells as the object of measurement itself can also be used as a positive control sample. In addition, regarding fluorescently labeled microspheres, it is not limited to microspheres directly bound to fluorescent dyes, and microspheres that have been bound to fluorescently labeled antibodies can also be used. It can also be a microsphere in which a fluorescent dye is embedded. Similarly, regarding fluorescently labeled cells, it is not limited to cells that have been directly bound to fluorescent dyes, and cells that have been bound to fluorescently labeled antibodies can also be used. Not only can it be a cell with a fluorescent dye bound to an antigen on the cell surface, but it can also be a cell with a fluorescent dye bound to an antigen (cytokine and transcription factor) present in the cytoplasm and nucleus. In addition, the object of fluorescent labeling is not limited to microspheres and cells, as long as it is a particle that can be fluorescently labeled. For example, droplets containing DNA and cells bound to fluorescent dyes can also be used as fluorescently labeled particles.

[0119] Number Description

[0120] 10: Flow chamber;

[0121] 20, 20a, 20b, 20c: light source;

[0122] 30, 30a, 30b, 30c, 30d, 30e, 30f, 30g, 30h, 30i, 30j, 30k, 30l, 30m, 30n, 30o, 30p: detector;

[0123] 33: Enlargement;

[0124] 40: Control unit;

[0125] 43: Input unit;

[0126] 100: Measuring device.

Claims

1. A measuring device comprising: A flow chamber for the sample containing particles to flow; a light source for irradiating light to the sample flowing in the flow chamber; a fluorescence detector for detecting fluorescence generated from the sample irradiated with light by the light source; A control unit adjusts the detection sensitivity of the fluorescence detector, wherein: setting a standard value of fluorescence intensity as a sensitivity adjustment target for the fluorescence detector, and an upper limit value and a lower limit value of an applied voltage to the fluorescence detector, adjusting the voltage applied to the fluorescence detector between the upper limit and the lower limit, allowing a positive control sample containing a fluorescent dye to flow into the flow chamber, The fluorescence detector measures the fluorescence generated from the positive control sample irradiated with light by the light source to obtain a measurement value. Comparing the obtained measured value with the set standard value, and adjusting the detection sensitivity of the fluorescence detector according to the comparison result; in, The adjustment of the detection sensitivity of the fluorescence detector includes: when the measured value is greater than the set standard value, changing the applied voltage of the fluorescence detector that obtains the measured value to the upper limit value; When the measured value is smaller than the standard value, the applied voltage of the fluorescence detector for obtaining the measured value is changed to the lower limit value.

2. The measuring device according to claim 1, characterized in that: The control unit repeatedly adjusts the detection sensitivity of the fluorescence detector according to the comparison result until the comparison result between the measured value and the standard value satisfies a condition.

3. The measuring device according to claim 2, characterized in that: When the difference between the measured value and the reference value is smaller than a threshold value, the control unit determines that the comparison result satisfies the condition.

4. The measuring device according to any one of claims 1 to 3, characterized in that: When the fluorescence generated from the positive control sample is measured by the fluorescence detector using the first detection sensitivity, and the comparison result between the measurement value obtained and the standard value does not satisfy the condition, the control unit measures the fluorescence generated from the positive control sample by the fluorescence detector using the second detection sensitivity in which the measurement value is closer to the standard value according to the comparison result than when the first detection sensitivity is used, and compares the obtained measurement value with the standard value.

5. The measuring device according to any one of claims 2 to 3, characterized in that: The control unit adjusts the detection sensitivity of the fluorescence detector using a lookup method.

6. The measuring device according to any one of claims 1 to 3, characterized in that: The positive control sample comprises a plurality of particles labeled with the fluorescent dye; The control unit measures the fluorescence generated from each particle in the positive control sample by the fluorescence detector, and compares a statistical value of the measurement value obtained from each particle with the standard value.

7. The measuring device according to claim 6, characterized in that: The statistical value is the median, mode or average of the measured values ​​obtained from each particle in the positive control sample.

8. The measuring device according to any one of claims 1 to 3, characterized in that: The control unit adjusts the detection sensitivity of the fluorescence detector by controlling the voltage applied to the fluorescence detector.

9. The measuring device according to any one of claims 1 to 3, characterized in that: The measuring device further comprises an input unit operated by a user; The control unit sets the value input by the user via the input unit as the standard value.

10. The measuring device according to any one of claims 1 to 3, characterized in that: The control unit adjusts the detection sensitivity of the fluorescence detector by switching the detection sensitivity of the fluorescence detector while the positive control sample is flowing into the flow cell.

11. The measuring device according to any one of claims 1 to 3, characterized in that: The flow chamber allows the sample containing particles multiply dyed with the fluorescent dye and other fluorescent dyes to flow; After adjusting the detection sensitivity of the fluorescence detector, the control unit allows the other positive control sample containing the other fluorescent dye to flow into the flow chamber, measures the fluorescence generated from the other positive control sample irradiated by the light source through the fluorescence detector, and determines a correction value for correcting the leakage of fluorescence from the other fluorescent dye into the fluorescence detector based on the obtained measurement value.

12. The measuring device according to claim 11, characterized in that: After adjusting the detection sensitivity of the fluorescence detector, the control unit allows a negative control sample that does not contain a fluorescent dye to flow into the flow chamber, measures the fluorescence generated from the negative control sample irradiated by the light source through the fluorescence detector, and determines the correction value based on the measurement value obtained from the negative control sample and the measurement value obtained from the other positive control samples.

13. The measuring device according to claim 12, characterized in that: The control unit allows a mixed sample of the negative control sample and the other positive control sample to flow into the flow cell.

14. A method for adjusting a measuring device, characterized in that: setting a standard value of fluorescence intensity as a sensitivity adjustment target for a fluorescence detector, and an upper limit value and a lower limit value of an applied voltage to the fluorescence detector; adjusting the voltage applied to the fluorescence detector between the upper limit and the lower limit; Allow a positive control sample containing a fluorescent dye to flow into the flow chamber; irradiating light to the positive control sample flowing in the flow chamber; measuring fluorescence generated from the positive control sample irradiated with light by a fluorescence detector; comparing the measured value obtained by the fluorescence detector with the standard value, and adjusting the detection sensitivity of the fluorescence detector according to the comparison result; Among them, the adjustment of the detection sensitivity of the fluorescence detector includes: when the measured value is greater than the set standard value, the applied voltage of the fluorescence detector that obtains the measured value is changed to the upper limit value; when the measured value is less than the standard value, the applied voltage of the fluorescence detector that obtains the measured value is changed to the lower limit value.

15. A computer storage medium that enables a computer to control the execution of the following processing steps: The step of setting a standard value of fluorescence intensity as a sensitivity adjustment target for a fluorescence detector, and an upper limit value and a lower limit value of an applied voltage to the fluorescence detector; The step of adjusting the voltage applied to the fluorescence detector between the upper limit value and the lower limit value; The step of supplying a positive control sample containing a fluorescent dye to the flow chamber; a step of irradiating light to the positive control sample flowing in the flow chamber; A step of measuring fluorescence generated from the positive control sample irradiated with light by a fluorescence detector; as well as, A step of comparing the measured value obtained by the fluorescence detector with a standard value, and adjusting the detection sensitivity of the fluorescence detector according to the comparison result; Among them, the adjustment of the detection sensitivity of the fluorescence detector includes: when the measured value is greater than the set standard value, the applied voltage of the fluorescence detector that obtains the measured value is changed to the upper limit value; when the measured value is less than the standard value, the applied voltage of the fluorescence detector that obtains the measured value is changed to the lower limit value.

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