Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/1012—Calibrating particle analysers; References therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/00584—Control arrangements for automatic analysers
  • G01N35/00594—Quality control, including calibration or testing of components of the analyser
  • G01N35/00693—Calibration
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/00584—Control arrangements for automatic analysers
  • G01N35/00594—Quality control, including calibration or testing of components of the analyser
  • G01N35/00712—Automatic status testing, e.g. at start-up or periodic

Definitions

• the present disclosure relates to a biological sample analyzer, a biological sample analysis system, and a method for verifying the state 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 light on particles flowing in a flow path, and a method for verifying the state of the biological sample analyzer.
• particle characteristics are measured by labeling a particle population such as cells, microorganisms, and liposomes with a fluorescent dye, irradiating each particle in the particle population with laser light, and measuring the intensity and/or pattern of fluorescence emitted from the excited fluorescent dye.
• a biological sample analyzer that performs such measurements is a flow cytometer.
• Another example of a biological sample analyzer configured to separate cells is a cell sorter.
• Flow cytometers and cell sorters can be configured to analyze multiple particles one by one by irradiating the particles flowing in a line through a flow path with laser light of a specific wavelength (excitation light) and detecting the fluorescent light and/or scattered light emitted by each particle.
• These devices convert the light detected by a photodetector into an electrical signal, digitize it, and perform statistical analysis to determine the characteristics of each particle, such as type, size, and structure.
• Patent Document 1 discloses a microparticle measuring device that includes a detection unit that detects light from microparticles and an information processing unit that corrects the value detected by the detection unit with a sensitivity correction coefficient to generate spectral data, the sensitivity correction coefficient being determined based on the value detected by the detection unit of light from fluorescent reference particles that emit fluorescence in a specified wavelength band.
• the same document also describes the application of the sensitivity correction coefficient to determining the possibility of using degraded fluorescent reference particles and the possibility of channel degradation.
• the state of the device may be calibrated or standardized using the fluorescence level obtained by irradiating fluorescent beads with light.
• fluorescent beads include Automatic Setup Beads (also known as ASB) and Align Check Beads.
• ASB Automatic Setup Beads
• Align Check Beads Align Check Beads.
• the fluorescence output at each specific wavelength obtained from the beads is used as a reference.
• the beads deteriorate, their fluorescence level changes significantly compared to before deterioration. Therefore, it is necessary to detect anomalies in the signal output from the beads. Also, incorrect data may be applied due to human error. If calibration or standardization is performed with such data applied, the device may be set up incorrectly.
• the output of only one of the multiple fluorescent channels may decrease due to optical or electrical reasons, for example. In these cases, it is necessary to detect device abnormalities.
• devices in which a chip or flow cell having a flow path through which light is irradiated onto particles in a sample is replaced. In such devices, it is conceivable that the signal output will change overall when the chip or flow cell is replaced. It is also important to detect such changes. Detecting such anomalies or changes with the device and/or detecting output anomalies in the fluorescent signal from the beads may be useful for better verification of the device status, particularly for calibration or standardization.
• the present disclosure aims to provide techniques for detecting changes or abnormalities in the devices or beads described above, particularly changes or abnormalities in the fluorescent signal output.
• the first index value may be a value calculated using at least the previous first output value obtained in an executed verification process executed before executing the verification process, and the current first output value obtained in the verification process (hereinafter referred to as the "current verification process").
• the first index value may be a value calculated using a ratio or difference between the previous first output value and the current first output value.
• the first index value may be a value calculated using a predetermined reference output value of the representative fluorescence channel in addition to the previous first output value and the current first output value. As the predetermined reference output value, a previous first reference output value obtained in the executed verification process and a first reference output value obtained in the current verification process may be used.
• the second index value may be a value calculated based on the first index value and second output values of the other fluorescence channels other than the representative fluorescence channel.
• the second index value may be a value calculated using at least the first index value, the previous second output value obtained in an executed verification process executed before executing the verification process, and the current second output value obtained in the verification process (hereinafter referred to as the "current verification process").
• the second index value may be a value calculated using the first index value, the previous second output value, and the current second output value, as well as predetermined reference output values for each of the other fluorescence channels.
• a previous second reference output value acquired in the executed verification process and a second reference output value acquired in the current verification process may be used.
• the biological sample analyzer can determine that the apparatus condition is appropriate when the first index value satisfies a predetermined first condition and the one or more second index values satisfy a predetermined second condition. After determining that the apparatus condition is appropriate, the biological sample analyzer can execute a gain adjustment process for each fluorescence channel. The biological sample analyzer can record the gain of each fluorescence channel that was set in the gain adjustment process. The biological sample analyzer can determine that the apparatus state is not appropriate if the first index value does not satisfy a predetermined first condition or if the one or more second index values do not satisfy a predetermined second condition.
• the biological sample analysis device may output one or more of the following: a display prompting the user to re-prepare the fluorescent beads, a display prompting the user to retry QC processing, a display prompting the user to inspect the chip, flow cell, or device, a display regarding changes or abnormalities in the fluorescent beads used in the verification process, a display regarding changes or abnormalities in the biological sample analysis device, and a display regarding the misapplication of reference value data.
• the biological sample analyzer can perform the verification process using fluorescent beads as the particles.
• the fluorescent beads may emit fluorescence over a range of fluorescent wavelengths that are detected by the multiple fluorescence channels.
• the biological sample analyzer may be a flow cytometer.
• the present disclosure also provides The flow path through which the particles flow is irradiated with light, and an information processing unit is provided for performing information processing using signal intensity data of light generated by the light irradiation of the flow path through which the particles flow, the information processing unit executes a verification process for verifying an apparatus state by using at least one or more first index values representing output level fluctuations across the plurality of fluorescence channels and one or more second index values representing output level fluctuations of each of the plurality of fluorescence channels;
• a biological sample analysis system is also provided.
• the present disclosure also provides a verification process for verifying a state of the device 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 light on particles flowing in the flow path; the first index value representing a power level variation across a plurality of fluorescent channels, and the second index value representing a power level variation for each of the plurality of fluorescent channels.
• a method for verifying the status of a biological sample analyzer is also provided.
• FIG. 4 is a schematic diagram illustrating fluctuations in the fluorescence level measured by the biological sample analyzer.
• FIG. 2 is a schematic diagram for explaining a determination method used to verify overall level and spectral variations.
• FIG. 1 is a diagram showing an example of the configuration of a biological sample analyzer according to the present disclosure.
• FIG. 4 is a flow diagram of an example verification process according to the present disclosure.
• 13 is a graph showing the results of index value calculation using judgment formulas A and B.
• 1 is a table showing an example configuration of a biological sample analyzer of the present disclosure.
• FIG. 1 is a diagram showing an example of a flow diagram of a bioparticle sorting process.
• FIG. 2 is a schematic enlarged view of a particle sorting section.
• FIG. 2 is a diagram illustrating a schematic configuration example of a control unit.
• First embodiment biological sample analyzer
• Basic Concept (1-1) Fluctuations in Overall Fluorescence Level and Fluctuations in a Partial Wavelength Range (1-2) Detection of Fluctuations in Overall Fluorescence Level and Fluctuations in a Partial Wavelength Range
• Configuration Example (3) Example of Verification Process (4)
• Example 1 Example of Verification Process
• Example 2 Actual Verification
• Third embodiment method of verifying a biological sample analyzer and a program for executing the method of verifying the biological sample analyzer
• the overall level fluctuation is a fluctuation in the output of the fluorescent signal over the entire wavelength range of the fluorescent spectrum.
• the overall level fluctuation can be caused, for example, when there is a defect or a fluctuation in the transmittance of a chip (e.g., a flow cell chip) having a flow path where light is irradiated to the beads, when there is an optical fluctuation in a light irradiating unit (e.g., a light emission system) that irradiates the flow path, or when there is a fluctuation in the laser power.
• a chip e.g., a flow cell chip
• a light irradiating unit e.g., a light emission system
• the overall level fluctuation is a fluctuation as shown by "High Level (gray line)” and “Standard (black line)” in the figure, and corresponds to a situation where the relationship of the output between each wavelength does not change, but a level difference occurs over the entire spectrum.
• the overall level fluctuation can be evaluated, for example, by the ratio (shown as Level Ratio in the figure) or difference in the signal output in the fluorescent channel ChA that detects the fluorescence of a specified wavelength.
• the fluorescence channel used to detect the overall level fluctuation is also referred to as "ChA".
• a biological sample analyzer such as a flow cytometer often has multiple fluorescence channels, and which of the multiple fluorescence channels is used to detect the overall level fluctuation may be appropriately selected by a person skilled in the art.
• the fluorescence channel used to detect the overall level fluctuation may be selected depending on the type or fluorescence characteristics of the beads used, or the configuration or characteristics of the device.
• the fluorescence channel ChA used to detect overall level fluctuations may also be called the "representative fluorescence channel.”
• the "representative" in this name is merely a denotation added for convenience in order to distinguish it from other fluorescence channels in terms of name.
• ChA is a fluorescence channel for detecting fluorescence on the short wavelength side of the wavelength range of fluorescence generated from the fluorescent beads, for example, fluorescence with a wavelength of 700 nm or less, preferably 650 nm or less, more preferably 600 nm or less. Fluorescence on the short wavelength side is particularly preferred as fluorescence representing overall fluorescence level fluctuations due to, for example, differences between chips or laser power anomalies.
• the fluorescence channel may be, for example, 300 nm or more, 350 nm or more, or 400 nm or more.
• ChA is preferably a fluorescence channel for detecting fluorescence in a wavelength range with a high signal intensity among the wavelength ranges of fluorescence generated from the fluorescent beads.
• ChA may be assigned to detect fluorescence in a wavelength range with the highest signal intensity among a plurality of fluorescence channels provided in the device, or may be assigned to detect fluorescence in a wavelength range with the second, third, fourth, or fifth highest signal intensity.
• a fluorescence channel that detects fluorescence in a wavelength range with a high signal intensity level to detect overall level fluctuations, the effect of noise on the verification process can be reduced.
• one or more fluorescence channels may be used to detect overall level variations. If one fluorescence channel is used for detection of global level fluctuations, that fluorescence channel may be ChA as described above. When two or more fluorescence channels are used for detecting overall level fluctuations, for example, in addition to ChA, one or more other fluorescence channels may be used. For example, the fluorescence level fluctuation in a certain wavelength region due to bead deterioration is noticeable in the long wavelength region and is not seen much in the short wavelength region. That is, the fluorescence channel assigned to detect the fluorescence in the short wavelength region is suitable for detecting the overall fluorescence level fluctuation.
• a fluorescence channel that detects fluorescence with a wavelength shorter than ChA for detecting the overall level fluctuation. That is, in the case where the biological sample analyzer has, in addition to ChA, one or more fluorescence channels that detect fluorescence with a shorter wavelength than ChA, any one or more of ChA and the one or more fluorescence channels may be used to detect the overall level fluctuation. Furthermore, in addition to ChA, all of the one or more fluorescence channels with a shorter wavelength than ChA may be used to detect the overall level fluctuation.
• the fluorescence level fluctuates as shown by "Standard (black line)” and “Spectral Deterioration (dashed line)” in the figure. That is, the fluorescence signal intensity fluctuates in a part of the wavelength range of the entire fluorescence spectrum generated by the beads. This fluctuation can be evaluated by the relative ratio (shown as Spectrum Ratio in the figure) between the output of each of the multiple fluorescence channels (especially the fluorescence channels other than ChA) and the output of ChA mentioned above.
• ChX the fluorescence channel used to detect fluctuations in the fluorescence level in a certain wavelength range of the entire fluorescence spectrum.
• ChX may mean a fluorescence channel other than ChA.
• the overall level and spectral variations described above are useful for detecting abnormalities or changes in the condition of the beads or biological sample analysis device or biological sample analysis system, or the chip or flow cell used in the device or system.
• the present disclosure provides a biological sample analysis device that verifies the device state based on the output level fluctuations across multiple fluorescent channels and the output level fluctuations of each of the multiple fluorescent channels.
• the present disclosure also provides a biological sample analysis system that performs the verification.
• the present disclosure also provides a biological sample analysis method that verifies the device state based on the output level fluctuations across multiple fluorescent channels and the output level fluctuations of each of the multiple fluorescent channels.
• the biological sample analysis device or the biological sample analysis system may include an information processing unit that performs information processing using light signal intensity data generated by irradiating light onto a flow path through which particles flow.
• the information processing unit may be configured to perform a verification process that verifies the device state using at least one or more first index values that represent output level fluctuations across multiple fluorescence channels and one or more second index values that represent output level fluctuations for each of the multiple fluorescence channels.
• the validation may involve one or more first index values representative of output level variations across the plurality of fluorescence channels.
• 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 the "first output value.”
• a second index value representing the output level fluctuation of each of the multiple fluorescence channels may also be used.
• the second index value may be a value calculated for each of the other fluorescence channels other than the representative fluorescence channel, for example, among the multiple fluorescence channels possessed by the biological sample analyzer.
• the second index value may be a value calculated based on the output value of each of the other fluorescence channels.
• the output value of the other fluorescence channel is also referred to as the "second output value.”
• the second index value may be a value calculated based on the first index value and the second output value.
• the particles used in the verification process according to the present disclosure may be, for example, fluorescent beads.
• the fluorescent beads may be appropriately selected by a person skilled in the art, but may preferably be particles that emit high levels of fluorescence over a wide wavelength range. Particularly preferably, the fluorescent beads emit fluorescence over the entire range of wavelengths of fluorescence detected by the multiple fluorescence channels. Examples of such fluorescent beads include the Automatic Setup Beads and Align Check Beads (both from Sony Group Corporation) described above.
• fluorescent beads including multiple types of particle groups having stepwise different fluorescence intensity levels, such as 8peakbeads and 6peakbeads may be used as such fluorescent beads.
• the fluorescent beads may be beads having a substantially uniform particle size.
• the particle size may be appropriately selected by a person skilled in the art depending on, for example, the size of the flow path, and may be, for example, 100 ⁇ m or less, 50 ⁇ m or less, or 30 ⁇ m or less.
• the particle size may be, for example, 0.1 ⁇ m or more, 0.3 ⁇ m or more, or 0.5 ⁇ m or more.
• the fluorescent beads may include one type or two or more types of beads.
• FIG. 2 shows a schematic example of a fluorescence spectrum generated by irradiating the beads described above with light.
• the horizontal axis of the figure is wavelength (Wavelength), and the vertical axis is signal level (LogHeight).
• a biological sample analyzer having four fluorescence channels Ch1, ChA, Ch2, and Ch3 is assumed, and the wavelength range of the fluorescence detected by each fluorescence channel (gray lined squares) is also shown in the figure.
• Detection of overall level fluctuations For example, in a biological sample analyzer in which a chip or flow cell having a flow path in which particles are irradiated with light is replaceable, when the chip or flow cell is replaced, a change in the fluorescence signal level may occur due to a difference in the transmittance of the chip or flow cell.
• the output value before the change and the output value after the change of the fluorescence channel ChA shown in the figure i.e., the output value of the fluorescence channel ChA related to the fluorescence generated by the light irradiation of particles flowing in the flow path of the chip or flow cell before the replacement and the output value of the fluorescence channel ChA related to the fluorescence generated by the light irradiation of particles flowing in the flow path of the chip or flow cell after the replacement
• the ratio or difference of these output values is used.
• the ratio or difference in the output value of ChA varies due to the difference in the laser power of the laser light incident on the chip or flow cell and the transmittance of the chip or flow cell, as well as the fluorescence level ratio for each bead. Therefore, a judgment formula for judging the presence or absence of an overall level fluctuation may be constructed using the reference value (adjustment target value, also called Gold Standard (GS)) in each fluorescence channel of the beads used in addition to the ratio or difference of the output value of ChA.
• GS Gold Standard
• An example of the judgment formula is the following judgment formula A.
• ChAold Indicates that this is the parameter for the previous ChA
• ChAnew Indicates that the parameters are for ChA.
• the reference value (Gold Standard) may be specified in advance, for example, by the manufacturer of the beads, and may be specified for each lot of beads.
• a reference biological sample analyzer may be prepared, and the reference value may be measured by the reference biological sample analyzer.
• the level ratio caused by, for example, different lots of beads is cancelled out, and it is possible to verify, for example, changes in laser power (such as an abnormality in laser power), changes in the characteristics of the chip or flow cell (such as defects (particularly scratches or dirt) that affect the transmittance of the chip or flow cell), or changes in beads.
• changes in laser power such as an abnormality in laser power
• changes in the characteristics of the chip or flow cell such as defects (particularly scratches or dirt) that affect the transmittance of the chip or flow cell
• changes in beads for example, changes in laser power (such as an abnormality in laser power), changes in the characteristics of the chip or flow cell (such as defects (particularly scratches or dirt) that affect the transmittance of the chip or flow cell), or changes in beads.
• a first index value such as (ChA ratio) may be used.
• the first index value is useful for detecting overall level fluctuations.
• the first index value may be calculated using first output values such as (ChAnew_Height) and (ChAold_Height).
• the first index value may be a value calculated using at least the previous first output value obtained in the executed verification process executed before executing 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 index value calculated using these first output values is useful for detecting overall level fluctuations.
• the first index value may be calculated using a ratio such as ⁇ (ChAnew_Height)/(ChAold_Height) ⁇ .
• the first index value may be calculated using a difference such as ⁇ (ChAnew_Height)-(ChAold_Height) ⁇ instead of the ratio. That is, the first index value may be a value calculated using a ratio or difference between the previous first output value and the current first output value, the ratio or difference being useful for obtaining a first index value suitable for detecting an overall level fluctuation.
• the first index value may be calculated using predetermined reference output values such as (ChAnew_GS) and (ChAold_GS) in addition to the first output value. That is, the first index value may be a value calculated using a predetermined reference output value of the representative fluorescence channel in addition to the previous first output value and the current first output value. Furthermore, the previous first reference output value (e.g., (ChAold_GS)) acquired in the executed verification process and the first reference output value (e.g., (ChAnew_GS)) acquired in the current verification process may be used as the predetermined reference output value.
• the predetermined reference output value it is possible to cancel the fluorescence level ratio caused by, for example, differences between fluorescent bead lots. This makes it possible to more appropriately detect fluorescence level fluctuations.
• the predetermined reference output value may be used as a ratio, such as ⁇ (ChAnew_GS) / (ChAold_GS) ⁇ , as described above. Also, instead of the ratio, a difference, such as ⁇ (ChAnew_GS) - (ChAold_GS) ⁇ , may be used. In other words, the first index value may be calculated using the ratio or difference between the previous first reference output value and the first reference output value obtained in the current verification process.
• a first index value based on the output value of one fluorescence channel may be used, but one first index value based on the output values of multiple fluorescence channels may be used, or multiple first index values based on each of the output values of multiple fluorescence channels may be used.
• the multiple fluorescence channels may be fluorescence channels assigned to detect fluorescence in a wavelength region that is less affected by bead deterioration.
• the multiple fluorescence channels may preferably include at least the above-mentioned ChA, and may further include one or more fluorescence channels that detect fluorescence at a wavelength shorter than that of the ChA.
• a first index value based on the output values of multiple fluorescence channels may be used to detect overall level fluctuations, for example, the average value of the output values of multiple fluorescence channels.
• a more specific example of the first index value in this embodiment is the average value of the output value of ChA and the output value of Ch1.
• An index value based on the ratio or difference of the average values may be used as the first index value.
• the first index value based on the ratio or difference of the average values may be (ChA1 ratio) calculated by the following judgment formula A1, for example.
• a ratio is used in the following judgment formula A1
• a difference may be used instead of the ratio, as described above for judgment formula A.
• the first index value calculated in this way may be used in a judgment using a standard described later.
• ChA1new_Height The average value of the median value of the height of ChA and the median value of the height of Ch1 obtained this time
• ChA1old_Height The average value of the previous median value of ChA's Height and the median value of Ch1's Height
• ChA1new_GS The average value of the Gold Standard of ChA and the Gold Standard of Ch1 obtained this time
• ChA1old_GS The average value of the previous Gold Standard of ChA and the Gold Standard of Ch1
• a plurality of first index values based on the output values of a plurality of fluorescence channels may be used to detect overall level fluctuations.
• the plurality of first index values in this embodiment include a first index value calculated based on the output value of ChA (hereinafter referred to as the "ChA first index value”) and a first index value calculated based on the output value of Ch1 (hereinafter referred to as the "Ch1 first index value").
• An example of the ChA first index value is the (ChA ratio) calculated by the above-mentioned judgment formula A.
• an example of the Ch1 first index value may be calculated by a formula similar to the judgment formula A, for example, the (Ch1 ratio) calculated by the following judgment formula 1.
• the larger first index value may be used in the determination using the standard described later.
• the smaller first index value may be used in the determination using the standard described below.
• ChX may mean a fluorescence channel other than ChA.
• ChA one of the four fluorescence channels used for verifying the overall level fluctuation
• ChX the remaining three fluorescence channels are indicated as ChX, where X is 1, 2, or 3. That is, the four fluorescence channels are indicated as ChA, Ch1, Ch2, and Ch3, respectively.
• the number of fluorescence channels that an apparatus has is N (N is any positive integer)
• the number of fluorescence channels ChX that the apparatus has is (N-1). That is, the fluorescence channels ChX of the apparatus can be expressed as Ch1, Ch2, ..., and Ch(N-1).
• the number N of fluorescence channels may be changed appropriately depending on the type of device, but may be, for example, 2 or more, particularly 3 or more, and more particularly 4 or more. Furthermore, there is no particular upper limit to the number N of fluorescence channels, but it may be, for example, 100 or less, 90 or less, or 80 or less.
• the biological sample analysis device or system may have, for example, 4 to 64 fluorescence channels, and may particularly have 4 to 32 fluorescence channels.
• a judgment formula for verifying the spectrum fluctuation may be constructed using a reference value (adjustment target value, also called Gold Standard (GS)) in ChX in addition to the ratio or difference in the output value of ChX.
• the judgment formula may include the ChA output ratio as a component.
• An example of the judgment formula is the following judgment formula B. Verification of the spectral variation using the judgment formula B may be performed for each of the fluorescence channels ChX.
• ChXold Indicates that this is the previous ChX parameter
• ChXnew Indicates that this is the newly acquired ChX parameter
• ChAold Indicates that this is the parameter for the previous ChA ChAnew: Indicates that the parameters are for ChA.
• the reference value (Gold Standard) may be specified in advance, for example, by the manufacturer of the beads, and may be specified for each lot of beads.
• a reference biological sample analyzer may be prepared, and the reference value may be measured by the reference biological sample analyzer.
• the variation in the spectral ratio caused by different bead lots is cancelled out, and only the spectral change can be verified, and for example, the presence or absence of degradation of the beads or the presence or absence of degradation in the output of a certain fluorescent channel in the device can be determined.
• the presence or absence of a change in the fluorescent spectrum can be verified purely (without being affected by the variation in the spectral ratio caused by different bead lots).
• a second index value such as (ChX output ratio) may be used.
• the second index value is useful for detecting level fluctuations of each of the other fluorescence channels.
• the second index value may be calculated using the second output values of other fluorescence channels, such as (ChXnew_Height) and (ChXold_Height) in addition to (ChA ratio).
• the second index value may be a value calculated using at least the first index value, the previous second output value obtained in the executed verification process executed before executing 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 calculated in this manner is useful for detecting relative level fluctuations of individual fluorescence channels.
• the second index value may be calculated using predetermined reference output values such as (ChXold_GS) and (ChXnew_GS) in addition to the first index value, the current second output value, and the previous second output value. That is, the second index value may be a value calculated using the first index value, the previous second output value, and the current second output value, as well as predetermined reference output values of the other fluorescent channels.
• the predetermined reference output value the previous second reference output value (e.g., (ChXold_GS)) acquired in the executed verification process and the second reference output value (e.g., (ChXnew_GS)) acquired in the current verification process may be used.
• predetermined reference output values it is possible to cancel the fluorescence level ratio caused by the difference between the lots of fluorescent beads, for example. This makes it possible to more appropriately detect the fluorescence level fluctuation.
• the above-mentioned judgment formula A in particular the (ChA ratio) calculated by the above-mentioned judgment formula A, may be incorporated into, for example, the following condition (standard A).
• condition standard A
• the presence or absence of an overall level fluctuation may be determined depending on whether such a condition is satisfied.
• the predetermined value ⁇ may be appropriately set by a person skilled in the art based on the measurement variation inherent to each device, the variation (in transmittance) of the chip or flow cell, and/or the variation of the device itself.
• a standard deviation may be calculated from a plurality of output value data obtained by a plurality of measurements, and ⁇ may be set based on the standard deviation.
• ⁇ may also be set based on a change in the beads or an abnormality in the transmittance of the chip or flow cell.
• ⁇ can be set as shown in the following specific example 1.
• the predetermined value ⁇ used in relation to the judgment formula A may be, for example, 0.01 to 0.3, and in particular, 0.05 to 0.25.
• (ChA1 ratio) may be used instead of (ChA ratio).
• (ChA ratio) a larger or smaller value among a plurality of first index values (e.g., a larger or smaller value among the ChA first index value and the Ch1 first index value) may be used.
• the above-mentioned judgment formula B in particular the (ChX output ratio) calculated by the above-mentioned judgment formula B, may be incorporated into, for example, the following condition (standard B).
• condition standard B
• the presence or absence of an overall level fluctuation may be determined depending on whether such a condition is satisfied.
• the predetermined value ⁇ may be appropriately set by a person skilled in the art based on the measurement variation inherent to each device, the variation (in transmittance) of the chip or flow cell, and/or the variation of the device itself.
• a standard deviation may be calculated from a plurality of output value data obtained by a plurality of measurements, and ⁇ may be set based on the standard deviation.
• ⁇ may also be set based on the change in beads.
• ⁇ can be set so as to satisfy the following specific example 2.
• ⁇ ⁇ ⁇ (value at which change in beads is judged to be abnormal) - Specific Example 2 ⁇ may be, for example, from 0.01 to 0.3, in particular from 0.05 to 0.25.
• the standard A may be relaxed or changed within the range of the specific example 1 described regarding the setting of ⁇ .
• either one of the upper limit and the lower limit of the standard A may not be limited. That is, the condition (1- ⁇ ) ⁇ (ChA ratio) or (ChA ratio) ⁇ (1+ ⁇ ) may be used.
• the setting of ⁇ in standard B may be relaxed or changed within the range of specific example 2 described above.
• either the upper limit or the lower limit in standard B may not be limited.
• the condition (1- ⁇ ) ⁇ (ChX output ratio) or (ChX output ratio) ⁇ (1+ ⁇ ) may be used.
• the variation is absolute and tends to be large for ⁇ in the judgment formula A.
• the judgment formula A is suitable for determining excessive change or deterioration of beads, or detecting large fluctuations in the transmittance of a chip or a flow cell.
• the variation in ⁇ in the judgment formula B is relative and tends to be small. Therefore, it is considered that the judgment formula B has a higher ability to detect bead abnormalities.
• standard B when the biological sample analyzer has multiple ChXs, standard B may be the same for each ChX. In another embodiment, when the biological sample analyzer has multiple ChXs, standard B may be set for each ChX, i.e., a different standard B may be set for each ChX. In other words, the value of ⁇ used in standard B may differ between ChXs.
• the biological sample analyzer may determine that the apparatus state is appropriate if the (ChA ratio) calculated according to the above-mentioned judgment formula A satisfies the above-mentioned standard A and the (ChX output ratio) calculated according to the above-mentioned judgment formula B satisfies the above-mentioned standard B. Satisfying the above-mentioned standard A means that there is no overall level fluctuation or it is within an acceptable range, and satisfying the above-mentioned standard B means that there is no relative level fluctuation in each of the fluorescence channels (particularly each of the fluorescence channels other than the representative fluorescence channel) or it is within an acceptable range.
• the biological sample analysis device of the present disclosure may determine that the device condition is appropriate when the first index value satisfies a predetermined first condition (e.g., standard A) and the one or more second index values satisfy a predetermined second condition (e.g., standard B).
• a predetermined first condition e.g., standard A
• a predetermined second condition e.g., standard B
• the biological sample analyzer may also execute a gain adjustment process for each fluorescence channel after determining that the device state is appropriate.
• the gain adjustment process may be executed so that the output value of each fluorescence channel is within a preset numerical range for each fluorescence channel. By adjusting the gain of each fluorescence channel after it has been confirmed that the device is in an appropriate state, it is possible to set an appropriate gain.
• the biological sample analyzer may be configured to record the gain of each fluorescence channel that was set in the gain adjustment process.
• the recording may be performed in the biological sample analyzer (particularly in a storage unit provided in the biological sample analyzer), or in a server to which the biological sample analyzer is connected. By recording the gains in this manner, the recorded gains can be used in the next verification process.
• the biological sample analyzer can determine that the apparatus state is inappropriate if the first index value does not satisfy a predetermined first condition or if the one or more second index values do not satisfy a predetermined second condition, thereby preventing a biological sample analyzer in an inappropriate state from performing biological sample analysis.
• the biological sample analysis device may be configured to output a display prompting the user to re-prepare the fluorescent beads, to retry the QC process, or to inspect the chip, flow cell, or device.
• the biological sample analyzer may be configured to output an indication regarding a change or abnormality in the fluorescent beads used in the verification process, or an indication regarding a change or abnormality in the biological sample analyzer, or an indication regarding a misapplication of reference value data. Such a display can prompt the user to check or adjust the device status.
• the biological sample analyzer 6100 shown in FIG. 3 includes a light irradiation unit 6101 that irradiates light onto the biological sample S flowing through a flow path C, a detection unit 6102 that detects light generated by irradiating the biological sample S with light, and an information processing unit 6103 that processes information related to the light detected by the detection unit.
• Examples of the biological sample analyzer 6100 include a flow cytometer and an imaging cytometer.
• the biological sample analyzer 6100 may include a fractionation unit 6104 that separates specific biological particles P from within the biological sample.
• An example of the biological sample analyzer 6100 that includes the fractionation unit is a cell sorter.
• the biological sample S may be a liquid sample containing biological particles.
• the biological particles are, for example, cells or non-cellular biological particles.
• the cells may be living cells, and more specific examples include blood cells such as red blood cells and white blood cells, and reproductive cells such as sperm and fertilized eggs.
• the cells may be directly collected from a specimen such as whole blood, or may be cultured cells obtained after culture.
• Examples of the non-cellular biological particles include extracellular vesicles, particularly exosomes and microvesicles.
• the biological particles may be labeled with one or more labeling substances (e.g., dyes (particularly fluorescent dyes) and fluorescent dye-labeled antibodies, etc.).
• the biological sample analyzer of the present disclosure may analyze particles other than biological particles, and beads, etc. may be analyzed for calibration, etc.
• the flow channel C is configured to allow the biological sample S to flow.
• the flow channel C can be configured to form a flow in which biological particles contained in the biological sample are arranged in a substantially straight line.
• the flow channel structure including the flow channel C may be designed to form a laminar flow.
• 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 may be appropriately selected by a person skilled in the art, and a known design may be adopted.
• the flow channel C may be formed in a flow channel structure such as a microchip (a chip having a flow channel on the order of micrometers) or a flow cell.
• the width of the flow channel C may be 1 mm or less, and in particular, 10 ⁇ m or more and 1 mm or less.
• the flow channel C and the flow channel structure including the flow channel C may be formed from a material such as plastic or glass.
• the biological sample analysis device of the present disclosure is configured so that the biological sample flowing through the flow path C, and in particular the biological particles in the biological sample, is irradiated with light from the light irradiation unit 6101.
• the biological sample analysis device of the present disclosure may be configured so that the interrogation point of light on the biological sample is in the flow path structure in which the flow path C is formed, or the interrogation point of light may be configured so that it is outside the flow path structure.
• An example of the former is a configuration in which the light is irradiated onto the flow path C in a microchip or flow cell. In the latter case, the light may be irradiated onto the biological particles after they have left the flow path structure (in particular its nozzle portion), and an example of this is a jet-in-air type flow cytometer.
• the light irradiation unit 6101 includes a light source unit that emits light and a light guide optical system that guides the light to an 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 the light emitted from each light source may be any of ultraviolet light, visible light, and infrared light.
• the light guide optical system includes optical components such as a beam splitter group, a mirror group, or an optical fiber.
• the light guide optical system may also include a lens group for collecting light, including, 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 may be configured to collect light irradiated from one or more different light sources to one irradiation point.
• the detection unit 6102 includes at least one photodetector that detects light generated by irradiating the bioparticles with light.
• the light to be detected is, for example, fluorescence or scattered light (for example, one or more of forward scattered light, back scattered light, and side scattered light).
• Each photodetector includes one or more light receiving elements, and has, for example, a light receiving element array.
• Each photodetector may include, as a light receiving element, one or more PMTs (photomultiplier tubes) and/or photodiodes such as APDs and MPPCs.
• 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 may also include an imaging element such as a CCD or CMOS.
• the detection unit 6102 may acquire an image of the bioparticle (for example, a bright field image, a dark field image, and a fluorescent image) using the imaging
• the detection unit 6102 includes a detection optical system that allows light of a specific detection wavelength to reach a corresponding photodetector.
• the detection optical system includes a spectroscopic section such as a prism or a diffraction grating, or a wavelength separation section such as a dichroic mirror or an optical filter.
• the detection optical system is configured to disperse light generated by irradiating a bioparticle with light, for example, and detect the dispersed light by a number of photodetectors greater than the number of fluorescent dyes with which the bioparticles are labeled.
• a flow cytometer that includes such a detection optical system is called a spectral flow cytometer.
• the detection optical system is also configured to separate light corresponding to the fluorescent wavelength range of a specific fluorescent dye from the light generated by irradiating a bioparticle with light, for example, and detect the separated light by a corresponding photodetector.
• the detection unit 6102 may also include a signal processing unit that converts the electrical signal obtained by the photodetector into a digital signal.
• the signal processing unit may include an A/D converter as a device that performs the conversion.
• the digital signal obtained by the conversion by the signal processing unit may be transmitted to the information processing unit 6103.
• the digital signal may be handled by the information processing unit 6103 as data related to light (hereinafter also referred to as "light data").
• the light data may be light data including, for example, fluorescent light data. More specifically, the light data may be light intensity data, and the light intensity may be light intensity data of light including fluorescent light (which may include feature quantities such as Area, Height, Width, etc.).
• the information processing unit 6103 includes, for example, a processing unit that executes processing of various data (for example, light data) and a storage unit that stores various data.
• the processing unit acquires light data corresponding to a fluorescent dye from the detection unit 6102
• the processing unit may perform a fluorescence leakage correction (compensation processing) on the light intensity data.
• the processing unit executes a fluorescence separation processing on the light data to acquire light intensity data corresponding to the fluorescent dye.
• the fluorescence separation processing may be performed according to, for example, the unmixing method described in JP 2011-232259 A.
• the processing unit may acquire morphological information of the bioparticles based on an image acquired by the image sensor.
• the storage unit may be configured to store the acquired light data.
• the storage unit may further be configured to store spectral reference data used in the unmixing processing.
• the information processing unit 6103 can execute a judgment as to whether to fractionate biological particles based on the optical data and/or morphological information. Then, the information processing unit 6103 can control the fractionation unit 6104 based on the result of the judgment, and the fractionation unit 6104 can fractionate the biological particles.
• the information processing unit 6103 may be configured to be able to output various data (e.g., optical data and images). For example, the information processing unit 6103 may output various data (e.g., two-dimensional plots, spectral plots, etc.) generated based on the optical data. The information processing unit 6103 may also be configured to be able to accept input of various data, for example, accepting gating processing on a plot by a user.
• the information processing unit 6103 may include an output unit (e.g., a display, etc.) or an input unit (e.g., a keyboard, etc.) for executing the output or input.
• the information processing unit 6103 may be configured as a general-purpose computer, for example, as an information processing device equipped with a CPU, RAM, and ROM.
• the information processing unit 6103 may be included in a housing in which the light irradiation unit 6101 and the detection unit 6102 are provided, or may be outside the housing.
• various processes or functions by the information processing unit 6103 may be realized by a server computer or cloud connected via a network.
• the sorting unit 6104 executes sorting of the bioparticles according to the determination result by the information processing unit 6103.
• the sorting method may be a method of generating droplets containing bioparticles by vibration, applying an electric charge to the droplets to be sorted, and controlling the direction of travel of the droplets by electrodes.
• the sorting method may be a method of controlling the direction of travel of the bioparticles in the flow path structure and sorting them.
• the flow path structure is provided with, for example, a control mechanism using pressure (spray or suction) or electric charge.
• An example of the flow path structure is a chip (for example, a chip described in JP 2020-76736 A) having a flow path structure in which a flow path C branches into a recovery flow path and a waste liquid flow path downstream thereof, and specific bioparticles are collected into the recovery flow path.
• FIG. 4 An example of a process for verifying the state of a biological sample analyzer using judgment formulas A and B is described below with reference to FIG. 4.
• the figure is a flow diagram of the process. Note that the process in this example is one of a series of QC (Quality Control, also called precision control or quality control) processes for the device, and involves determining whether there is a change or abnormality in the output (particularly the fluorescent output).
• QC Quality Control
• step S100 the biological sample analyzer starts the verification process.
• the biological sample analyzer acquires reference value data for the fluorescent beads to be used.
• the reference value data may include at least the ChA reference value and the ChX reference value used to calculate the above-mentioned (ChA ratio) and (ChX output ratio).
• the reference value data may be stored in advance in the biological sample analyzer (e.g., stored in a storage medium provided in the device), or the biological sample analyzer may acquire reference value data held by, for example, the manufacturer of the fluorescent beads via a network.
• the reference value data may be acquired via a network, for example, by using a code (e.g., a number or a one-dimensional or two-dimensional code) attached to a container in which the fluorescent beads are used.
• a code e.g., a number or a one-dimensional or two-dimensional code
• the biological sample analyzer performs optical adjustment.
• the optical adjustment may be, for example, adjustment of the laser light irradiation position by the light irradiation unit and/or the chip or flow cell.
• the method of the optical adjustment may be appropriately selected by a person skilled in the art depending on the configuration of the biological sample analyzer.
• step S103 the biological sample analyzer (particularly the information processing unit) determines whether the apparatus has passed QC processing at least once. That is, in this step, it may be determined whether the apparatus state has been determined at least once by QC processing to be acceptable for biological sample analysis. By making this determination and executing the following steps S104 to S107, it is possible to calculate determination values A and B using the gain settings that passed QC processing, and thus the most recent apparatus state is reflected in these determination values. This contributes to more appropriately performing apparatus state evaluation. If it is determined that the QC process has been passed at least once, the device proceeds to step S104. If it is determined that the QC process has not been passed at least once (i.e., the device has not passed the QC process even once, e.g., the device is being used for the first time), the device proceeds to step S109.
• step S104 the biological sample analyzer sets the light receiving element gain recorded in the previously passed QC process (i.e. the light receiving element gain recorded the most recently passed QC process) to each light receiving element.
• the light receiving element gain recorded in the previously passed QC process may be, for example, the gain recorded in step S111 described below. This allows the processing from step S105 onwards to be performed using the light receiving element gain that reflects the latest device status. This contributes to more appropriately evaluating the device status.
• the biological sample analyzer acquires singlet data relating to a specific particle from the event data acquired in step S105.
• the specific particle is a bead for calculating the determination value A and the determination value B, and may be determined in advance.
• the fluorescent beads for verifying the device state may include multiple types of fluorescent beads. In this case, it may be determined in advance which of these multiple types of fluorescent beads the event data of which fluorescent bead is to be used for the verification process according to the present disclosure.
• the biological sample analyzer can acquire only singlet data of a specific particle determined in advance from the event data by using predetermined software, which may be, but is not limited to, AutoGate.
• the biological sample analyzer may further acquire a signal intensity representative value of each fluorescence channel based on the acquired singlet data.
• the signal intensity representative value may be, for example, a representative value for Height or Area.
• the representative value may be a median value, a mode value, or an average value, and is particularly a median value.
• the signal intensity representative value is a median value of Height.
• the biological sample analyzer can obtain a median value of the height of each fluorescence 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 of ChA and ChX can be obtained.
• the biological sample analyzer calculates the ChA ratio and the ChX output ratio using the singlet data (particularly the representative signal intensity values of each fluorescent channel) of the specific particle acquired in step S106.
• the calculated ChA ratio and ChX output ratio are used in the determination process in step S108.
• the (ChX output ratio) can be calculated for each ChX. For example, if the biological sample analyzer has three fluorescence channels, Ch1, Ch2, and Ch3, as described above as the fluorescence channels ChX, then the (Ch1 output ratio), (Ch2 output ratio), and (Ch3 output ratio) are calculated.
• the biological sample analyzer has (N-1) fluorescence channels, Ch1, Ch2, ..., and Ch(N-1), as the fluorescence channels ChX, then the (Ch1 output ratio), (Ch2 output ratio), ..., and (Ch(N-1) output ratio) are calculated.
• the (ChA ratio) may be calculated, for example, by using the above-mentioned judgment formula A.
• the values used as the components of the judgment formula A are as follows.
• the median value of the Height of ChA recorded in the previously passed QC process is used as (ChAold_Height).
• the standard value (GoldStandard) of ChA recorded in the previously passed QC process is used as (ChAold_GS).
• the median value of the Height of ChA obtained in step S106 is used as (ChAnew_GS).
• the reference value (GoldStandard) of ChA acquired in step S101 is used as (ChAnew_GS).
• the (ChX output ratio) may be calculated, for example, by using the above-mentioned judgment formula B.
• the values used as the components of the judgment formula B are as follows.
• the median value of the Height of ChX obtained in step S106 is used as (ChXnew_Height).
• As (ChAnew_Height) the median value of the Height of ChA obtained in step S106 is used.
• the median value of the Height of ChX recorded in the previously passed QC process is used as (ChXold_Height).
• the median value of the Height of ChA recorded in the previously passed QC process is used as (ChAold_Height).
• the reference value (GoldStandard) of ChX acquired in step S101 is used as (ChXnew_GS).
• the reference value (GoldStandard) of ChA acquired in step S101 is used as (ChAnew_GS). If the (ChA ratio) has already been calculated using the judgment formula A, the calculated (ChA ratio) may be used. If the (ChA ratio) is used, as is clear from the judgment formula B, some of the above values may not be used.
• step S108 the biological sample analyzer (particularly the information processing unit) determines whether the (ChA ratio) satisfies standard A, and whether the (ChX output ratio) satisfies standard B. If the device has multiple fluorescence channels as ChX, it is determined whether the (ChX output ratio) of each of the multiple fluorescence channels satisfies standard B set for each fluorescence channel. If the (ChA ratio) meets standard A and the (ChX output ratio) meets standard B (if there are multiple ChXs, all ChXs meet standard B), the biological sample analyzer proceeds to step S109. In other words, if all fluorescence channels meet the standards set for each fluorescence channel, the biological sample analyzer proceeds to step S109.
• step S113 If the (ChA ratio) does not meet standard A or the (ChX output ratio) does not meet standard B (if there are multiple ChXs, one or more of the multiple ChXs do not meet standard B), the biological sample analyzer proceeds to step S113. In other words, if at least one fluorescence channel does not meet the standard set for that channel, the biological sample analyzer proceeds to step S113.
• the standards A and B used in step S108 may be the standards described above.
• step S109 the biological sample analyzer flows the fluorescent beads and acquires a predetermined number of event data.
• the number may be, for example, but is not limited to, 10,000, and may be, for example, 1,000 to 1,000,000, 2,000 to 500,000, or 5,000 to 100,000.
• the event data may be acquired by detecting fluorescence generated by irradiating light onto each of the measurement target particles (fluorescent beads) flowing through the flow path, as will be described elsewhere in this specification.
• the acquisition of event data in step S109 is preferably performed in the same manner as in step S105.
• the supply rate of the particle-containing sample and the wavelength and intensity of the irradiated light in step S109 may be the same as those in step S105.
• step S110 the biological sample analyzer (particularly the information processing unit) acquires singlet data relating to a 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.
• the biological sample analyzer can acquire only the singlet data of the specific particle from the event data by using predetermined software, which may be, but is not limited to, AutoGate.
• the biological sample analyzer obtains the median value of the height for each fluorescence channel based on the acquired singlet data, i.e., the median value of the height for ChA and the median value of the height for each ChX.
• the biological sample analyzer adjusts the gain of each fluorescence channel so that the acquired median value of the height of each fluorescence channel falls within a predetermined range.
• the predetermined range may be set in advance for each fluorescence channel.
• the biological sample analyzer records the median value of the Height of each fluorescence channel after the gain adjustment in step S110, the reference value (GoldStandard) of each fluorescence channel, and the light receiving element gain of each fluorescence channel.
• These data are preferably recorded after passing a QC process such as an instrument condition verification process based on the robust coefficient of variation (rCV) of each fluorescence channel, i.e., after it is confirmed that the instrument condition satisfies a predetermined quality control condition, these data are recorded.
• the data recorded in this manner may be used to perform the verification process according to the present disclosure next time, particularly in steps S104 and S107.
• the biological sample analyzer ends the verification process according to the present disclosure, meaning that there is no variation in the overall fluorescence level or it is within a predetermined acceptable range, and there is no variation in the fluorescence level of each fluorescence channel or it is within a predetermined acceptable range. Therefore, upon completion of the analysis, the biological sample analyzer may display, for example on a display device, a message indicating that there is no variation in the overall fluorescence level or that it is within an acceptable range and that there is also no variation in the fluorescence level of each fluorescence channel or that it is within an acceptable range. Furthermore, upon completion of the analysis, the biological material analyzer may cause the display device to display a message prompting the user to perform an operation for carrying out biological sample analysis. After this is completed, the biological sample analyzer can perform biological sample analysis using the light receiving element gain recorded in step S111.
• the biological sample analyzer terminates the verification process according to the present disclosure, meaning that the overall fluorescence level variation is unacceptable, or the fluorescence level variation in at least one fluorescence channel is unacceptable, or both. Therefore, following the termination, the biological sample analyzer may display, for example, on a display device, a message indicating that the overall fluorescence level fluctuation is unacceptable, or that the fluorescence level fluctuation of at least one fluorescence channel is unacceptable, or both. That is, after the termination, the biological sample analyzer may output, for example, on a display device, a message indicating that the verification process was not passed. This can prompt the user to inspect or replace the biological sample analyzer, chip, or flow cell.
• the biological sample analyzer may change the display content output in step S113 depending on the result of the determination in step S108. For example, if the (ChA ratio) according to the judgment formula A does not satisfy the standard A, this may mean that there is a change in the overall fluorescence level. Therefore, in this case, the biological sample analyzer may output an indication indicating that there is a change in the overall fluorescence level. In this case, an indication suggesting a change or abnormality in the chip or flow cell, an indication suggesting a change or abnormality in the light irradiation unit, or an indication suggesting a change or abnormality in the laser power may be output.
• the biological sample analyzer may output a display indicating the fluorescent channel that does not satisfy standard B. Also, in this case, a display may be output indicating that the fluorescent beads have deteriorated.
• a cell sorter is used as the biological sample analysis device, but the verification process according to the present disclosure may be performed not only in a flow cytometer having a fractionation section such as a cell sorter, but also in a flow cytometer that does not have a fractionation section.
• fluorescent beads ASB (Sony Group Corporation) are used as particles used for the verification process.
• the fluorescent beads ASB contain two types of fluorescent particles, one type being fluorescent particles having a particle diameter of 3 ⁇ m and the other type being fluorescent particles having a particle diameter of 10 ⁇ m.
• the verification process is performed using singlet data of the fluorescent particles having a particle diameter of 3 ⁇ m out of these two types of fluorescent particles, but the verification process according to the present disclosure may also be performed using fluorescent particles having a particle diameter of 10 ⁇ m. In addition, the verification process according to the present disclosure may also be performed using other fluorescent beads.
• the cell sorter was configured to perform the verification process described with reference to Figure 3. Details of each step in the verification process are as follows:
• step S100 the cell sorter begins the verification process in accordance with this disclosure.
• step S103 the cell sorter determines whether the cell sorter has passed one or more QC processes, which are a series of processes for managing the performance of the device, including the fluorescence level variation verification process according to the present disclosure as well as other verification processes (e.g., device status verification process based on rCV, etc.). If it is determined that the QC process has been passed at least once, the cell sorter advances the process to step S104. If it is determined that the QC process has not been passed at least once, the cell sorter proceeds to step S109.
• QC processes are a series of processes for managing the performance of the device, including the fluorescence level variation verification process according to the present disclosure as well as other verification processes (e.g., device status verification process based on rCV, etc.). If it is determined that the QC process has been passed at least once, the cell sorter advances the process to step S104. If it is determined that the QC process has not been passed at least once, the cell sorter proceeds to step S109.
• step S104 the cell sorter sets the light receiving element gain recorded in the previously passed QC process for each fluorescence channel.
• step S105 the cell sorter flows the fluorescent beads into the flow path where light irradiation is performed, and acquires 10,000 event data.
• step S106 the cell sorter uses AutoGate to acquire singlet data of 3 ⁇ m fluorescent beads from the 10,000 event data, and then uses the singlet data to calculate the median value of the height of each fluorescence channel.
• the median value of the height of the singlet data is calculated for each of all the fluorescence channels provided in the cell sorter.
• the cell sorter calculates (ChA ratio) according to the above-mentioned judgment formula A using the median value of the height of that fluorescence channel and the reference value (Gold Standard) of that fluorescence channel recorded when the QC process was passed last time, the reference value (Gold Standard) of that fluorescence channel acquired in step S101, and the median value of the height of that fluorescence channel acquired in step S106.
• the cell sorter calculates a (ChX output ratio) according to judgment formula B using the median value of the height of each fluorescence channel and the reference value (GoldStandard) of the fluorescence channel recorded when the QC process was passed the previous time, the reference value (GoldStandard) of each fluorescence channel acquired in step S101, and the median value of the height of each fluorescence channel acquired in step S106.
• step S108 the cell sorter determines whether the judgment value (ChA ratio) satisfies standard A and whether the judgment value (ChX output ratio) satisfies standard B.
• the judgment value (ChX output ratio) is calculated for each fluorescence channel, and the Ch1 output ratio, Ch2 output ratio, and Ch3 output ratio are calculated for each of the three fluorescence channels Ch1 to Ch3.
• Standard B is also set for each fluorescence channel.
• the standards used to judge the Ch1 output ratio, Ch2 output ratio, and Ch3 output ratio are also referred to as standard B1, standard B2, and standard B3, respectively.
• step S108 if the (ChA ratio) meets standard A and the (ChX output ratio) meets standard B (i.e., if the Ch1 output ratio meets standard B1, the Ch2 output ratio meets standard B2, and the Ch3 output ratio meets standard B3), the cell sorter proceeds to step S109.
• step S108 if the (Ch A ratio) does not satisfy standard A or the (Ch X output ratio) does not satisfy standard B (i.e., if one or more of the Ch1 output ratio, Ch2 output ratio, and Ch3 output ratio do not satisfy the set standard), the cell sorter proceeds to step S113.
• step S109 the cell sorter flows the fluorescent beads and acquires 10,000 event data.
• the event data is acquired in the same manner as in step S105.
• step S110 the cell sorter acquires singlet data of fluorescent beads having a particle size of 3 ⁇ m from the event data acquired in step S109, in the same manner as in step S106. Furthermore, in step S110, the cell sorter acquires the median value of the height for each fluorescence channel based on the acquired singlet data. Then, in step S110, the cell sorter adjusts the light receiving element gain of each fluorescence channel so that the acquired median value of the height of each fluorescence channel falls within a predetermined range.
• step S111 if the cell sorter passes a series of QC processes, such as device state verification processes based on the rCV (robust coefficient of variation) of each fluorescence channel, it records the median value of the height of each fluorescence channel after the gain adjustment in step S110, the reference value (GoldStandard) of each fluorescence channel, and the light receiving element gain of each fluorescence channel.
• a series of QC processes such as device state verification processes based on the rCV (robust coefficient of variation) of each fluorescence channel.
• step S112 the cell sorter ends the verification process according to the present disclosure. This means that there is no variation in the overall fluorescence level or it is within a predetermined acceptable range, and there is no variation in the fluorescence level of each fluorescence channel or it is within a predetermined acceptable range. After this end, the cell sorter can perform biological sample analysis using the light receiving element gain recorded in step S111.
• step S113 the cell sorter terminates the verification process according to the present disclosure.
• This termination means that the overall fluorescence level fluctuation is unacceptable, or that the fluorescence level fluctuation in at least one fluorescence channel is unacceptable, or both. Therefore, unlike step S112, no biological sample analysis is performed after this termination.
• the verification process was carried out under conditions A to E shown in Table 1 below. Among these conditions, the verification process under conditions A to C was carried out as follows. 1. Using undegraded AP05, the GoldStandard of AP05 was applied and the verification process described in Example 1 above was performed, and the QC was passed. 2. The verification process described in Example 1 above was carried out using the verification beads (AP05 that had been left at room temperature for a long time under condition A, and AN183 that had not deteriorated under conditions B and C) and GoldStandard as shown in the table. 3. The calculation results of the judgment formula A and the judgment formula B in the verification process in 2 above were confirmed.
• Condition C A verification process was performed on AN183 using the GS of AN183. Under these conditions, the correspondence between the GS and the fluorescent beads was appropriate, and the beads were used normally. Under these conditions, no abnormalities were detected for either judgment formula A or B. In other words, no abnormalities were detected when appropriate beads were used.
• condition D In the verification process under condition D, the laser light output irradiated to the beads is reduced by 30% compared to the QC performed before the verification process, resulting in an overall decrease in the fluorescence level. This decrease was detectable by decision formula A. However, this decrease was not detected by decision formula B.
• Condition E The verification process for condition E used data from 10 ⁇ m beads, but the QC performed prior to the verification process used data from 3 ⁇ m beads. Due to the different types of beads used in the verification process, the fluorescence levels differed across the spectrum and the fluorescence levels of each fluorescence channel also differed. These differences were detectable by both judgment formula A and judgment formula B.
• the verification process according to the present disclosure can appropriately evaluate the device state for all five conditions. That is, the presence or absence of fluctuations in the fluorescence level can be appropriately verified by a biological sample analyzer (particularly a flow cytometer) that executes the verification process according to the present disclosure.
• the verification process can detect variations in the overall fluorescence level and can also detect variations in the fluorescence level in each fluorescence channel. It can also determine whether there is an overall fluorescence level variation or an individual fluorescence channel variation. This can detect, for example, degradation of the beads, application of erroneous data, anomalies or changes in the optical system, or anomalies or changes in the flow system (particularly the chip or flow cell having the flow path through which the particles are irradiated).
• the biological sample analysis device of the present disclosure may be configured as a biological particle sorting device, for example as a cell sorting device.
• the biological particle sorting device may be a device that performs analysis and/or sorting of biological particles in a microchip without forming droplets.
• the biological particle sorting device may be configured to perform the verification process described above. This embodiment will be described below with reference to Figures 6 and 7.
• FIG. 6 shows a schematic diagram of an example of the configuration of a microchip for bioparticle sorting and a bioparticle analyzer including the microchip.
• FIG. 7 shows an example of a flow diagram of the bioparticle sorting operation using the bioparticle analyzer.
• FIG. 6 has a sample liquid flow path 152 and a sheath liquid flow path 154 that joins with the sample liquid flow path 152 at a joining portion 162.
• the microchip 150 for sorting bioparticles is further provided with a sample liquid inlet 151 and a sheath liquid inlet 153.
• a part of the sheath liquid flow path 154 is shown by a dotted line.
• the part shown by the dotted line is located at a lower position (a position shifted in the optical axis direction described later) than the sample liquid flow path 152 shown by the solid line, and the flow paths shown by the dotted line and the flow path shown by the solid line do not communicate with each other at the position where they intersect.
• Fig. 6 has a sample liquid flow path 152 and a sheath liquid flow path 154 that joins with the sample liquid flow path 152 at a joining portion 162.
• the microchip 150 for sorting bioparticles is further provided with a sample liquid inlet 151 and a shea
• the sample liquid flow path 152 is shown to bend twice between the sample liquid inlet 151 and the junction 162, but this is to make it easy to distinguish between the sample liquid flow path 152 and the sheath liquid flow path 154.
• the sample liquid flow path 152 may be configured linearly between the sample liquid inlet 151 and the junction 162 without bending in this way.
• sample liquid containing bioparticles is introduced into the sample liquid flow path 152 from the sample liquid inlet 151, and sheath liquid not containing bioparticles is introduced into the sheath liquid flow path 154 from the sheath liquid inlet 153.
• the bioparticle sorting microchip 150 has a junction flow path 155 having a junction portion 162 at one end.
• the sample liquid and the sheath liquid join at the junction 162 and flow through the junction flow channel 155 toward the particle sorting section 157.
• the sample liquid and the sheath liquid join at the junction 162 to form a laminar flow in which the sample liquid is surrounded by the sheath liquid.
• the bioparticles are arranged in a line in the laminar flow.
• the sample liquid flow channel 152 and the two sheath liquid flow channels 154 join at the junction 162, and the flow channel structure has a junction flow channel 155 with the junction 162 at one end, forming a laminar flow including the bioparticles flowing in a line. This makes it easier to distinguish between light generated by light irradiation of one bioparticle and light generated by light irradiation of another bioparticle in the light irradiation in the detection region 156 described below.
• the bioparticle sorting microchip 150 further has a particle sorting section 157 at the other end of the junction flow channel 155.
• Fig. 8 shows an enlarged view of the particle sorting section 157.
• the junction flow channel 155 is connected to a bioparticle recovery flow channel 159 via a connection flow channel 170.
• the junction flow channel 155, the connection flow channel 170, and the bioparticle recovery flow channel 159 may be coaxial.
• a flow is formed that passes from the junction flow channel 155 through the connection flow channel 170 and enters the bioparticle collection flow channel 159, and the particles to be collected are collected into the bioparticle collection flow channel 159.
• the particles to be collected flow into the bioparticle collection flow channel 159 through the connection flow channel 170.
• the bioparticle recovery channel 159 is formed so as to extend linearly from the particle sorting section 157, make a U-turn, and reach the same plane as the plane formed by the sample liquid inlet 151 and the sheath liquid inlet 153.
• the liquid flowing through the bioparticle recovery channel 159 is discharged from the recovery channel end 163 to the outside of the chip.
• the two branch channels 158 also extend linearly from the particle sorting section 157, make a U-turn, and are formed so as to reach the same plane as the plane formed by the sample liquid inlet 151 and the sheath liquid inlet 153.
• the liquid flowing through the branch channel 158 is discharged from the branch channel end 166 to the outside of the chip.
• the way in which the bioparticle recovery channel 159 is displayed is changed to a solid line and a dotted line at the U-turn point. This change indicates that the position in the optical axis direction changes along the way.
• the bioparticle recovery channel 159 and the branch channel 158 do not communicate with each other at the point where they intersect with the branch channel 158.
• Both the recovery flow path end 163 and the two branch flow path ends 166 are formed on the surface on which the sample liquid inlet 151 and the sheath liquid inlet 153 are formed. Furthermore, the introduction flow path inlet 164 for introducing liquid into the introduction flow path 161 is also formed on this surface. In this manner, in the bioparticle sorting microchip 150, the inlets for introducing liquid and the outlets for discharging liquid are all formed on one surface. This makes it easier to attach the chip to the bioparticle analysis device 100. For example, compared to a case in which the inlets and/or outlets are formed on two or more surfaces, it becomes easier to connect the flow paths provided in the bioparticle analysis device 100 to the flow paths of the bioparticle sorting microchip 150. As shown in FIGS.
• the bioparticle sorting microchip 150 has an introduction flow path 161 for introducing a liquid into the connection flow path 170 .
• introduction flow path 161 for introducing a liquid into the connection flow path 170 .
• the microchip 150 for sorting bioparticles has two branch channels 158 connected to the other end of the junction channel 155.
• the junction channel may be branched into the connection channel and the at least one branch channel. Bioparticles other than the target particles for collection do not enter the bioparticle collection channel 159 but flow to one of the two branch channels 158 .
• the bioparticle sorting microchip 150 constitutes part of a bioparticle analysis device 100 that includes, in addition to the microchip, a light irradiation unit 101, a detection unit 102, and a 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) above, respectively, and the explanations thereof also apply to this configuration example.
• the control unit 103 of the bioparticle analysis device 100 can include a signal processing unit 104, a determination unit 105, and a sorting control unit 106, as shown in FIG. 9.
• the bioparticle sorting operation using the bioparticle sorting microchip 150 described above includes a flow process S101 in which a liquid containing bioparticles is flowed into the junction flow channel 155, a determination process S102 in which it is determined whether the bioparticles flowing through the junction flow channel 155 are particles to be collected, and a recovery process S103 in which the particles to be collected are collected into the bioparticle collection flow channel 159.
• a flow process S101 in which a liquid containing bioparticles is flowed into the junction flow channel 155
• a determination process S102 in which it is determined whether the bioparticles flowing through the junction flow channel 155 are particles to be collected
• a recovery process S103 in which the particles to be collected are collected into the bioparticle collection flow channel 159.
• the sample liquid containing bioparticles and the sheath liquid not containing bioparticles are introduced from the sample liquid inlet 151 and the sheath liquid inlet 153 into the sample liquid flow path 152 and the sheath liquid flow path 154, respectively.
• the sample liquid and the sheath liquid join at the joining portion 162 to form a laminar flow in which the sample liquid is surrounded by the sheath liquid, for example.
• the bioparticles are aligned substantially in a line in the laminar flow. That is, in the flowing step S101, a laminar flow including the bioparticles flowing substantially in a line can be formed.
• the liquid containing the bioparticles is passed through the junction flow channel 155, particularly as a laminar flow.
• the liquid flows through the junction flow channel 155 from the junction portion 162 toward the particle sorting portion 157.
• the determination step S102 it is determined whether the bioparticles flowing through the junction flow path 155 are particles to be collected. This determination can be made by the determination unit 105.
• the determination unit 105 can make this determination based on light generated by the light irradiation unit 101 irradiating the bioparticles with light.
• the signal processing unit 104 included in the control unit 103 may process the waveform of the digital electric signal obtained by the detection unit 102 to generate information (data) regarding the characteristics of the light used for the judgment by the judgment unit 105. As the information regarding the characteristics of the light, the signal processing unit 104 may acquire, for example, one, two, or three of the width, height, and area of the waveform from the waveform of the digital electric signal.
• the information regarding the characteristics of the light may include, for example, the time when the light was detected.
• the above processing by the signal processing unit 104 may be performed particularly in an embodiment in which the scattered light and/or fluorescence is detected.
• the judgment unit 105 included in the control unit 103 judges whether the bioparticles flowing in the flow path are particles to be collected based on the light generated by irradiating the bioparticles with light.
• the bioparticles determined to be collection target particles in the determination step S102 are collected into the bioparticle collection channel 159.
• the collection step S103 is performed in the particle sorting section 157 in the microchip 150.
• the particle sorting section 157 the laminar flow flowing through the junction channel 155 is separated into two branch channels 158.
• the particle sorting section 157 shown in FIG. 6 has two branch channels 158, but the number of branch channels is not limited to two.
• the particle sorting section 157 may be provided with, for example, one or more (e.g., two, three, or four) branch channels.
• the branch channels may be configured to branch in a Y-shape on one plane as shown in FIG.
• the collection target particles are collected into the bioparticle collection channel through the connection channel due to pressure fluctuations in the bioparticle collection channel 159.
• the recovery may be performed, for example, by generating a negative pressure in the bioparticle recovery channel 159 as described above.
• the negative pressure may be generated by, for example, the actuator 107 (particularly a piezoelectric actuator) attached to the outside of the microchip 150, deforming the wall that defines the bioparticle recovery channel 159.
• the flow that enters the bioparticle recovery channel 159 may be formed by the negative pressure.
• the actuator 107 may be attached to the outside of the microchip 150 so as to be able to deform the wall of the bioparticle recovery channel 159.
• the deformation of the wall may change the inner space of the bioparticle recovery channel 159, thereby generating a negative pressure.
• the actuator 107 may be, for example, a piezoelectric actuator.
• connection flow path 170 In order to prevent bioparticles that are not the target particles for recovery from entering the bioparticle recovery flow path 159 through the connection flow path 170, the connection flow path 170 is provided with an introduction flow path 161. A liquid is introduced from the introduction flow path 161 into the connection flow path 170. The introduction of the liquid fills the connection flow path 170 with the liquid. Furthermore, a flow from the connection flow path 170 toward the junction flow path 155 is formed by a part of the liquid, so that bioparticles other than the target particles for recovery are prevented from entering the bioparticle recovery flow path 159.
• the liquid that forms the flow from the connection flow path 170 toward the junction flow path 155 flows through the branch flow path 158 in the same manner as the liquid, without flowing through the junction flow path 155, due to the flow of the liquid flowing through the junction flow path 155 flowing to the branch flow path 158.
• the remainder of the liquid introduced into the connection flow path 170 flows into the bioparticle recovery flow path 159.
• the bioparticle recovery flow path 159 can be filled with the liquid.
• the flow that has flowed into the branch flow channel 158 can be discharged to the outside of the microchip at the branch flow channel end 160.
• the recovery target particles that have been recovered into the bioparticle recovery flow channel 159 can be discharged to the outside of the microchip at the recovery flow channel end 163.
• a container can be connected to the recovery flow channel end 163 via a flow channel such as a tube. The recovery target particles may be recovered in the container.
• the present disclosure also provides a biological sample analysis system configured to execute the verification process described in 1 above. That is, the system includes an information processing unit that executes information processing using light signal intensity data generated by irradiating light onto a flow path through which particles flow, and the information processing unit may be configured to execute a verification process that verifies the device state using at least a first index value that represents the output level fluctuation across multiple fluorescence channels and one or more second index values that represent the output level fluctuation of each of the multiple fluorescence channels.
• the biological sample analysis system may have the light irradiation unit and detection unit described in 1. above in addition to the information processing unit, and may further have a fractionation unit. These components may be provided in one device, or may be separated into multiple devices.
• the biological sample analysis system may have an information processing device configured as the information processing unit.
• the biological sample analysis system may be configured to include an analysis device having the light irradiation unit and the detection unit (as well as the fractionation unit) in addition to the information processing device. These devices may be connected to each other by wire or wirelessly. Furthermore, these devices may be connected via a network.
• the system may execute the verification process according to the present disclosure, for example, as follows: Details of each step are as described in 1 above.
• step S101 of the flow chart of Fig. 4 described in 1 above the information processing device acquires reference value data.
• step S102 the biological sample analyzing system (particularly the analyzing device) performs optical adjustment.
• step S103 the biological sample analyzing system (particularly the information processing device) determines whether the analyzer has passed the QC process one or more times.
• the biological sample analyzing system (particularly the information processing device) sets the light receiving element gain recorded in the previously passed QC process in each light receiving element of the analyzer.
• step S105 the biological sample analyzing system (particularly the analyzer) causes fluorescent beads to flow and acquires a predetermined number of event data items, which are then transmitted to the information processing device.
• step S106 the biological sample analyzing system (particularly the information processing device) acquires singlet data relating to a specific particle from the event data.
• step S107 the biological sample analyzing system (particularly the information processing device) calculates the ChA ratio and the ChX output ratio using the singlet data of the specific particle.
• step S108 the biological sample analyzing system (particularly the information processing device) determines whether (ChA ratio) satisfies standard A and whether (ChX output ratio) satisfies standard B.
• step S109 the biological sample analyzing system (particularly the analyzer) causes fluorescent beads to flow and acquires a predetermined number of event data items, which are then transmitted to the information processing device.
• step S110 the biological sample analyzing system (particularly the information processing device) acquires singlet data relating to a specific particle from the event data, and the information processing unit adjusts the gain of each fluorescence channel based on the acquired singlet data.
• step S111 the biological sample analysis system (particularly the information processing device), for example after passing a predetermined QC process, records the median value of the height of each fluorescence channel after the gain adjustment, the reference value of each fluorescence channel, and the light receiving element gain of each fluorescence channel.
• step S112 the biological sample analysis system (particularly the analysis device) completes the verification process according to the present disclosure. After completion, the system can perform biological sample analysis.
• step S113 the biological sample analyzing system (particularly the information processing device) ends the verification process according to the present disclosure. After the end of the process, the biological sample analyzing system may output, for example, on a display device, a message indicating that the verification process was not passed.
• the present disclosure also provides a method for verifying the state of a biological sample analyzer, which includes performing the verification process described in 1. above. That is, the verification method includes a verification process for verifying the state of the apparatus using at least a first index value and one or more second index values generated from signal intensity data of light generated by irradiating light on particles flowing in a flow path, the first index value representing output level fluctuations across multiple fluorescence channels, and the second index value representing output level fluctuations of each of the multiple fluorescence channels.
• the verification process may be performed, for example, as described in 1. above with reference to FIG. 4, and the description also applies to the verification method.
• the present disclosure also provides a program for executing the verification method in a biological sample analysis device or a biological sample analysis system.
• the program may be stored, for example, in a biological sample analysis device or an information processing device (particularly a storage unit), or may be stored in an information storage medium.
• the information storage medium may be, for example, an SD card, a micro SD card, a CD, a DVD, a flash memory, or a magnetic recording medium.
• the present disclosure may be configured as follows.
• [1] The flow path through which the particles flow is irradiated with light, and an information processing unit is provided for performing information processing using signal intensity data of light generated by the light irradiation of the flow path through which the particles flow, the information processing unit executes a verification process for verifying an apparatus state by using at least one or more first index values representing output level fluctuations across the plurality of fluorescence channels and one or more second index values representing output level fluctuations of each of the plurality of fluorescence channels; Biological sample analyzer.
• [2] The biological sample analyzer according to [1], wherein the first index value is a value calculated based on a first output value of a representative fluorescence channel that represents an output level of all of a plurality of fluorescence channels.
• the biological sample analyzer according to any one of [1] to [15], wherein the biological sample analyzer performs the verification process using fluorescent beads as the particles.
• the flow path through which the particles flow is irradiated with light, and an information processing unit is provided for performing information processing using signal intensity data of light generated by the light irradiation of the flow path through which the particles flow, the information processing unit executes a verification process for verifying an apparatus state by using at least one or more first index values representing output level fluctuations across the plurality of fluorescence channels and one or more second index values representing output level fluctuations of each of the plurality of fluorescence channels; Biological sample analysis system.
• a verification process for verifying a state of the device 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 light on particles flowing in the flow path; the first index value representing a power level variation across a plurality of fluorescent channels, and the second index value representing a power level variation for each of the plurality of fluorescent channels.

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• 2023
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