Biological sample analyzer, biological sample analysis system, and method for verifying state of biological sample analyzer

Abstract

An object of the present disclosure is to provide techniques for detecting any changes or abnormalities related to a device or bead, particularly any changes or abnormalities related to the output of a fluorescent signal. The present disclosure provides a biological sample analysis apparatus including an information processing unit that performs information processing using signal intensity data on light generated at the time of irradiation of a flow channel through which particles flow. The information processing unit performs verification by using at least one or more first index values representing fluctuations in output levels on all of the plurality of fluorescent channels and one or more second index values representing fluctuations in output levels of each of the plurality of fluorescent channels and verifying the state of the device. The invention further provides a biological sample analysis system comprising the information processing unit. The present disclosure also provides a method for verifying the status of a biological sample analysis device, the method comprising the aforementioned verification process.

Description

Technical Field

[0001] The present disclosure relates to a biological sample analyzer, a biological sample analysis system, and a method for verifying the status of a biological sample analyzer. More specifically, the present disclosure relates to a biological sample analyzer and a biological sample analysis system that perform analysis based on light generated by irradiating particles flowing in a flow channel with light, and to a method for verifying the status of a biological sample analyzer. Background Art

[0002] For example, a characteristic of the particles has been measured by labeling a group of particles such as cells, microorganisms, and liposomes with a fluorescent dye, irradiating each particle of the group with a laser, and measuring the intensity and / or pattern of fluorescence emitted from the fluorescent dye excited by the laser irradiation. An example of a biological sample analyzer for performing this measurement may be a flow cytometer. In addition, an example of a biological sample analyzer designed to sort cells may be a cell sorter.

[0003] Flow cytometers and cell sorters can be designed to analyze multiple particles individually by irradiating particles flowing in a line in a flow channel with laser light (excitation light) of a specific wavelength and detecting the fluorescence and / or scattered light emitted from each particle. These devices convert the light detected by the photodetector into an electrical signal for quantification and statistically analyze the electrical signal to determine the characteristics of individual particles, such as type, size, and structure.

[0004] Regarding this type of biological sample analyzer, for example, Patent Document 1 described below discloses a particle measurement device comprising a detection unit that detects light emitted from particles and an information processing unit that uses a sensitivity correction coefficient to correct the value detected by the detection unit and generate spectral data. The sensitivity correction coefficient is determined based on the value of light detected by the detection unit from fluorescent-labeled particles that emit fluorescence having a predetermined wavelength bandwidth. Patent Document 1 also describes how the sensitivity correction coefficient can be applied to determine the likelihood of using degraded fluorescent-labeled particles and the likelihood of channel degradation.

[0005] [Citation List]

[0006] [Patent Document]

[0007] [Patent Document 1]

[0008] JP 2017-026556 A Summary of the Invention

[0009] [Technical Issues]

[0010] In some cases, such biological sample analyzers (such as flow cytometers and cell separation systems) are calibrated or standardized using the fluorescence level obtained by irradiating fluorescent beads with light. Examples of such fluorescent beads include Automatic Setup Beads (also referred to as ASBs) and Align Check Beads.

[0011] In this calibration or standardization operation, the fluorescence output obtained from each bead at a specific wavelength is used as a standard. However, as the beads degrade, their fluorescence levels vary significantly from those of undegraded beads. Therefore, it is necessary to detect abnormalities in signal output caused by such beads.

[0012] Furthermore, erroneous data may be applied due to human error. If calibration or standardization is performed using such incorrect data, the analyzer may not be properly established. Furthermore, the output of only one of the multiple fluorescence channels may be reduced, for example due to optical or electrical problems. For these situations, it is also necessary to detect abnormalities in the analyzer.

[0013] Furthermore, some analyzers use a replacement chip or flow cell containing a flow channel that illuminates particles in a sample with light. With such analyzers, overall changes in signal output may occur as the chip or flow cell is replaced. Detecting such changes is also important.

[0014] Detection of such anomalies or changes in the analyzer and / or detection of output anomalies in the fluorescent signal caused by the beads is believed to be useful in order to more appropriately verify the status of the analyzer, and more specifically, calibrate or standardize the status of the analyzer.

[0015] It is an object of the present disclosure to provide techniques for detecting changes or anomalies in an analyzer or microbead as described above, and in particular for detecting changes or anomalies in the fluorescent signal output.

[0016] [Solution to the problem]

[0017] According to the present disclosure, a biological sample analyzer is provided, including: an information processing unit that performs information processing using signal intensity data of light generated by irradiating a flow channel through which particles flow. The information processing unit performs verification processing for verifying the status of the analyzer using at least one or more first index values and one or more second index values, the first index value indicating an overall output level change in a plurality of fluorescence channels, and the second index value indicating an output level change in each of the plurality of fluorescence channels.

[0018] The first index value may be calculated based on a first output value of a representative fluorescent channel representing an overall output level of the plurality of fluorescent channels.

[0019] The first indicator value can be calculated by using at least a previous first output value and a current first output value, wherein the previous first output value is obtained in a completed verification process that has been executed before the verification process is executed, and the current first output value is obtained in a verification process (hereinafter referred to as "current verification process").

[0020] The first index value may be calculated by using a ratio or a difference between a previous first output value and a current first output value.

[0021] The first index value may be calculated by using a predetermined standard output value of a representative fluorescence channel in addition to the previous first output value and the current first output value.

[0022] A previous first standard output value acquired in a verification process to be performed and a first standard output value acquired in a current verification process may be used as the predetermined standard output value.

[0023] The second index value may be calculated based on the first index value and a second output value of each of the other fluorescent channels except the representative fluorescent channel.

[0024] The second indicator value can be calculated by using at least the first indicator value, the previous second output value and the current second output value, wherein the previous second output value is obtained in the verification process that has been performed before the verification process is performed, and the current second output value is obtained in the verification process (hereinafter referred to as the "current verification process").

[0025] The second index value may be calculated by further using a predetermined standard output value of each of the other fluorescent channels in addition to the first index value, the previous second output value, and the current second output value.

[0026] A previous second standard output value acquired in the verification process that has been executed and a second standard output value acquired in the current verification process may be used as the predetermined standard output value.

[0027] The biological sample analyzer may determine that the status of the analyzer is appropriate if the first indicator value satisfies a predetermined first condition and the one or more second indicator values satisfy a predetermined second condition.

[0028] The biological sample analyzer may perform a gain adjustment process for each of the fluorescence channels after determining that the state of the analyzer is appropriate.

[0029] The biological sample analyzer may record the gain of each of the fluorescence channels, the gain being set in the gain adjustment process.

[0030] In the event that the first indicator value does not satisfy a predetermined first condition or the one or more second indicator values do not satisfy a predetermined second condition, the biological sample analyzer may determine that the status of the analyzer is unsuitable.

[0031] The biological sample analyzer may, after determining that the status of the analyzer is inappropriate, output one or more items selected from the group consisting of: a display prompting reloading of fluorescent beads, a display prompting re-performing QC processing, a display prompting inspection of a chip, a circulation cell or an analyzer, a display related to changes or abnormalities in fluorescent beads used in a validation process, a display related to changes or abnormalities in the biological sample analyzer, and a display related to incorrect application of standard value data.

[0032] The biological sample analyzer can perform a verification process by using fluorescent beads as particles.

[0033] The fluorescent beads can emit fluorescence covering the entire wavelength range of fluorescence to be detected by multiple fluorescence channels.

[0034] The biological sample analyzer may include a flow cytometer.

[0035] Furthermore, the present disclosure provides a biological sample analysis system including an information processing unit configured to perform information processing using signal intensity data of light generated by irradiating a flow channel through which particles flow. The information processing unit performs verification processing to verify the status of an analyzer using at least one or more first index values indicating overall output level changes in a plurality of fluorescence channels and one or more second index values indicating output level changes in each of the plurality of fluorescence channels.

[0036] Furthermore, according to the present disclosure, a method for verifying the status of a biological sample analyzer is provided, the method comprising: verifying the status of the analyzer by using at least one or more first index values and one or more second index values, the first index value and the second index value being generated from signal intensity data of light generated by irradiating light onto particles flowing in a flow channel, the first index value indicating an overall output level change in a plurality of fluorescence channels, and the second index value indicating an output level change for each of the plurality of fluorescence channels. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a schematic diagram for explaining changes in fluorescence levels measured by a biological sample analyzer.

[0038] Figure 2 is a schematic diagram for explaining a determination method for verifying overall level changes and spectrum changes.

[0039] Figure 3is a diagram schematically showing the overall configuration of a biological sample analyzer.

[0040] Figure 4 is a flow chart of an embodiment of a verification process according to the present disclosure.

[0041] Figure 5 is a graph indicating the results of index value calculations performed by determination formula A and determination formula B.

[0042] Figure 6 is a table showing a configuration example of a biological sample analyzer according to the present disclosure.

[0043] Figure 7 is a schematic diagram illustrating an embodiment of a flow chart for a biological particle sorting process.

[0044] Figure 8 An enlarged view of the particle sorting section is schematically shown.

[0045] Figure 9 is a diagram showing a schematic configuration example of a control unit. DETAILED DESCRIPTION

[0046] Hereinafter, preferred embodiments for implementing the present disclosure will be described. It should be noted that the following embodiments are described as typical embodiments of the present disclosure. Therefore, the scope of the present disclosure should not be limited to these embodiments. It should be noted that the description of the present disclosure will be presented in the following order.

[0047] 1. First embodiment (biological sample analyzer)

[0048] (1) Basic concepts

[0049] (1-1) Overall fluorescence level changes and fluorescence level changes in certain wavelength ranges

[0050] (1-2) Detecting Overall Fluorescence Level Changes and Fluorescence Level Changes in Partial Wavelength Ranges (2) Configuration Example

[0051] (3) Example of Verification Processing

[0052] (4) Example 1 (Example of verification processing)

[0053] (5) Example 2 (actual verification)

[0054] (6) Example of configuration of biological particle separation device

[0055] 2. Second embodiment (biological sample analysis system)

[0056] 3. Third Embodiment (Biological Sample Analyzer Verification Method and Program for Executing the Verification Method)

[0057] 1. First embodiment (biological sample analyzer)

[0058] (1) Basic concepts

[0059] (1-1) Overall fluorescence level changes and fluorescence level changes in certain wavelength ranges

[0060] Changes or abnormalities in the above analyzer or microbeads can be detected based on changes in fluorescence levels. Figure 1 Describe changes in fluorescence levels. Figure 1 An example of a fluorescence spectrum generated by irradiating the above beads with light is schematically shown. Figure 1 In FIG, the horizontal axis represents wavelength (Wavelength), while the vertical axis represents signal level (LogHeight). The overall level change and relative spectral change of fluorescence obtained from beads will be described in more detail below, where the output at a specific wavelength is used as a standard.

[0061] (Overall level change)

[0062] The overall level change is a change in the output of the fluorescence signal over the entire wavelength range of the fluorescence spectrum. For example, when a chip having a flow channel with beads irradiated with light (e.g., a flow cell chip) has a defect or a transmittance change, when there is an optical change in a light irradiation unit (e.g., a light emitting system) that irradiates the flow channel with light, when the laser power changes, or in other cases, the overall level change can be caused. The overall level change is caused by Figure 1 This change is represented by the "high level (high level, gray line)" and "standard (standard, black line)" in the figure. This change corresponds to a state in which a level difference appears in the entire spectrum although the output relationship between the individual wavelengths does not change. For example, it can be based on the ratio (in Figure 1 The overall level change is evaluated by the difference between the signal outputs in the fluorescence channel ChA for detecting fluorescence having a predetermined wavelength (indicated as a level ratio in FIG).

[0063] In this specification, the fluorescence channel used to detect changes in overall levels will also be referred to as "ChA". In many cases, biological sample analyzers (such as flow cytometers) each include multiple fluorescence channels. Those skilled in the art can appropriately select which of the multiple fluorescence channels to use for detecting changes in overall levels. For example, the fluorescence channel used to detect changes in overall levels can be selected based on the type or fluorescence characteristics of the beads to be used, the configuration or characteristics of the analyzer, or other factors.

[0064] In addition, in this specification, the fluorescence channel ChA for detecting changes in the overall level may also be referred to as a “representative fluorescence channel.” For convenience, the term “representative” in this name is merely an indication given to distinguish it from other fluorescence channels by name.

[0065] Preferably, ChA is a fluorescence channel for detecting fluorescence having a short wavelength within the wavelength range of fluorescence generated by fluorescent beads. For example, this fluorescence channel is for detecting fluorescence having a wavelength shorter than or equal to 700 nm, preferably shorter than or equal to 650 nm, and more preferably shorter than or equal to 600 nm. Fluorescence having a short wavelength is particularly preferred as fluorescence that indicates changes in overall fluorescence levels due to, for example, differences between chips or abnormal laser power.

[0066] Note that, for example, the fluorescent channel may be 300 nm or higher, 350 nm or higher, or 400 nm or higher.

[0067] Furthermore, ChA is preferably a fluorescence channel for detecting fluorescence within a wavelength range with high signal intensity within the wavelength range of fluorescence generated by fluorescent beads. For example, ChA can be assigned to a channel for detecting fluorescence within a wavelength range with the highest signal intensity among the multiple fluorescence channels included in the analyzer, or can be assigned to a channel for detecting fluorescence within a wavelength range with the second, third, fourth, or fifth highest signal intensity. In this way, the fluorescence channel for detecting fluorescence within a wavelength range with high signal intensity levels is used to detect overall level changes, thereby reducing the adverse effects of noise on the verification process.

[0068] Additionally, one or more fluorescence channels can be used to detect changes in overall levels.

[0069] In the case where one fluorescent channel is used to detect changes in global levels, the fluorescent channel may be ChA as described above.

[0070] For example, in the case where two or more fluorescence channels are used to detect changes in overall levels, in addition to ChA, different one or more fluorescence channels may be further used.

[0071] For example, when the fluorescence level changes within a portion of the wavelength range due to degradation of beads, etc., the fluorescence level change is noticeable in the long wavelength range but is barely discernible in the short wavelength range. That is, the fluorescence channel assigned to the channel for detecting fluorescence in the short wavelength range is suitable for detecting overall fluorescence level changes. Therefore, it is preferable that, in addition to ChA, a fluorescence channel for detecting fluorescence with a wavelength shorter than that detected by ChA is also used to detect overall level changes.

[0072] That is, when the biological sample analyzer has one or more fluorescence channels for detecting fluorescence having a wavelength shorter than that detected by ChA in addition to ChA, any one or more of the channels can be selected from ChA and the one or more fluorescence channels for shorter wavelengths and used to detect overall level changes. Furthermore, in addition to ChA, all one or more fluorescence channels for wavelengths shorter than that detected by ChA can be used to detect overall level changes.

[0073] (spectral changes)

[0074] For example, in the case where the beads have deteriorated or where beads from a different batch are used as standard spectra, a change occurs as indicated by Figure 1 This fluorescence level change is indicated by the "standard (standard, black line)" and "spectral degradation (degraded, dotted line)" in FIG. That is, the fluorescence signal intensity changes in a portion of the wavelength range of the entire fluorescence spectrum generated from the bead. The relative ratio of each output from a plurality of fluorescence channels (specifically, fluorescence channels other than ChA) to the output of the above-mentioned ChA can be used. Figure 1 This change was evaluated by spectral ratios (expressed in ).

[0075] In this specification, a fluorescence channel for detecting changes in fluorescence levels in a portion of the wavelength range of the entire fluorescence spectrum will also be referred to as "ChX." In other words, "ChX" can represent a fluorescence channel other than ChA. ChX will be described in detail later.

[0076] The above-described overall level changes and spectral changes are useful for detecting abnormalities or changes in the state of beads, a biological sample analyzer, a biological sample analysis system, or a chip or flow cell used in the analyzer or system.

[0077] Specifically, the present disclosure provides a biological sample analyzer that verifies the analyzer status based on changes in the overall output level of multiple fluorescence channels and changes in the output level of each of the multiple fluorescence channels. Furthermore, the present disclosure provides a biological sample analysis system that performs this verification. Furthermore, the present disclosure provides a biological sample analysis method that verifies the analyzer status based on changes in the overall output level of multiple fluorescence channels and changes in the output level of each of the multiple fluorescence channels.

[0078] According to one embodiment, a biological sample analyzer or biological sample analysis system may include an information processing unit that performs information processing using signal intensity data of light generated by irradiating a flow channel through which particles flow. The information processing unit may be configured to perform verification processing for verifying the status of the analyzer using at least one or more first indicator values indicating overall output level changes in a plurality of fluorescence channels and one or more second indicator values indicating output level changes in each of the plurality of fluorescence channels.

[0079] One or more first indicator values each indicating a change in the total output level in the plurality of fluorescent channels may be used for verification.

[0080] The first index value may be a value calculated based on the output value of the representative fluorescence channel. Note that in this specification, the output value of the representative fluorescence channel is also referred to as a "first output value."

[0081] Additionally, a second indicator value indicating a change in the output level of each of the multiple fluorescence channels can also be used for verification. For example, the second indicator value can be a value calculated for each of the multiple fluorescence channels included in the biological sample analyzer, excluding the representative fluorescence channel. That is, the second indicator value can be a value calculated based on each output value of the other fluorescence channels. It should be noted that each output value of the other fluorescence channels is also referred to as a "second output value" in this specification. As will be described later, the second indicator value can be a value calculated based on the first indicator value and the second output value.

[0082] (beads)

[0083] The particles used in the verification process according to the present disclosure can be, for example, fluorescent beads. Those skilled in the art can appropriately select these fluorescent beads. However, it is preferred that these fluorescent beads are particles that emit high levels of fluorescence over an entire wide wavelength range. It is particularly preferred that the fluorescent beads are fluorescent beads that fluoresce over the entire wavelength range of the fluorescence detected by the multiple fluorescent channels. As such fluorescent beads, for example, the above-mentioned automatic setting beads and alignment detection beads (both manufactured by Sony Group Corporation) are available. In addition, as such fluorescent beads, for example, fluorescent beads containing a plurality of types of particle groups (such as 8-peak beads and 6-peak beads) with gradually changing fluorescence intensity levels can be used.

[0084] The fluorescent beads may be beads having a substantially uniform particle size. For example, the particle size may be appropriately selected by a person skilled in the art based on the flow channel size, etc. For example, the particle size may be 100 μm or less, 50 μm or less, or 30 μm or less. Furthermore, for example, the particle size may be 0.1 μm or greater, 0.3 μm or greater, or 0.5 μm or greater. The fluorescent beads may comprise one type or two or more types of beads.

[0085] (1-2) Detecting changes in overall fluorescence levels and changes in fluorescence levels within a certain wavelength range

[0086] In the following we will refer to Figure 2 Embodiments of a determination method for detecting overall level changes and spectral changes are described. Figure 2 An example of a fluorescence spectrum generated by irradiating the above beads with light is schematically shown. Figure 2 , the horizontal axis represents wavelength (Wavelength), and the vertical axis represents signal level (LogHeight). In this specification, it is assumed that a biological sample analyzer including four fluorescence channels Ch1, ChA, Ch2, and Ch3 is used. Figure 2 Also shown are the wavelength ranges of the fluorescence detected by the corresponding fluorescence channels (the wavelength ranges are represented by the gray-lined rectangles).

[0087] (Detection of overall level changes)

[0088] For example, in a biological sample analyzer that allows replacement of a chip or a flow cell having a flow channel in which particles are irradiated with light, when the chip or flow cell is replaced, the fluorescence signal level may change due to the difference in transmittance of the chip or flow cell. In order to verify this change, Figure 2 The output value of the fluorescence channel ChA shown in is yet to be changed and the output value thereof has already been changed (i.e., the output value of the fluorescence channel ChA associated with fluorescence generated by irradiating light onto particles flowing in the flow channel of the chip or flow cell that is still to be replaced, and the output value of the fluorescence channel ChA associated with fluorescence generated by irradiating light onto particles flowing in the chip or flow cell that has already been replaced). Specifically, the ratio or difference between these output values is used.

[0089] Here, not only the difference in laser power of the laser entering the chip or flow cell and the transmittance of the chip or flow cell, but also the ratio of the fluorescence level of each bead is a factor in the change in the ratio or difference between the output values of ChA. Therefore, a determination equation for determining the presence or absence of overall level changes can be established by using the standard value (adjustment target value, standard gold value (GoldStandard) (also called GS)) of the beads to be used for each fluorescent channel and the ratio or difference between the output values of ChA. The following determination equation A is an example of this determination equation.

[0090] <Deterministic Formula A>

[0091] (ChA ratio)

[0092] ={(ChAnew_Height) / (ChAold_Height)} / {(ChAnew_GS) / (ChAold_GS)}

[0093] =(ChAnew_Height)×(ChAold_GS) / {(ChAold_Height)×(ChAnew_GS)}

[0094] The constituent elements of formula A are expressed as follows.

[0095] ChAold: represents the previous parameters of ChA

[0096] ChAnew: indicates the parameters of the currently acquired ChA

[0097] Height: median height

[0098] GS: Standard gold value = standard value of beads used in the corresponding channel (ChA) (= adjustment target value)

[0099] (ChAnew_Height): The median value of the height of the currently obtained ChA

[0100] (ChAold_Height): Previous median value of the height of ChA

[0101] (ChAnew_GS): The standard gold value of the currently obtained ChA

[0102] (ChAold_GS): Previous standard gold value of ChA

[0103] For example, the standard value (gold standard value GoldStandard) may be pre-identified by the bead manufacturer and may be identified for each batch of beads. To obtain the standard value, for example, a biological sample analyzer used as a standard may be prepared to measure the standard value using the biological sample analyzer used as a standard.

[0104] For example, by using the above-described determination formula A, and in particular the (ChA ratio) obtained by determination formula A, the level ratio caused by differences among a large number of beads is eliminated. Therefore, for example, it is possible to verify changes in laser power (e.g., abnormalities in laser power), changes in chip or flow cell characteristics (e.g., defects that affect the transmittance of the chip or flow cell (specifically, defects, dirt, etc.)), or changes in beads. In other words, the presence or absence of overall level changes in the fluorescence spectrum can be truly verified (without being affected by the level ratio caused by differences among a large number of beads).

[0105] In this manner, the first index value such as (ChA ratio) can be used in the verification process according to the present disclosure as described above. The first index value is useful for detection of changes in the overall level.

[0106] As described above, for example, the first indicator value can be calculated using first output values such as (ChAnew_Height) and (ChAold_Height). That is, the first indicator value can be calculated by using at least the previous first output value obtained in the verification process that was executed before the verification process (hereinafter also referred to as the "current verification process") and the current first output value obtained in the verification process. The first indicator value calculated by using these first output values is useful for detecting changes in the overall level.

[0107] As described above, for example, the first index value may be calculated by using a ratio such as {(ChAnew_Height) / (ChAold_Height)} Alternatively, for example, the first index value may be calculated by using a difference value such as {(ChAnew_Height)-(ChAold_Height)} instead of a ratio.

[0108] That is, the first index value may be a value calculated by using a ratio or difference between a previous first output value and a current first output value. This ratio or difference is useful for obtaining a first index value suitable for detecting changes in the overall level.

[0109] As described above, for example, in addition to the first output value, the first index value can also be calculated by using predetermined standard output values such as (ChAnew_GS) and (ChAold_GS). That is, the first index value can be a value calculated by using the predetermined standard output value of the representative fluorescence channel in addition to the previous first output value and the current first output value. In addition, the previous first standard output value (e.g., (ChAold_GS)) obtained in the verification process being performed and the first standard output value (e.g., (ChAnew_GS)) obtained in the current verification process can be used as the predetermined standard output value. For example, by using the predetermined standard output value, the fluorescence level ratio caused by differences between fluorescent bead batches can be eliminated. Therefore, changes in the fluorescence level can be more appropriately detected.

[0110] For example, a predetermined standard output value may be used as a ratio, such as {(ChAnew_GS) / (ChAold_GS)} as described above. Alternatively, a difference value such as {(ChAnew_GS)-(ChAold_GS)} may be used instead of a ratio. That is, the first indicator value may be calculated using the ratio or difference between the previous first standard output value and the first standard output value obtained in the current verification process.

[0111] In addition, as described above, a first indicator value based on the output value of one fluorescence channel can be used to detect the overall level change. In addition, a first indicator value based on the output values of multiple fluorescence channels or multiple first indicator values based on the corresponding output values of multiple fluorescence channels can also be used for the detection.

[0112] When the output values of multiple fluorescence channels are used to detect changes in the overall level, the multiple fluorescence channels are preferably assigned to detect fluorescence within a wavelength range less affected by microbead degradation. Preferably, the multiple fluorescence channels include at least the aforementioned ChA and further include one or more fluorescence channels that detect fluorescence having a wavelength shorter than that detected by the ChA.

[0113] According to one embodiment, a first index value based on the output values of multiple fluorescence channels can be used to detect overall level changes. For example, the average value of the output values of multiple fluorescence channels can be used. A more specific example of the first index value in this embodiment includes the average value of the output value of ChA and the output value of Ch1. An index value based on the ratio or difference between the average values can be used as the first index value. For example, the first index value based on the ratio or difference between the average values can be calculated by the following determination formula A1 (ChA1 ratio). In addition, although the ratio is used in the following determination formula A1, the difference can be used as described in conjunction with the above determination formula A, rather than the ratio. The first index value calculated in this way can be used for the determination of the specifications described later.

[0114] <Determination formula A1>

[0115] (ChA1 ratio)

[0116] ={(ChA1new_Height) / (ChA1old_Height)} / {(ChA1new_GS) / (ChA1old_GS)}

[0117] =(ChA1new_Height)×(ChA1old_GS) / {(ChA1old_Height)×(ChA1hAnew_GS)}

[0118] The constituent elements of the formula A1 are expressed as follows.

[0119] (ChA1new_Height): The average of the median value of the current ChA height and the median value of the Ch1 height

[0120] (ChA1old_Height): Average of the previous median value of ChA height and the previous median value of Ch1 height

[0121] (ChA1new_GS): The average of the currently obtained ChA standard gold value and the Ch1 standard gold value

[0122] (ChA1old_GS): Average of the previous standard gold value of ChA and the previous standard gold value of Ch1

[0123] In another embodiment, a plurality of first index values based on the respective output values of a plurality of fluorescent channels can also be used to detect changes in the overall level. For example, the plurality of first index values in the embodiment include a first index value calculated based on the output value of ChA (hereinafter, the first index value will be referred to as the "ChA first index value") and a first index value calculated based on the output value of Ch1 (hereinafter, the first index value will be referred to as the "Ch1 first index value"). The (ChA ratio) calculated by the above-mentioned determination formula A is an embodiment of the ChA first index value. In addition, an embodiment of the Ch1 first index value can be calculated by an equation similar to the determination formula A. For example, the Ch1 first index value can be the (Ch1 ratio) calculated by the following determination formula 1.

[0124] <Determination formula 1>

[0125] (Ch1 ratio)

[0126] ={(Ch1new_Height) / (Ch1old_Height)} / {(Ch1new_GS) / (Ch1old_GS)}

[0127] =(Ch1new_Height)×(Ch1old_GS) / {(Ch1old_Height)×(Ch1new_GS)}

[0128] The constituent elements of Formula 1 are expressed as follows.

[0129] (Ch1new_Height): The median value of the height of the current Ch1

[0130] (Ch1old_Height): Previous median value of Ch1's height

[0131] (Ch1new_GS): The current standard gold value of Ch1

[0132] (Ch1old_GS): Previous standard gold value of Ch1

[0133] The larger of the two first index values calculated in this manner (the ChA first index value and the Ch1 first index value) can be used for determination using a specification described later.

[0134] Alternatively, the smaller of the two first indicator values may be used for determination using the specifications described below.

[0135] (Detection of spectral changes)

[0136] The ratio of the output of the fluorescent channel ChA to the output of the other fluorescent channel ChX (in Figure 2 In the case where ChX corresponds to one of Ch1, Ch2, and Ch3) is defined as the spectral ratio.

[0137] In this specification, "ChX" may refer to a fluorescence channel other than ChA. For example, in a biological sample analyzer including Figure 2 In the case of the four fluorescent channels shown in , one of the four fluorescent channels used to verify the overall level change is represented as ChA, and each of the other three fluorescent channels is represented as ChX, where X is 1, 2, or 3. In other words, the four fluorescent channels are represented as ChA, Ch1, Ch2, and Ch3. In this way, when the number of fluorescent channels included in the analyzer is N (N is any positive integer), the number of fluorescent channels ChX included in the analyzer is (N-1). In other words, the fluorescent channels ChX in the analyzer can be represented as Ch1, Ch2, ..., and Ch(N-1).

[0138] The number N of fluorescent channels can be appropriately changed depending on the type of analyzer. For example, the number N of fluorescent channels is two or more, particularly three or more, and more particularly four or more. In addition, the upper limit of the number N of fluorescent channels may not be particularly set, but may be 100 or less, 90 or less, or 80 or less. For example, a biological sample analyzer or system may include 4 to 64 fluorescent channels, particularly 4 to 32 fluorescent channels.

[0139] For example, if the beads have degraded, the spectrum may change from a state where the beads have not degraded. Furthermore, the level ratio of each bead also affects the spectrum. Therefore, in addition to the ratio or difference between the output values of ChX, by using a standard value in ChX (adjustment target value, standard gold value (also called GS)). Furthermore, from the perspective of level changes in other fluorescent channels relative to each other, this determination formula can include the ChA output ratio as a component. Determination formula B below is an example of this determination formula.

[0140] Spectral change verification using determination formula B can be performed for each fluorescent channel ChX.

[0141] <Deterministic formula B>

[0142] (ChX output ratio)

[0143] =[{(ChXnew_Height) / (ChAnew_Height)} / {(ChXold_Height) / (ChAold_Height)}] / [{(ChXnew_GS) / (ChAnew_GS)} / {(ChXold_GS) / (ChAold_GS)}]

[0144] ={(ChXnew_Height)×(ChXold_GS)} / {(ChXold_Height)×(ChA ratio)×(ChXnew_GS)}

[0145] The constituent elements of the deterministic formula B are expressed as follows.

[0146] ChXold: represents the previous parameter of ChX

[0147] ChXnew: indicates the currently obtained ChX parameters

[0148] ChAold: represents the previous parameters of ChA

[0149] ChAnew: indicates the parameters of the currently acquired ChA

[0150] Height: median height

[0151] GS: Standard value of beads used in the corresponding channel (= adjustment target value)

[0152] (ChXnew_Height): The median value of the height of the current ChX

[0153] (ChAnew_Height): The median value of the height of the currently obtained ChA

[0154] (ChXold_Height) : Previous median value of the height of ChX

[0155] (ChAold_Height): Previous median value of the height of ChA

[0156] (ChXnew_GS): The standard gold value of the currently obtained ChX

[0157] (ChAnew_GS): The standard gold value of the currently obtained ChA

[0158] (ChXold_GS): Previous gold standard value of ChX

[0159] (ChAold_GS): Previous standard gold value of ChA

[0160] (ChA ratio): obtained by the above determination formula A

[0161] For example, the standard value (gold standard value GoldStandard) may be pre-identified by the bead manufacturer and may be identified for each batch of beads. To obtain the standard value, for example, a biological sample analyzer used as a standard may be prepared to measure the standard value using the biological sample analyzer used as a standard.

[0162] By using the above-described determination formula B, and in particular the (Ch x output ratio) obtained by determination formula B, variations in the spectral ratio caused by differences between a large number of beads are eliminated. Therefore, only spectral variations can be verified, and thus, for example, the presence or absence of bead degradation, the presence or absence of output degradation of a specific fluorescence channel included in the analyzer, etc. can be determined. In other words, the presence or absence of fluorescence spectral variations can be truly verified (without being affected by variations in the spectral ratio caused by differences between bead batches).

[0163] In this manner, a second index value such as (ChX output ratio) can be used in the verification process according to the present disclosure as described above. The second index value can be used to detect level changes in each of the other fluorescent channels.

[0164] As described above, for example, the second index value can be calculated by using the second output values of other fluorescence channels in addition to the (ChA ratio), such as (ChXnew_Height) and (ChXold_Height). That is, the second index value can be calculated by using at least the first index value, the previous second output value obtained in the verification process that was completed before the verification process (hereinafter referred to as the "current verification process"), and the current second output value obtained in the current verification process. The second index value thus calculated can be used to detect relative level changes in each fluorescence channel.

[0165] As described above, in addition to the first index value, the current second output value, and the previous second output value, the second index value may be calculated by using predetermined standard output values such as (ChXold_GS) and (ChXnew_GS).

[0166] In addition to the first indicator value, the previous second output value, and the current second output value, each of the predetermined standard output values of other fluorescence channels can be used as the calculated value. The previous second standard output value (e.g., (ChXold_GS)) obtained in the verification process being executed and the second standard output value (e.g., (ChXnew_GS)) obtained in the current verification process can be used as the predetermined standard output value. For example, by using such predetermined standard output values as described above, it is possible to eliminate the fluorescence level ratio caused by differences between fluorescent bead batches. Therefore, changes in fluorescence levels can be more appropriately detected.

[0167] (Determination method using determination formula A)

[0168] For example, the above determination formula A, specifically, the (ChA ratio) obtained by the above determination formula A can be incorporated into the following condition (Specification A). According to the present disclosure, the presence or absence of the overall level change can be determined based on whether the condition is satisfied.

[0169] <Specification A>

[0170] (1-α)<(ChA ratio)<(1+α)

[0171] In the above-mentioned specification A, the predetermined value α can be appropriately set by a person skilled in the art based on measurement variations unique to a single analyzer, variations (in transmittance) of the chip or flow cell, and / or variations in the analyzer itself. For example, a standard deviation can be calculated from multiple pieces of output value data obtained through multiple measurements, and α can be set based on this standard deviation. α can also be set based on variations in beads or abnormalities in the transmittance of the chip or flow cell.

[0172] For example, α may be set as described in Specific Example 1 below.

[0173] (measurement change or change in transmittance of the chip or flow cell) < α < (value at which the change in the indicator bead is determined to be abnormal or value at which the transmittance of the indicator chip or flow cell is determined to be abnormal). Specific Example 1

[0174] For example, the predetermined value α used to determine the formula A may be a value ranging from 0.01 to 0.3, specifically, from 0.05 to 0.25.

[0175] In addition, in specification A, the above-mentioned (ChA1 ratio) can be used instead of (ChA ratio).

[0176] Alternatively, a larger value or a smaller value among the plurality of first index values (eg, the larger or smaller of the following two values: the ChA first index value and the Ch1 first index value) may be used in specification A instead of (the ChA ratio).

[0177] (Use the determination method of determination formula B)

[0178] For example, the above determination formula B, specifically, the (ChX output ratio) obtained by the above determination formula B may be incorporated into the following condition (specification B). According to the present disclosure, the presence or absence of the overall level change may be determined based on whether this condition is satisfied.

[0179] <Specification B>

[0180] (1-β)<(ChX output ratio)<(1+β)

[0181] In the above-mentioned specification B, the predetermined value β can be appropriately set by a person skilled in the art based on measurement variations unique to individual analyzers, variations (in light transmittance) of the chip or flow cell, and / or variations in the analyzer itself. For example, a standard deviation can be calculated from multiple pieces of output value data obtained through multiple measurements, and β can be set based on this standard deviation. β can also be set based on variations in beads.

[0182] For example, β can be set to satisfy the following specific embodiment 2.

[0183] (Measurement variation or variation in transmittance of the chip or flow cell) < β < (value at which variation of the indicator bead is determined to be abnormal) ... Specific Example 2

[0184] For example, β may be a value ranging from 0.01 to 0.3, in particular from 0.05 to 0.25.

[0185] Preferably, the various elements of the above specifications are applied without contradiction in terms of large-small relationship. However, in the case where it is difficult to achieve the above, part of the specifications may be relaxed or ignored.

[0186] For example, specification A may be relaxed or changed within the scope of Specific Example 1 describing the setting of α. In addition, the upper or lower limit defined in specification A may be eliminated. That is, conditions such as (1-α) < (ChA ratio) or (ChA ratio) < (1+α) may be used.

[0187] Furthermore, specification B may be relaxed or changed within the scope of Specific Example 2 describing the setting of β. Furthermore, it is also possible to eliminate the upper or lower limit defined in specification B. That is, conditions such as (1-β) < (ChX output ratio) or (ChX output ratio) < (1+β) may be used.

[0188] Specifically, the change in α in Determination Formula A is an absolute change and tends to have a large value. Determination Formula A is suitable for determining excessive changes or degradation of beads, or large changes in transmittance of a detection chip or flow cell.

[0189] Furthermore, the change in β in the determination formula B is a relative change and tends to have a small value. Therefore, the determination formula B is considered to have a high ability to identify bead abnormalities.

[0190] In one embodiment, when the biological sample analyzer includes a plurality of channels ChX, the same specification B may be set for each channel ChX.

[0191] In another embodiment, when the biological sample analyzer includes a plurality of channels ChX, a specification B may be set for each channel ChX, that is, different specifications B may be set for the respective channels ChX. In other words, the value of β used in the specification B may be different between the channels ChX.

[0192] The biological sample analyzer can determine that the analyzer status is appropriate when the (ChA ratio) calculated according to the above determination formula A and the (ChX output ratio) calculated according to the above determination formula B respectively meet specifications A and B. A status that meets specification A means that the overall level change does not exist or falls within the allowable range, and a status that meets specification B means that the relative level change of each fluorescent channel (especially fluorescent channels other than the representative fluorescent channel) does not exist or falls within the allowable range.

[0193] In this way, when the first indicator value satisfies a predetermined first condition (e.g., specification A) and one or more second indicator values satisfy a predetermined second condition (e.g., specification B), the biological sample analyzer according to the present disclosure can determine that the state of the analyzer is appropriate.

[0194] Furthermore, the biological sample analyzer can perform gain adjustment processing on each fluorescence channel after confirming that the analyzer is in a proper state. This gain adjustment processing can be performed so that the output value of each fluorescence channel falls within a predetermined numerical range for each fluorescence channel. By adjusting the gain of each fluorescence channel after confirming that the state is proper, the gain can be appropriately set.

[0195] Furthermore, the biological sample analyzer may be designed to record the gain of the corresponding fluorescence channel set during the gain adjustment process. The gain may be recorded in the biological sample analyzer (specifically, a storage unit included in the biological sample analyzer) or in a server to which the biological sample analyzer is connected. The gain recorded in this manner may be used in the next verification process.

[0196] If the first indicator value does not satisfy the predetermined first condition or one or more second indicator values do not satisfy the predetermined second condition, the biological sample analyzer may determine that the analyzer state is inappropriate. This configuration can prevent the biological sample analyzer in an inappropriate state from performing biological sample analysis.

[0197] The biological sample analyzer may be designed to output a display prompting reloading of fluorescent beads, a display prompting re-performing of QC processing, or a display prompting inspection of the chip, flow cell, or analyzer after determining that the status is unsuitable.

[0198] Furthermore, the biological sample analyzer may be designed to output a display related to changes or abnormalities in fluorescent beads used in the verification process, changes or abnormalities in the biological sample analyzer, or incorrect application of standard value data after determining that the status is inappropriate.

[0199] The above display can prompt the user to check or adjust the status of the analyzer.

[0200] (2) Configuration Example

[0201] Figure 3 An exemplary configuration of the biological sample analyzer of the present disclosure is shown. Figure 3 The biological sample analyzer 6100 shown in FIG. 1 includes a light irradiation unit 6101 for irradiating a biological sample S flowing in a flow channel C with light; a detection unit 6102 for detecting light generated by irradiating the biological sample S with light; and an information processing unit 6103 for processing information regarding the light detected by the detection unit. For example, the biological sample analyzer 6100 is a flow cytometer or an imaging cytometer. The biological sample analyzer 6100 may also include a sorting unit 6104 for sorting specific biological particles P from the biological sample. A biological sample analyzer 6100 including a sorting unit is, for example, a cell sorter.

[0202] (Biological Samples)

[0203] The biological sample S can be a liquid sample containing biological particles. Biological particles are, for example, cells or non-cellular biological particles. Cells can be living cells, and more specific embodiments thereof include blood cells (such as red blood cells and white blood cells) and reproductive cells (such as semen and fertilized eggs). In addition, cells can be those directly collected from samples such as whole blood, or can be cultured cells obtained after cultivation. For example, non-cellular biological particles are extracellular vesicles, or in particular, exosomes and microvesicles. Biological particles can be labeled with one or more marker substances (such as dyes (in particular, fluorescent dyes) and fluorescent dye-labeled antibodies). It should be noted that particles other than biological particles can be analyzed by the biological sample analyzer of the present disclosure, and beads, etc. can be analyzed for calibration, etc.

[0204] (Runner)

[0205] The flow channel C is designed so as to form a flow of the biological sample S. Specifically, the flow channel C can be designed so as to form a flow in which the biological particles contained in the biological sample are basically aligned in a row. The flow channel structure including the flow channel C can be designed to form a laminar flow. Specifically, the flow channel structure is designed to form a laminar flow in which the flow of the biological sample (sample flow) is surrounded by the flow of the sheath liquid. The design of the flow channel structure can be appropriately selected by those skilled in the art, or a known design can be adopted. The flow channel C can be formed into a flow channel structure such as a microchip (a chip with a flow channel at the micron level) or a flow cell. The width of the flow channel C is less than 1 mm, specifically, it can be more than 10 μm and less than 1 mm. The flow channel C and the flow channel structure including the flow channel C can be made of materials such as plastic or glass.

[0206] The biological sample analyzer of the present disclosure is designed so that the biological sample flowing in the flow channel C, or specifically, the biological particles in the biological sample, are irradiated with light from the light irradiation unit 6101. The biological sample analyzer of the present disclosure can be designed so that the irradiation point of the light on the biological sample is located in the flow channel structure in which the flow channel C is formed, or can be designed so that the irradiation point is located outside the flow channel structure. An embodiment of the former case can be a configuration in which light is emitted onto the flow channel C in a microchip or a flow cell. In the latter case, the biological particles after leaving the flow channel structure (specifically, its nozzle portion) can be irradiated with light, and, for example, an air jet type flow cytometer can be used.

[0207] (Light irradiation unit)

[0208] The light irradiation unit 6101 includes a light source unit that emits light and a light-guiding optical system that guides the light to the irradiation point. The light source unit includes one or more light sources. The type of light source is, for example, a laser light source or an LED. The wavelength of light emitted from each light source can be any wavelength of ultraviolet light, visible light, and infrared light. For example, the light-guiding optical system includes optical components such as a beam splitter, a reflector, or an optical fiber. The light-guiding optical system may also include a lens group for focusing light, and may include, for example, an objective lens. There may be one or more irradiation points where the biological sample and the light intersect. The light irradiation unit 6101 can be designed to collect light emitted from one light source or different light sources to one irradiation point.

[0209] (Detection unit)

[0210] The detection unit 6102 includes at least one photodetector that detects light generated by emitting light onto the biological particles. For example, the light to be detected can be fluorescence or scattered light (such as one or more of the following: forward scattered light, backscattered light, and side scattered light). For example, each photodetector includes one or more light receiving elements and has an array of light receiving elements. Each photodetector can include one or more photomultiplier tubes (PMTs) and / or photodiodes such as APDs and MPPCs as light receiving elements. The photodetector includes, for example, a PMT array in which a plurality of PMTs are arranged in a one-dimensional direction. The detection unit 6102 can also include an image sensor such as a CCD or a CMOS. Through the image sensor, the detection unit 6102 can obtain an image of the biological particles (for example, such as a bright field image, a dark field image, or a fluorescent image).

[0211] The detection unit 6102 includes a detection optical system that allows light of a predetermined detection wavelength to reach a corresponding photodetector. The detection optical system includes a spectroscopic unit such as a prism or a diffraction grating, or a wavelength separation unit such as a dichroic mirror or a filter. For example, the detection optical system is designed to disperse light generated by light irradiation to biological particles and detect the dispersed light using a larger number of photodetectors than the number of fluorescent dyes used to label the biological particles. A flow cytometer that includes such a detection optical system is called a spectral flow cytometer. In addition, for example, the detection optical system is designed to separate light corresponding to a fluorescent wavelength band of a specific fluorescent dye from light generated by light irradiation to biological particles and cause the corresponding photodetector to detect the separated light.

[0212] The detection unit 6102 may further include a signal processing unit that converts the electrical signal obtained by the light detector into a digital signal. The signal processing unit may include an A / D converter as a device for performing the conversion. The digital signal obtained by the conversion performed by the signal processing unit may be sent to the information processing unit 6103. The digital signal may be processed by the information processing unit 6103 into data related to light (hereinafter also referred to as "light data"). For example, the light data may be light data including fluorescence data. More specifically, the light data may be light intensity data, and the light intensity may be light intensity data of light including fluorescence (the light intensity data may include feature quantities such as area, height, and width).

[0213] (Information Processing Unit)

[0214] For example, the information processing unit 6103 includes a processing unit that performs processing of various types of data (e.g., light data) and a storage unit that stores various types of data. When the processing unit obtains light data corresponding to a fluorescent dye from the detection unit 6102, the processing unit can perform fluorescence leakage correction (compensation processing) on the light intensity data. In the case of a spectral flow cytometer, the processing unit also performs fluorescence separation processing on the light data and obtains light intensity data corresponding to the fluorescent dye. For example, fluorescence separation processing can be performed by the unmixing method disclosed in JP 2011-232259 A. When the detection unit 6102 includes an image sensor, the processing unit can obtain morphological information about biological particles based on the image obtained by the image sensor. The storage unit can be designed to be able to store the acquired light data. The storage unit can be designed to be able to further store spectral reference data to be used in the unmixing process.

[0215] When the biological sample analyzer 6100 includes a sorting unit 6104 described later, the information processing unit 6103 can determine whether to sort the biological particles based on the optical data and / or morphological information. The information processing unit 6103 then controls the sorting unit 6104 based on the determination result, and the biological particles can be sorted by the sorting unit 6104.

[0216] The information processing unit 6103 can be designed to output various types of data (e.g., such as light data and images). For example, the information processing unit 6103 can output various types of data generated based on the light data (e.g., such as two-dimensional curves or spectral curves). For example, the information processing unit 6103 can also be designed to accept input of various types of data and accept user-controlled gating of drawings. The information processing unit 6103 can include an output unit (e.g., such as a display) or an input unit (e.g., such as a keyboard) for performing output or input.

[0217] The information processing unit 6103 can be designed as a general-purpose computer, and can be designed as an information processing device including, for example, a CPU, RAM, and ROM. The information processing unit 6103 can be included in the housing including the light irradiation unit 6101 and the detection unit 6102, or can be located outside the housing. In addition, various processes or functions to be performed by the information processing unit 6103 can be implemented by a server computer or cloud connected via a network.

[0218] (Sorting unit)

[0219] The sorting unit 6104 sorts the biological particles according to the determination result performed by the information processing unit 6103. The sorting method can be a method of generating droplets containing biological particles by vibration, applying electric charge to the droplets to be sorted, and controlling the moving direction of the droplets by electrodes. The sorting method can be a method for sorting by controlling the direction of travel of biological particles in a flow channel structure. For example, the flow channel structure has a control mechanism based on pressure (injection or suction) or charge. An embodiment of the flow channel structure can be a chip having a flow channel structure in which the flow channel C branches into a recovery flow channel and a waste liquid flow channel on the downstream side, and collects specific biological particles in the recovery flow channel (for example, the chip disclosed in JP 2020-76736A).

[0220] (3) Example of Verification Processing

[0221] refer to Figure 4 , an embodiment of a process of verifying the state of a biological sample analyzer by using determination formula A and determination formula B will be described below. Figure 4 Note that the process in this embodiment is a process for determining a change or abnormality in the output (especially the fluorescence output) in a series of QC (Quality Control, also called precision control or quality management) processes of the analyzer.

[0222] In step S100 , the biological sample analyzer starts a verification process.

[0223] In step S101, the biological sample analyzer (specifically, the information processing unit) obtains the standard value data of the fluorescent beads to be used. As described above, the standard value data may include at least the standard value of ChA and the standard value of ChX used to calculate the (ChA ratio) and (ChX output ratio). The standard value data can be pre-stored in the biological sample analyzer (for example, in a storage medium included in the analyzer), or the biological sample analyzer can obtain the standard value data retained by the manufacturer of the fluorescent beads, for example, via a network. For example, the standard value data can be obtained via a network using a code (for example, a number or a one-dimensional or two-dimensional code) attached to a container containing the fluorescent beads.

[0224] In step S102, the biological sample analyzer performs optical adjustments. For example, the optical adjustments may involve adjusting the irradiation point of laser light applied from the light irradiation unit and / or the chip or flow cell. Furthermore, those skilled in the art may appropriately select a method for performing the optical adjustments based on the configuration of the biological sample analyzer.

[0225] In step S103, the biological sample analyzer (specifically, the information processing unit) determines whether the analyzer has passed the QC process one or more times. That is, in this step, it can be determined whether the analyzer's status has been determined to allow one or more biological sample analyses to be performed through the QC process. This determination is then performed, and subsequent steps S104 to S107 are then executed to calculate the determination values A and B using the gain settings that have passed the QC process. Thus, the latest status of the analyzer is reflected in these determination values. This helps to more appropriately perform the evaluation of the analyzer's status.

[0226] In the case where it is determined that the QC process has been passed once or more times, the analyzer advances the process to step S104.

[0227] In the case where it is determined that the QC process has not been passed one or more times (ie, the case where the analyzer has never passed the QC process before, for example, the case where the analyzer is used for the first time), the analyzer advances the process to step S109.

[0228] In step S104, the biological sample analyzer sets the light receiving element gain recorded during the previous pass of the QC process (i.e., the light receiving element gain recorded during the last pass of the QC process) for each light receiving element. For example, the light receiving element gain recorded during the previous pass of the QC process may be the gain recorded in step S111 described below. In this way, the processing in step S105 and subsequent steps can be performed using the light receiving element gain that reflects the latest state of the analyzer. This helps to more appropriately perform the evaluation of the analyzer's state.

[0229] In step S105, the biological sample analyzer causes the fluorescent beads to flow to acquire a predetermined number of event data. For example, the number of event data items may be 10,000, but is not limited to this number. For example, the number of event data items may range from 1,000 to 1,000,000, 2,000 to 500,000, or 5,000 to 100,000, etc. Alternatively, as described separately below in this specification, event data may be acquired by detecting fluorescence generated by light irradiation of each measurement target particle (fluorescent bead) flowing in the flow channel.

[0230] More specifically, the light irradiation unit irradiates the particles flowing in the flow channel with light, and the detection unit detects light (specifically, fluorescence) generated by the light irradiation. Then, the information processing unit acquires a signal intensity data set (event data) of the detected light.

[0231] In step S106, the biological sample analyzer (specifically, the information processing unit) obtains singlet data associated with specific particles from the event data obtained in step S105. These specific particles are beads used to calculate the determination value A and the determination value B, and can be predetermined. For example, in some cases, the fluorescent beads used to verify the status of the analyzer may include multiple types of fluorescent beads. In this case, it can be predetermined which of the multiple types of fluorescent beads is used to obtain the event data used in the verification process according to the present disclosure.

[0232] The biological sample analyzer can obtain only singlet data related to predetermined specific particles from the event data by using predetermined software. For example, the predetermined software may be, but is not limited to, AutoGate.

[0233] In step S106, the biological sample analyzer can further obtain a signal intensity representative value for each fluorescence channel based on the obtained singlet data. For example, the signal intensity representative value can be a representative value related to height or area. In addition, the representative value can be a median (median value), a mode value (mode value) or an average value (average value). In particular, the representative value can be a median (intermediate value). The signal intensity representative value is preferably the median value of the height.

[0234] According to one embodiment, the biological sample analyzer may obtain the median value of the height of each fluorescent channel based on the obtained singlet data, that is, the median value of the height of ChA and the median value of the height of each ChaX may be obtained.

[0235] In step S107, the biological sample analyzer (specifically, the information processing unit) calculates the (ChA ratio) and (ChX output ratio) using the singlet data (specifically, the signal intensity representative value of each fluorescent channel) associated with the specific particle acquired in step S106. The thus calculated (ChA ratio) and (ChX output ratio) are used in the determination process in step S108.

[0236] The (ChX output ratio) can be calculated for each channel ChX. For example, if the biological sample analyzer includes three fluorescence channels, Ch1, Ch2, and Ch3, as the fluorescence channel ChX described above, the (Ch1 output ratio), (Ch2 output ratio), and (Ch3 output ratio) are calculated. In other words, if the biological sample analyzer includes (N-1) fluorescence channels, Ch1, Ch2, ..., and Ch(N-1), as the fluorescence channel ChX described above, the (Ch1 output ratio), (Ch2 output ratio), ..., and (Ch(N-1) output ratio) are calculated.

[0237] For example, the (ChA ratio) can be calculated by using the above-mentioned determination formula A. The following values are used as the respective constituent elements of the determination formula A.

[0238] The median value of the height of ChA recorded at the time of previous QC processing was used as (ChAold_Height).

[0239] The standard value of ChA (GoldStandard) recorded previously during QC processing was used as (ChAold_GS).

[0240] The median value of the heights of ChA acquired in step S106 is used as (ChAnew_Height).

[0241] The standard value (GoldStandard) of ChA acquired in step S101 is used as (ChAnew_GS).

[0242] For example, (ChX output ratio) can be calculated by using the above-mentioned determination formula B. The following values are used as the respective constituent elements of determination formula B.

[0243] The median value of the heights of ChX acquired in step S106 is used as (ChXnew_Height).

[0244] The median value of the heights of ChA acquired in step S106 is used as (ChAnew_Height).

[0245] The median value of the height of ChX recorded at the previous QC process was used as (ChXold_Height).

[0246] The median value of the height of ChA recorded at the time of previous QC processing was used as (ChAold_Height).

[0247] The standard value (GoldStandard) of ChX acquired in step S101 is used as (ChXnew_GS).

[0248] The standard value (GoldStandard) of ChA acquired in step S101 is used as (ChAnew_GS).

[0249] In the case where the (ChA ratio) has been calculated by using the determination formula A, the calculated (ChA ratio) may be used. In the case where the (ChA ratio) is used, as is apparent from the determination formula B, some of the above values may not be used.

[0250] In step S108 , the biological sample analyzer (specifically, the information processing unit) determines whether the (ChA ratio) satisfies specification A, and also determines whether the (ChX output ratio) satisfies specification B. In the case where the analyzer has a plurality of fluorescence channels as ChX, it determines whether the (ChX output ratio) of each of the plurality of fluorescence channels satisfies specification B set for the corresponding fluorescence channel.

[0251] If the (ChA ratio) and (ChX output ratio) satisfy specifications A and B, respectively (if all channels ChX using an analyzer including multiple channels ChX satisfy specification B), the biological sample analyzer proceeds with the process to step S109. In other words, if all fluorescence channels satisfy the specifications set for the corresponding fluorescence channels, the biological sample analyzer proceeds with the process to step S109.

[0252] If the (ChA ratio) does not meet specification A or the (ChX output ratio) does not meet specification B (when using an analyzer including multiple channels ChX, one or more of the multiple channels ChX do not meet specification B), the biological sample analyzer advances the process to step S113. In other words, if at least one of the fluorescence channels does not meet the specifications set for the corresponding channel, the biological sample analyzer advances the process to step S113.

[0253] The above-described specifications can be used as the specification A and the specification B used in step S108 .

[0254] In step S109, the biological sample analyzer causes the fluorescent beads to flow to acquire a predetermined number of event data. For example, the number of event data items may be 10,000, but is not limited to this number. For example, the number of event data items may be 1,000 to 1,000,000, 2,000 to 500,000, or 5,000 to 100,000. Alternatively, as described separately below in this specification, event data may be acquired by detecting fluorescence generated by light irradiation of each measurement target particle (fluorescent bead) flowing in the flow channel.

[0255] Preferably, the acquisition of event data in step S109 is performed in a similar manner to that in step S105. For example, the supply speed of the sample containing particles and the wavelength and intensity of the applied light in step S109 may be similar to those in step S105.

[0256] In step S110, the biological sample analyzer (specifically, the information processing unit) acquires singlet data related to the specific particle from the event data acquired in step S109. The specific particle may be the same as the specific particle described in step S106.

[0257] The biological sample analyzer can obtain only singlet data related to specific particles from the event data by using predetermined software. For example, the predetermined software may be, but is not limited to, AutoGate.

[0258] In step S110 , the biological sample analyzer further obtains the median value of the height of each fluorescent channel based on the obtained singlet data, that is, obtains the median value of the height of ChA and the median value of the height of each ChX.

[0259] Then, in step S110 , the biological sample analyzer adjusts the gain of each fluorescent channel so that the median value of the height of each fluorescent channel obtained falls within a predetermined range. The predetermined range can be pre-set for each fluorescent channel.

[0260] In step S111 , the biological sample analyzer records the median value of the height of each fluorescent channel whose gain has been adjusted in step S110 , the standard value (GoldStandard) of each fluorescent channel, and the light receiving element gain of each fluorescent channel.

[0261] Preferably, these data items are recorded after a QC process (such as a process for checking the status of the analyzer based on the rCV (robust coefficient of variation) of each fluorescent channel) has passed. That is, these data are recorded after confirming that the status of the analyzer meets predetermined precision control conditions.

[0262] The data thus recorded may be used to perform the next verification process according to the present disclosure, and specifically used in steps S104 and S107 .

[0263] In step S112 , the biological sample analyzer ends the verification process according to the present disclosure, which means that the overall fluorescence level variation does not exist or falls within a predetermined allowable range, and the fluorescence level variation in each fluorescence channel does not exist or falls within a predetermined allowable range.

[0264] Therefore, upon completion of the processing, the biological sample analyzer can cause the display device to display, for example, that the overall fluorescence level variation does not exist or falls within a predetermined allowable range, and that the fluorescence level variation in each fluorescence channel also does not exist or falls within a predetermined allowable range.

[0265] Furthermore, following completion of the processing, the bioregion analyzer may cause the display device to display an instruction for prompting the user to perform an operation for performing the biological sample analysis.

[0266] After the processing ends, the biological sample analyzer can perform biological sample analysis by using the light receiving element gain recorded in step S111 .

[0267] In step S113 , the biological sample analyzer ends the verification process according to the present disclosure, which means that the overall fluorescence level variation exceeds the allowable range, the fluorescence level variation in at least one fluorescence channel exceeds the allowable range, or both variations exceed the allowable range.

[0268] Therefore, upon completion of the process, the biological sample analyzer can cause the display device to display, for example, that the overall fluorescence level change exceeds the allowable range, that the fluorescence level change in at least one fluorescence channel exceeds the allowable range, or that both changes exceed the allowable range. In other words, after the process is completed, the biological sample analyzer can cause the display device to output, for example, that the verification process has not passed. This can prompt the user to inspect or replace the biological sample analyzer, chip, or flow cell.

[0269] The biological sample analyzer may change the display details output in step S113 according to the determination result in step S108 .

[0270] For example, if the (ChA ratio) obtained by determining formula A does not meet specification A, it can be said that there is a change in the overall fluorescence level. Therefore, in this case, the biological sample analyzer can output a display indicating that there is a change in the overall fluorescence level. In addition, in this case, a display indicating that there may be a change or abnormality in the chip or flow cell, a display indicating that there may be a change or abnormality in the light irradiation unit, or a display indicating that there may be a change or increase in laser power can be further output.

[0271] Furthermore, if the (Ch x output ratio) obtained by determining equation B does not satisfy specification B, it can be said that there is a fluorescent channel that does not satisfy specification B or that the fluorescent beads have deteriorated. Therefore, in this case, the biological sample analyzer can output a display indicating that the fluorescent channel does not satisfy specification B. Furthermore, in this case, a display indicating that the fluorescent beads have deteriorated can also be output.

[0272] (4) Example 1 (Example of verification processing)

[0273] Hereinafter, another specific embodiment of the verification process according to the present disclosure will be described.

[0274] It should be noted that in the following embodiments, a cell sorter is used as a biological sample analyzer, but the verification processing of the present disclosure can be performed not only by a flow cytometer having a sorting unit (such as a cell sorter), but also by a flow cytometer without a sorting unit.

[0275] In addition, in the following embodiments, fluorescent beads ASB (Sony Group Corporation) are used as particles for verification processing. Fluorescent beads ASB contain two types of fluorescent particles. One of the two types of particles is a fluorescent particle with a particle size of 3 μm, and the other type of particle is a fluorescent particle with a particle size of 10 μm. Although the verification processing is performed by using singlet data related to the fluorescent particle with a particle size of 3 μm of the two types of fluorescent particles in the following embodiments, the verification processing according to the present disclosure can also be performed by using fluorescent particles with a particle size of 10 μm. In addition, the verification processing according to the present disclosure can also be performed by using other types of fluorescent beads.

[0276] The cell sorter is designed to perform reference Figure 3 Each step of the verification process is described below.

[0277] In step S100 , the cell sorter starts a verification process according to the present disclosure.

[0278] In step S101, a cell sorter reads standard value data related to a batch containing fluorescent beads ASB. The standard value data includes standard values of all fluorescent channels included in the cell sorter.

[0279] In step S102 , the cell sorter performs optical adjustment. In the optical adjustment, the position of the chip having the flow channel for irradiating the fluorescent beads with light and / or the irradiation point of the light irradiation unit for performing the light irradiation are adjusted.

[0280] In step S103, the cell sorter determines whether the cell sorter has passed the QC process one or more times. The QC process is a series of processes for controlling the performance of the analyzer and includes not only the fluorescence level change verification process according to the present disclosure but also other verification processes (e.g., a process for verifying the status of the analyzer based on rCV).

[0281] In the case where it is determined that the QC process has passed one or more times, the cell sorter advances the process to step S104.

[0282] In the case where it is determined that the QC process has not been passed one or more times, the cell sorter advances the process to step S109.

[0283] In step S104 , for each fluorescent channel, the cell sorter sets the light receiving element gain recorded at the previous pass of the QC process.

[0284] In step S105 , the cell sorter causes the fluorescent beads to flow in a flow channel that is irradiated with light to acquire 10,000 pieces of event data.

[0285] In step S106, the cell sorter uses AutoGate to obtain singlet data associated with 3 μm fluorescent beads from 10,000 pieces of event data, and then calculates the median height of each fluorescent channel using this singlet data. That is, the median height of the singlet data is calculated for each of all fluorescent channels included in the cell sorter.

[0286] In step S107, the cell sorter uses the Ledian value and the standard value (GoldStandard) of the height of each fluorescent channel recorded at the previous pass of the QC process, the standard value (GoldStandard) of each fluorescent channel obtained in the above step S101, and the median value of the height of each fluorescent channel obtained in the above step S106 to calculate the determination value (ChA ratio) and the determination value (ChX output ratio) according to the above determination formula A and determination formula B.

[0287] More specifically, the cell sorter includes a plurality of fluorescent channels, one of which is allocated to a fluorescent channel for calculating the (ChA ratio), and the other fluorescent channels are allocated to a fluorescent channel for calculating the (ChX output ratio).

[0288] That is, for one fluorescent channel ChA in the former case, the cell sorter uses the median and standard value (GoldStandard) of the height of one fluorescent channel recorded in the previous pass of the QC processing, the standard value (GoldStandard) of one fluorescent channel obtained in the above step S101, and the median of the height of one fluorescent channel obtained in the above step S106 to calculate (ChA ratio) according to the above determination formula A.

[0289] In addition, for each other fluorescent channel ChX in the latter case, the cell sorter uses the median and standard value (GoldStandard) of the height of each fluorescent channel recorded at the previous pass of the QC process, the standard value (GoldStandard) of each fluorescent channel obtained in the above step S101, and the median of the height of each fluorescent channel obtained in the above step S106 to calculate (ChX output ratio) according to determination formula B.

[0290] In step S108 , the cell sorter determines whether the determination value (ChA ratio) satisfies specification A, and determines whether the determination value (ChX output ratio) satisfies specification B.

[0291] Note that in this embodiment, the determination value (ChA ratio) is one value.

[0292] Furthermore, in this embodiment, a determination value (ChX output ratio) is calculated for each fluorescent channel. Therefore, the Ch1 output ratio, Ch2 output ratio, and Ch3 output ratio are calculated for each of the three fluorescent channels, Ch1 to Ch3. Furthermore, a specification B is set for each fluorescent channel. The specifications used to determine the Ch1 output ratio, Ch2 output ratio, and Ch3 output ratio are also referred to as specifications B1, B2, and B3, respectively.

[0293] When (ChA ratio) and (ChX output ratio) satisfy specifications A and B respectively (i.e., Ch1 output ratio, Ch2 output ratio and Ch3 output ratio satisfy specifications B1, B2 and B3 respectively), in step S108, the cell sorter advances the processing to step S109.

[0294] In step S108, if (ChA ratio) does not satisfy specification A or (ChX output ratio) does not satisfy specification B (i.e., if one or more of Ch1 output ratio, Ch2 output ratio, and Ch3 output ratio do not satisfy the set specifications), the cell sorter proceeds to step S113.

[0295] In step S109, the cell sorter flows the fluorescent beads to acquire 10,000 pieces of event data. The acquisition of this event data is performed in a similar manner to the acquisition in step S105.

[0296] In step S110 , similarly to step S106 , the cell sorter acquires singlet data associated with fluorescent beads having a particle size of 3 μm from the event data acquired in step S109 .

[0297] In step S110 , the cell sorter further obtains a median value of the height of each fluorescent channel based on the obtained singlet data.

[0298] Then, the cell sorter adjusts the light receiving element gain of each fluorescent channel in step S110 so that the median value of the height of each fluorescent channel obtained falls within a predetermined range.

[0299] In step S111, in the case of passing QC processing, which is a series of processing such as processing for checking the state of the analyzer based on the rCV (robust coefficient of variation) of each fluorescent channel, the cell sorter records the median value of the height of each fluorescent channel, the standard value (GoldStandard) of each fluorescent channel, and the light receiving element gain of each fluorescent channel after the gain adjustment in step S110.

[0300] In step S112, the cell sorter completes the verification process according to the present disclosure, which means that the overall fluorescence level change does not exist or falls within the predetermined allowable range, and the fluorescence level change in each fluorescence channel does not exist or falls within the predetermined allowable range. After the process is completed, the cell sorter can perform biological sample analysis by using the light receiving element gain recorded in step S111.

[0301] In step S113, the cell sorter ends the verification process according to the present disclosure, which means that the overall fluorescence level change exceeds the allowable range, the fluorescence level change in at least one fluorescence channel exceeds the allowable range, or both of these changes exceed the allowable range. Therefore, unlike step S112, biological sample analysis is not performed after the process ends.

[0302] (5) Example 2 (actual verification)

[0303] Two different batches of fluorescent beads ASB (Sony Group Corporation) were prepared. One batch had the batch number AP05, and the other had the batch number AN183. Using these two batches of fluorescent beads, the cell sorter was subjected to the validation process described in Example 1.

[0304] This verification process is performed under conditions A to E shown in Table 1 below.

[0305] For conditions A to C among these conditions, the verification process is performed as follows.

[0306] 1. The validation process in Example 1 above was performed using non-degraded AP05 and using Gold Standard of AP05 applied. The cell sorter passed QC.

[0307] 2. The verification process in Example 1 above was carried out using the beads shown in Table 1 (AP05 was left at room temperature for a long time in condition A, and AN183 did not deteriorate in conditions B and C) and GoldStandard.

[0308] 3. Check the calculation results of the determination formula A and the determination formula B in the verification process in 2 above.

[0309] For conditions D and E among these conditions, the verification process was performed as follows. First, data for 3 μm beads in AP05 was acquired, and then the state (state of the analyzer or bead type) was changed as shown in the table. In this way, the verification process described in Example 1 was performed.

[0310] [Table 1]

[0311] Table 1: Details of the verification process

[0312]

[0313]

[0314]

[0315] For example, the determination values calculated by determination formula A and determination formula B vary depending on individual differences in fluorescent beads or chips (flow cell chips) having a flow channel in which the fluorescent beads flow. These variations are predetermined and are 15% and 13% for determination formula A and determination formula B, respectively. Therefore, in consideration of these variations, α and β for specifications A and B are set such that α = 20% = 0.20 and β = 15% = 0.15.

[0316] That is, the specification A used in step S108 is as follows.

[0317] 0.80 (= 1 - 0.20) < (ChA ratio) < 1.20 (= 1 + 0.20)

[0318] Furthermore, the specification B used in step S108 is as follows.

[0319] 0.85 (= 1-0.15) < (ChX output ratio) < 1.15 (= 1+0.15)

[0320] The following Table 2 and Figure 5 The results calculated by the determination formula A and the determination formula B are shown.

[0321] [Table 2]

[0322] Table 2: Results

[0323] aisle dye Deterministic application A B C D E Ch1 Fett Deterministic formula B 95.6％ 94.2％ 100.4％ 97.0％ 124.0％ ChA PE Deterministic formula A 109.1％ 110.3％ 101.4％ 72.5％ 888.2％ Ch2 PerCP Deterministic formula B 86.9％ 90.0％ 99.0％ 97.9％ 172.5％ Ch3 PE-Cy7 Deterministic formula B 82.1％ 84.6％ 98.9％ 98.2％ 164.8％

[0324] The results of the verification process will be described in more detail below.

[0325] Condition A: The fluorescence spectrum of AP05, after being left in the room for 18 hours, changes. More specifically, the signal intensity decreases across a portion of the wavelength range. This spectral change is detectable based on the (ChX output ratio) associated with the fluorescence channels Ch2 and Ch3 using Equation B. Note that this spectral change is undetectable using the (ChA ratio) using Equation A.

[0326] Condition B: AP05 and AN183 GS have different fluorescence spectra, and the signal intensity changes in a certain wavelength range. This change can be detected based on the determination formula B (ChX output ratio) associated with the fluorescence channel Ch3. It should be noted that this spectral change cannot be detected based on the determination formula A (ChA ratio).

[0327] Condition C: AN183 was validated using GS of AN183. Under this condition, the correlation between GS and fluorescent beads was adequate, and the beads were typically used. No abnormalities were detected under this condition for either Formula A or Formula B. In other words, no abnormalities were detected using the appropriately used beads.

[0328] Condition D. During the validation process under Condition D, the laser output applied to the beads was reduced by 30% from the laser output during the QC performed before the validation process. This resulted in an overall decrease in fluorescence levels. This decrease was detected by Determination A. On the other hand, this decrease was not detected by Determination B.

[0329] Condition E. The validation process under Condition E used data from 10 μm beads. However, the QC performed before this validation process used data from 3 μm beads. Using different types of beads for validation results in different fluorescence levels across the spectrum and in different fluorescence channels. These differences can be detected using Determination Formulas A and B.

[0330] As described above, by performing the verification process according to the present disclosure, the status of the analyzer can be appropriately evaluated under all five conditions. That is, the presence or absence of a change in fluorescence level can be appropriately verified by a biological sample analyzer (specifically, a flow cytometer) performing the verification process according to the present disclosure.

[0331] Furthermore, the overall fluorescence level change can be detected by the verification process, and the fluorescence level change in each fluorescence channel can also be detected. In addition, it is also possible to determine whether an overall fluorescence level change has occurred in each fluorescence channel, or whether a fluorescence level change has occurred. This makes it possible to detect, for example, degradation of beads, application of incorrect data, anomalies or changes in the optical system, or anomalies or changes in the flow system (specifically, a chip or flow cell having a flow channel in which particles are illuminated with light).

[0332] (6) Example of configuration of biological particle separation device

[0333] According to one embodiment, the biological sample analyzer of the present disclosure can be configured as a biological particle sorting device and can be configured as, for example, a cell sorting device. The biological particle sorting device can be a device that analyzes and / or sorts biological particles in a microchip without forming droplets. The biological particle sorting device can be designed to perform the above-mentioned verification process. Figure 6 and Figure 7 This embodiment is described.

[0334] Figure 6 is a schematic diagram showing a configuration example of a microchip for sorting biological particles and a configuration example of a biological particle analyzer including the microchip. Figure 7 An embodiment of a flow chart illustrating a biological particle sorting operation performed by the biological particle analyzer is shown.

[0335] Figure 6 The biological particle sorting microchip 150 shown in FIG. 1 includes a sample liquid flow channel 152 and a sheath liquid flow channel 154 joined to the sample liquid flow channel 152 at a junction 162. The biological particle sorting microchip 150 further includes a sample liquid inlet 151 and a sheath liquid inlet 153.

[0336] It should be noted that some portions of the sheath fluid channel 154 are Figure 6 The portion indicated by the dotted line is located below the sample liquid flow channel 152 indicated by the solid line (a position offset in the optical axis direction as described below), and the flow channel indicated by the dotted line and the flow channel indicated by the solid line are not connected to each other at the intersection of these channels. Figure 6 The sample liquid flow path 152 is shown to bend twice between the sample liquid inlet 151 and the junction 162, so as to easily distinguish between the sample liquid flow path 152 and the sheath liquid flow path 154. The sample liquid flow path 152 may be designed to extend linearly between the sample liquid inlet 151 and the junction 162 without bending.

[0337] In the biological particle sorting operation, a sample liquid containing biological particles is introduced into the sample liquid flow channel 152 from the sample liquid inlet 151 , and a sheath liquid not containing biological particles is introduced into the sheath liquid flow channel 154 from the sheath liquid inlet 153 .

[0338] The biological particle sorting microchip 150 includes a junction flow channel 155 having a junction portion 162 formed at one end thereof.

[0339] The sample liquid and the sheath liquid are joined together at the junction 162 and then flow toward the particle sorting section 157 in the junction flow channel 155. Specifically, for example, the sample liquid and the sheath liquid are joined at the junction 162, and a laminar flow is formed in which the sample liquid is surrounded by the sheath liquid. Preferably, the biological particles are arranged substantially in a row in the laminar flow. The sample liquid flow channel 152 and the two sheath liquid flow channels 154 are joined at the junction 162, and the junction flow channel 155 has a junction 162 formed at one end thereof. In such a flow channel structure as described above, a laminar flow is formed containing biological particles flowing substantially in a row. Thus, when the detection area 156 described later is irradiated with light, it is easy to distinguish between light generated by irradiating one biological particle and light generated by irradiating other biological particles.

[0340] The biological particle sorting microchip 150 further includes a particle sorting section 157 located at the other end of the junction flow channel 155 . Figure 8 FIG. 1 shows an enlarged view of the particle sorting section 157. Figure 8 As shown in FIG. 1A , the other end of the joining channel 155 is connected to the biological particle collection channel 159 via the connecting channel 170. Figure 8 As shown in FIG. 1A , the joining flow channel 155 , the connecting flow channel 170 , and the biological particle collecting flow channel 159 may be on the same axis.

[0341] When the collected target particles flow into the particle sorting section 157, as shown in FIG. Figure 8 As shown in FIG. 1B , a flow is formed from the joining flow channel 155 through the connecting flow channel 170 into the biological particle collection flow channel 159. Therefore, the collection target particles are collected in the biological particle collection flow channel 159. In this way, the collection target particles flow into the biological particle collection flow channel 159 through the connecting flow channel 170.

[0342] On the other hand, in the case where biological particles other than the collection target particles flow into the particle sorting section 157, the biological particles flow into the particle sorting section 157. Figure 8 A branch flow channel 158 is shown in FIG. In this case, no flow is formed to enter the biological particle collection flow channel 159.

[0343] like Figure 6 As shown, the biological particle collection flow channel 159 is formed to extend linearly from the particle sorting section 157, make a U-shaped turn, and then reach the same plane as the plane formed by the sample liquid inlet 151 and the sheath liquid inlet 153. The liquid flowing in the biological particle collection flow channel 159 is discharged to the outside of the chip from the collection flow channel distal end 163.

[0344] like Figure 6As shown in FIG, each of the two branch flow channels 158 is also formed to extend linearly from the particle sorting section 157, make a U-turn, and then reach the same plane as the plane formed by the sample liquid inlet 151 and the sheath liquid inlet 153. The liquid flowing in the branch flow channel 158 is discharged from the branch flow channel distal end 166 to the outside of the chip.

[0345] exist Figure 6 , portions of the biological particle collection channel 159 that form a U-shaped turn are shown differently using solid and dashed lines, indicating that the position of the biological particle collection channel 159 is offset in the optical axis direction between these portions. In this manner, the biological particle collection channel 159 is offset in the optical axis direction and, therefore, does not communicate with the branch channel 158 at the portion intersecting the branch channel 158.

[0346] The collection flow channel distal end 163 and the two branch flow channel distal ends 166 are each formed in a plane formed by the sample liquid inlet 151 and the sheath liquid inlet 153. In addition, the introduction flow channel inlet 164, through which the liquid is introduced into the introduction flow channel 161, is also formed in this plane. In this way, the biological particle sorting microchip 150 is configured so that the inlet through which the liquid is introduced and the outlet through which the liquid is discharged are both formed in one plane. This configuration facilitates attachment of the chip to the biological particle analyzer 100. For example, compared to a case where the inlet and / or outlet are formed in two or more different planes, the flow channel formed in the biological particle analyzer 100 and the flow channel formed in the biological particle sorting microchip 150 are easily connected to each other.

[0347] like Figure 6 and Figure 8 As shown, the biological particle sorting microchip 150 includes an introduction flow channel 161 for introducing liquid into the connecting flow channel 170 .

[0348] The interior of the connecting flow channel 170 is filled with liquid introduced into the connecting flow channel 170 from the introduction flow channel 161. This configuration can prevent biological particles other than the target from entering the biological particle collection flow channel 159.

[0349] The biological particle sorting microchip 150 includes two branch flow channels 158 connected to the other end of the junction flow channel 155. As in this configuration, the junction flow channel of the biological particle sorting microchip used in the present technology can be branched into a connection flow channel and at least one branch flow channel.

[0350] Biological particles other than the collection target particles flow to one of the two branch flow channels 158 without entering the biological particle collection flow channel 159 .

[0351] In addition, if Figure 6As shown, in addition to the microchip, the biological particle sorting microchip 150 constitutes a part of the biological particle analyzer 100 including the light irradiation unit 101, the detection unit 102, and the control unit 103. The light irradiation unit 101, the detection unit 102, and the control unit 103 correspond to the light irradiation unit 6101, the detection unit 6102, and the information processing unit 6103 described in (2), respectively, and the explanation of these units can be applied to this configuration embodiment. Figure 9 As shown, the control unit 103 of the biological particle analyzer 100 may include a signal processing unit 104 , a determination unit 105 and a sorting control unit 106 .

[0352] like Figure 7 As shown, the biological particle sorting operation using the above-described biological particle sorting microchip 150 includes: a liquid supplying step S101 for supplying a liquid containing biological particles to the junction flow channel 155; a determining step S102 for determining whether the biological particles flowing in the junction flow channel 155 are collection target particles; and a collecting step S103 for collecting the collection target particles into the biological particle collecting flow channel 159. Each step will be described below.

[0353] (7-1) Liquid supply step

[0354] In the liquid supply step S101 , a sample liquid containing biological particles and a sheath liquid not containing biological particles are introduced into the sample liquid flow channel 152 and the sheath liquid flow channel 154 from the sample liquid inlet 151 and the sheath liquid inlet 153 , respectively.

[0355] For example, the sample liquid and the sheath liquid are joined at the joint 162, forming a laminar flow in which the sample liquid is surrounded by the sheath liquid. Preferably, the biological particles are arranged substantially in a line in the laminar flow. That is, in the liquid supply step S101, a laminar flow containing biological particles flowing substantially in a line can be formed.

[0356] In this manner, in the liquid supply step S101 , the liquid containing the biological particles is supplied, in particular as a laminar flow, through the junction flow channel 155 . The liquid flows in the junction flow channel 155 from the junction portion 162 toward the particle sorting portion 157 .

[0357] (7-2) Determine the steps

[0358] In the determination step S102, it is determined whether the biological particles flowing in the joining flow channel 155 are collection target particles. This determination can be made by the determination unit 105. The determination unit 105 can make this determination based on the light generated by light irradiation from the light irradiation unit 101 on the biological particles.

[0359] The signal processing unit 104 included in the control unit 103 can process the waveform of the digital electrical signal obtained by the detection unit 102 to generate information (data) related to the characteristics of the light for determination by the determination unit 105. The signal processing unit 104 can obtain one, two, or three factors selected from the waveform of the digital electrical signal, for example, the width of the waveform, the height of the waveform, and the area of the waveform as information related to the characteristics of the light. In addition, for example, the information related to the characteristics of the light may include the detection time of the light. Specifically, the above-mentioned processing performed by the signal processing unit 104 can be performed in a mode of detecting scattered light and / or fluorescence. The determination unit 105 included in the control unit 103 determines whether the biological particle is a collection target particle based on the light generated by irradiating the biological particle flowing in the flow channel with light.

[0360] (7-3) Collection steps

[0361] In the collecting step S103, the biological particles determined as the collection target particles in the determining step S102 are collected in the biological particle collecting flow channel 159. The collecting step S103 is performed by the particle sorting section 157 included in the microchip 150. The laminar flow that has passed through the joining flow channel 155 is divided into two branch flow channels 158 at the particle sorting section 157. Figure 6 The particle sorting section 157 shown in FIG has two branch flow channels 158, but the number of branch flow channels is not limited to two. For example, the particle sorting section 157 may have one or more (e.g., two, three, or four) branch flow channels. Figure 6 As shown, the branch flow channel can be designed to branch in a Y shape in a plane, or can be designed to branch in three dimensions. In the collection step S103, the target particles are collected by connecting the flow channels and are collected in the biological particle collection flow channel when the pressure in the biological particle collection flow channel 159 changes. For example, the target particles can be collected by generating negative pressure in the biological particle collection flow channel 159, as described above. This negative pressure can be generated by deforming the wall of the biological particle collection flow channel 159 using, for example, an actuator 107 (specifically, a piezoelectric actuator) attached to the outside of the microchip 150. This negative pressure can form a flow that enters the biological particle collection flow channel 159. For example, in order to generate this negative pressure, the actuator 107 can be attached to the outside of the microchip 150 so that the wall of the biological particle collection flow channel 159 is deformed. This deformation of the wall can change the internal space of the biological particle collection flow channel 159 and generate negative pressure. For example, the actuator 107 can be a piezoelectric actuator. When the collection target particles are drawn into the biological particle collection flow channel 159, the sample liquid forming a laminar flow, or the sample liquid and sheath fluid forming a laminar flow, may also flow into the biological particle collection flow channel 159. In this manner, the collection target particles are sorted at the particle sorting section 157 and collected in the biological particle collection flow channel 159.

[0362] Connecting channel 170 includes an introduction channel 161 to prevent biological particles other than the target particles from entering biological particle collection channel 159 from connecting channel 170. Liquid is introduced into connecting channel 170 from introduction channel 161. Connecting channel 170 is filled with the liquid introduced in this manner. In addition, a portion of the liquid forms a flow from connecting channel 170 toward junction channel 155. This flow prevents biological particles other than the target particles from entering biological particle collection channel 159. The liquid that forms a flow from connecting channel 170 toward junction channel 155 is prevented from flowing into junction channel 155 by flowing through junction channel 155 and into branch channel 158, and similarly flows into branch channel 158. It should be noted that the remaining liquid introduced into connecting channel 170 flows toward biological particle collection channel 159. In this way, the interior of biological particle collection channel 159 can be filled with this liquid.

[0363] The flow entering the branch flow channel 158 can be discharged from the branch flow channel distal end 160 to the outside of the chip. Furthermore, the target particles collected in the biological particle collection flow channel 159 can be discharged from the collection flow channel distal end 163 to the outside of the microchip. A container can be connected to the collection flow channel distal end 163 via a flow channel such as a tube. The target particles can be collected in the container.

[0364] 2. Second embodiment (biological sample analysis system)

[0365] The present disclosure also provides a biological sample analysis system designed to perform the verification process described in Section 1 above. Specifically, the system includes an information processing unit that performs information processing using signal intensity data generated by irradiating a flow channel through which particles flow. The information processing unit can be designed to perform verification processing for verifying the status of an analyzer using at least a first indicator value indicating overall output level changes in multiple fluorescence channels and one or more second indicator values indicating output level changes in each of the multiple fluorescence channels.

[0366] In addition to the information processing unit, the biological sample analysis system may also include the light irradiation unit and the detection unit described above in part 1, and may further include a sorting unit. These components may be included in one device, or may be divided into multiple devices. For example, the biological sample analysis system may have an information processing device configured as an information processing unit. In addition to the information processing device, the biological sample analysis system may include an analyzer equipped with a light irradiation unit and a detection unit (and a sorting unit). These devices may be connected to each other in a wired or wireless manner. In addition, these devices may be connected to each other via a network.

[0367] For example, the system may perform the following processing as the verification processing according to the present disclosure. Details of each step are similar to those described in Section 1 above.

[0368] Described in Section 1 above Figure 4 In step S101 in the flowchart of FIG. 1 , the information processing apparatus acquires standard value data.

[0369] In step S102 , the biological sample analysis system (specifically, the analyzer) performs optical adjustment.

[0370] In step S103 , the biological sample analysis system (specifically, the information processing apparatus) determines whether the analyzer has passed one or more QC processes.

[0371] In step S104 , the biological sample analysis system (specifically, the information processing apparatus) sets the light-receiving element gain recorded at the previous pass of the QC process for each light-receiving element of the analyzer.

[0372] In step S105, the biological sample analysis system (specifically, the analyzer) causes the fluorescent beads to flow to acquire a predetermined amount of event data. The event data thus acquired is sent to the information processing device.

[0373] In step S106, the biological sample analysis system (specifically, the information processing device) obtains singlet data associated with the specific particle from the event data. In step S107, the biological sample analysis system (specifically, the information processing device) calculates the (ChA ratio) and (ChX output ratio) using the singlet data associated with the specific particle.

[0374] In step S108 , the biological sample analysis system (specifically, the information processing apparatus) determines whether the (ChA ratio) satisfies specification A, and determines whether the (ChX output ratio) satisfies specification B.

[0375] In step S109, the biological sample analysis system (specifically, the analyzer) causes the fluorescent beads to flow to acquire a predetermined amount of event data. The event data thus acquired is sent to the information processing device.

[0376] In step S110 , the biological sample analysis system (specifically, the information processing device) acquires singlet data related to a specific particle from the event data and adjusts the gain of each fluorescence channel based on the acquired singlet data.

[0377] In step S111, for example, after passing a predetermined QC process, the biological sample analysis system (specifically, the information processing device) records the median value of the height of each fluorescent channel whose gain has been adjusted, the standard value of each fluorescent channel, and the light receiving element gain of each fluorescent channel.

[0378] In step S112 , the biological sample analysis system (specifically, the analyzer) ends the verification process according to the present disclosure. After the process ends, the system can perform biological sample analysis.

[0379] In step S113 , the biological sample analysis system (specifically, the information processing device) ends the verification process according to the present disclosure. After the process ends, the biological sample analysis system can cause the display device to output, for example, that the verification process has not been passed.

[0380] 3. Third Embodiment (Biological Sample Analyzer Verification Method and Program for Executing the Verification Method)

[0381] The present disclosure also provides a method for verifying the status of a biological sample analyzer, the method including performing the verification process described in Section 1. That is, the verification method includes a verification process for verifying the status of the analyzer by using at least a first indicator value and one or more second indicator values, each of which is generated by signal intensity data of light generated by irradiating light on particles flowing in a flow channel. The first indicator value indicates a change in output level in a plurality of fluorescence channels, and the second indicator value indicates a change in output level for each of the plurality of fluorescence channels. For example, as described above in Section 1, Figure 4 The verification process is performed as described. The explanation of the verification process also applies to the verification process in this article.

[0382] The present disclosure also provides a program for causing a biological sample analyzer or biological sample analysis system to perform the verification method. For example, the program can be stored in the biological sample analyzer or information processing device (specifically, a storage unit), or can also be stored in an information storage medium. For example, the information storage medium can be an SD card, micro SD card, CD, DVD, flash memory, or magnetic recording medium.

[0383] It should be noted that the present disclosure can also adopt the following configurations. (1)

[0385] A biological sample analyzer, comprising:

[0386] an information processing unit that performs information processing by using signal intensity data of light generated by irradiating light to a flow channel in which particles flow, wherein

[0387] The information processing unit performs verification processing for verifying the state of the analyzer by using at least one or more first indicator values and one or more second indicator values, the first indicator value indicating the overall output level change in the multiple fluorescent channels and the second indicator value indicating the output level change of each of the multiple fluorescent channels. (2)

[0389] The biological sample analyzer according to (1) above, wherein

[0390] A first index value is calculated based on a first output value of a representative fluorescent channel indicating an overall output level of the plurality of fluorescent channels. (3)

[0392] The biological sample analyzer according to (2) above, wherein

[0393] The first indicator value is calculated by using at least a previous first output value and a current first output value, wherein the previous first output value is obtained in a verification process that has been performed before the verification process is performed, and the current first output value is obtained in a verification process (hereinafter referred to as "current verification process"). (4)

[0395] The biological sample analyzer according to (3) above, wherein

[0396] The first index value is calculated by using a ratio or a difference between a previous first output value and a current first output value. (5)

[0398] The biological sample analyzer according to (3) or (4) above, wherein

[0399] The first index value is calculated by further using a predetermined standard output value of a representative fluorescence channel in addition to the previous first output value and the current first output value. (6)

[0401] The biological sample analyzer according to (5) above, wherein

[0402] The previous first standard output value acquired in the verification process whose execution is completed and the first standard output value acquired in the current verification process are used as the predetermined standard output value. (7)

[0404] The biological sample analyzer according to any one of (2) to (6) above, wherein

[0405] The second index value is calculated based on the first index value and the second output value of each of the other fluorescent channels except the representative fluorescent channel. (8)

[0407] The biological sample analyzer according to (7) above, wherein

[0408] The second indicator value is calculated by using at least a first indicator value, a previous second output value, and a current second output value, wherein the previous second output value is obtained in a verification process that has been performed before the verification process is performed, and the current second output value is obtained in a verification process (hereinafter referred to as "current verification process"). (9)

[0410] The biological sample analyzer according to (8) above, wherein

[0411] The second index value is calculated by further using a predetermined standard output value of each of the other fluorescent channels in addition to the first index value, the previous second output value, and the current second output value. (10)

[0413] The biological sample analyzer according to (9) above, wherein

[0414] The previous second standard output value acquired in the verification process whose execution is completed and the second standard output value acquired in the current verification process are used as the predetermined standard output value. (11)

[0416] The biological sample analyzer according to any one of (1) to (10) above, wherein

[0417] In a case where the first indicator value satisfies a predetermined first condition and the one or more second indicator values satisfy a predetermined second condition, the biological sample analyzer determines that the status of the analyzer is appropriate. (12)

[0419] The biological sample analyzer according to (11) above, wherein

[0420] The biological sample analyzer performs a gain adjustment process for each of the fluorescence channels after determining that the state of the analyzer is appropriate. (13)

[0422] The biological sample analyzer according to (12) above, wherein

[0423] The biological sample analyzer records the gain of each of the fluorescence channels, the gain being set in the gain adjustment process. (14)

[0425] The biological sample analyzer according to any one of (1) to (13) above, wherein

[0426] In the event that the first indicator value does not satisfy the predetermined first condition or the one or more second indicator values do not satisfy the predetermined second condition, the biological sample analyzer determines that the status of the analyzer is unsuitable. (15)

[0428] The biological sample analyzer according to (14) above, wherein

[0429] After determining that the status of the analyzer is inappropriate, the biological sample analyzer outputs one or more items selected from the group consisting of: a display prompting re-replication of fluorescent beads, a display prompting re-execution of QC processing, a display prompting inspection of the chip, circulation cell or analyzer, a display associated with changes or abnormalities in the fluorescent beads used in the verification process, a display associated with changes or abnormalities in the biological sample analyzer, and a display associated with incorrect application of standard value data. (16)

[0431] The biological sample analyzer according to any one of (1) to (15) above, wherein

[0432] The biological sample analyzer performs a verification process by using fluorescent beads as particles. (17)

[0434] The biological sample analyzer according to (16) above, wherein

[0435] The fluorescent beads emit fluorescent light covering the entire wavelength range of fluorescence to be detected by multiple fluorescent channels. (18)

[0437] The biological sample analyzer according to any one of (1) to (17) above, wherein

[0438] Biological sample analyzers include flow cytometers. (19)

[0440] A biological sample analysis system, comprising:

[0441] an information processing unit that performs information processing by using signal intensity data of light generated by irradiating light to a flow channel in which particles flow, wherein

[0442] The information processing unit performs verification processing for verifying the state of the analyzer by using at least one first index value and one or more second index values, the first index value indicating the overall output level change in the multiple fluorescent channels and the second index value indicating the output level change of each of the multiple fluorescent channels. (20)

[0444] A method for verifying a status of a biological sample analyzer, the method comprising:

[0445] a verification process for verifying the state of the analyzer by using at least one or more first index values and one or more second index values, the first index value and the second index value being generated from signal intensity data of light generated by irradiating light to particles flowing in a flow channel, wherein

[0446] The first index value indicates an overall output level variation in the plurality of fluorescent channels, and the second index value indicates an output level variation in each of the plurality of fluorescent channels.

[0447] [List of Reference Numbers]

[0448] 6100: Biological Sample Analyzer

[0449] 6101: Light irradiation unit

[0450] 6102: Detection unit

[0451] 6103: Information Processing Unit

Claims

1. A biological sample analyzer comprising: an information processing unit that performs information processing by using signal intensity data of light generated by irradiating light to a flow channel in which particles flow, wherein The information processing unit performs verification processing for verifying the state of the analyzer by using at least one or more first index values and one or more second index values, wherein the first index values indicate the overall output level change in the plurality of fluorescent channels and the second index values indicate the output level change of each of the plurality of fluorescent channels.

2. The biological sample analyzer according to claim 1, wherein The first index value is calculated based on a first output value of a representative fluorescent channel indicating an overall output level of the plurality of fluorescent channels.

3. The biological sample analyzer according to claim 2, wherein: The first indicator value is calculated by using at least a previous first output value and a current first output value, wherein the previous first output value is obtained in a completed verification process that has been executed before the verification process is executed, and the current first output value is obtained in the verification process (hereinafter referred to as "current verification process").

4. The biological sample analyzer according to claim 3, wherein: The first index value is calculated by using a ratio or a difference between the previous first output value and the current first output value.

5. The biological sample analyzer according to claim 3, wherein: The first index value is calculated by further using a predetermined standard output value of the representative fluorescence channel in addition to the previous first output value and the current first output value. The biological sample analyzer according to claim 5 , wherein: A previous first standard output value acquired in the verification process that has been executed and a first standard output value acquired in the current verification process are used as the predetermined standard output value.

7. The biological sample analyzer according to claim 2, wherein: The second index value is calculated based on the first index value and a second output value of each of the other fluorescent channels except the representative fluorescent channel.

8. The biological sample analyzer according to claim 7, wherein: The second indicator value is calculated by using at least the first indicator value, a previous second output value, and a current second output value, wherein the previous second output value is obtained in a completed verification process that has been performed before the verification process is performed, and the current second output value is obtained in the verification process (hereinafter referred to as "current verification process").

9. The biological sample analyzer according to claim 8, wherein: The second index value is calculated by further using a predetermined standard output value of each of the other fluorescent channels in addition to the first index value, the previous second output value, and the current second output value.

10. The biological sample analyzer according to claim 9, wherein The previous second standard output value acquired in the verification process that has been executed and the second standard output value acquired in the current verification process are used as the predetermined standard output value.

11. The biological sample analyzer according to claim 1, wherein The biological sample analyzer determines that the status of the analyzer is appropriate in a case where the first indicator value satisfies a predetermined first condition and the one or more second indicator values satisfy a predetermined second condition.

12. The biological sample analyzer according to claim 11, wherein The biological sample analyzer performs a gain adjustment process for each of the fluorescence channels after determining that the state of the analyzer is appropriate.

13. The biological sample analyzer according to claim 12, wherein: The biological sample analyzer records a gain of each of the fluorescence channels, the gain being set in the gain adjustment process.

14. The biological sample analyzer according to claim 1, wherein In a case where the first indicator value does not satisfy a predetermined first condition or the one or more second indicator values do not satisfy a predetermined second condition, the biological sample analyzer determines that the status of the analyzer is unsuitable.

15. The biological sample analyzer according to claim 14, wherein After determining that the status of the analyzer is inappropriate, the biological sample analyzer outputs one or more items selected from the group consisting of: a display prompting re-replication of fluorescent beads, a display prompting re-execution of QC processing, a display prompting inspection of a chip, a circulation pool or the analyzer, a display associated with changes or abnormalities in the fluorescent beads used in the verification process, a display associated with changes or abnormalities in the biological sample analyzer, and a display associated with incorrect application of standard value data.

16. The biological sample analyzer according to claim 1, wherein The biological sample analyzer performs the verification process by using fluorescent beads as particles.

17. The biological sample analyzer according to claim 16, wherein: The fluorescent beads emit fluorescent light covering the entire wavelength range of fluorescent light to be detected by the plurality of fluorescent channels.

18. The biological sample analyzer according to claim 1, wherein The biological sample analyzer includes a flow cytometer.

19. A biological sample analysis system comprising: an information processing unit that performs information processing by using signal intensity data of light generated by irradiating light to a flow channel in which particles flow, wherein The information processing unit performs verification processing for verifying the state of the analyzer by using at least one or more first index values and one or more second index values, wherein the first index values indicate the overall output level change in the plurality of fluorescent channels and the second index values indicate the output level change of each of the plurality of fluorescent channels.

20. A method for verifying a status of a biological sample analyzer, the method comprising: a verification process for verifying the state of the analyzer by using at least one or more first index values and one or more second index values generated from signal intensity data of light generated by irradiating the light to particles flowing in the flow channel, wherein The first index value indicates an overall output level variation in a plurality of fluorescent channels, and the second index value indicates an output level variation in each of the plurality of fluorescent channels.

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