WO2015099116A1 - Procédé de comptage de particules et appareil de comptage de particules - Google Patents

Procédé de comptage de particules et appareil de comptage de particules Download PDF

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Publication number
WO2015099116A1
WO2015099116A1 PCT/JP2014/084488 JP2014084488W WO2015099116A1 WO 2015099116 A1 WO2015099116 A1 WO 2015099116A1 JP 2014084488 W JP2014084488 W JP 2014084488W WO 2015099116 A1 WO2015099116 A1 WO 2015099116A1
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pulse waveform
particle
counting
pulse
waveform
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PCT/JP2014/084488
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English (en)
Japanese (ja)
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勝敏 石塚
寛子 松林
正志 西森
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株式会社堀場製作所
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    • 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
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0266—Investigating particle size or size distribution with electrical classification
    • 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
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
    • 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
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/012—Red blood cells
    • 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
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/016—White blood cells
    • 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
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/018—Platelets
    • 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
    • G01N2015/1006—Investigating individual particles for cytology
    • 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
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
    • G01N2015/135—Electrodes
    • G01N2015/136—Scanning electrodes

Definitions

  • the present invention relates to a method for counting particles such as blood cells based on an electric resistance method or an optical method and a counting device therefor, and in particular, an individual obtained based on the electric resistance method or an optical method. It is related with the technique which determines whether the pulse waveform of particle
  • a particle counting device for counting particles (for example, blood cells such as red blood cells, white blood cells, and platelets) in a sample liquid
  • particles for example, blood cells such as red blood cells, white blood cells, and platelets
  • an electric resistance method also called an impedance method
  • a device configured to measure a voltage between the electrodes and obtain a pulsed electric signal (usually a pulse voltage) corresponding to the size of each particle is known.
  • a pulsed electric signal usually a pulse voltage
  • a sample liquid for example, a predetermined sample blood dispersed in a diluent
  • An aperture (small hole) 200 in which the cross-sectional area of the flow path becomes small like an orifice is provided, and electrodes 300 and 310 are provided on the upstream side and the downstream side of the aperture 200, respectively, so that particles passing through the aperture 200 (blood cells) ) Is measured based on a change in electrical characteristics between the electrodes.
  • the electrical resistance or impedance between the electrodes changes in a pulse shape. Therefore, when a voltage is applied from the constant current power source 400 to the pair of electrodes 300 and 310, the voltage also changes in accordance with the impedance change as described above, as shown in FIG. As an electrical signal).
  • the number of particles can be known by counting the number of pulse-like electric signals by, for example, the arithmetic unit 500 connected to a constant current power source.
  • the peak height (usually, the maximum voltage value of the pulse voltage) Vp of the pulse waveform of the pulsed electric signal when the particle passes through the aperture is proportional to the size of the particle.
  • the size of the particle volume can be determined.
  • the width W of the pulse waveform increases in proportion to the size of the particle, so that the size of the particle volume can be determined to some extent also by the width of the pulse waveform. Therefore, when counting particles by the electric resistance method, not only knowing the number of particles, but also how many blood cells are present in the sample solution based on the obtained pulse waveform.
  • the volume frequency distribution for can also be obtained.
  • an average red blood cell volume (Mean Corpuscular Volume) is used as one of indices for pathological diagnosis such as anemia.
  • the average red blood cell volume is an average value of the red blood cell volume obtained by creating a frequency distribution from the height information (volume information) of the pulse waveform of each red blood cell measured by the electric resistance method.
  • Patent Documents 1 to 3 describe in detail the configuration of the apparatus such as a flow path, aperture, and electrode arrangement for counting blood cells based on the electrical resistance method.
  • the flow path on the downstream side of the aperture is branched into two by an original configuration, and in the device of Patent Document 3, a pair of electrodes is provided on the downstream side of the aperture by the unique configuration.
  • the basic principle of the electrical resistance method for determining the size of blood cells is the same as described above.
  • FIG. 12 a particle counter based on an optical technique is also known.
  • a photoelectric sensor in the example shown, a light projecting element E1
  • a pair of light receiving elements E2 When each particle X10 in the sample solution flowing through the pipe 201 crosses the light L1, the amount of received light changes (decrease from 100% received amount to cut off to return to 100% received amount).
  • a pulsed electric signal corresponding to the size of each particle is obtained.
  • Vp is the height of the pulse.
  • the time required for the particle to cross the light L1 becomes long, so that the width W of the obtained signal pulse becomes large. Therefore, in the case of such an optical method, as in the electrical resistance method described above, based on the obtained pulse waveform, how many blood cells are present in the sample solution at what frequency. The volume frequency distribution for can be obtained.
  • the pulse waveform schematically shown as the pulse voltage in FIG. 11B is a waveform in an ideal passing state in which one particle passes through the center of the aperture linearly at a constant speed (hereinafter referred to as “normal waveform”). ").
  • normal waveform a waveform in which one particle passes through the center of the aperture linearly at a constant speed
  • FIG. 13 (b) shows various passing states and disturbances in which a plurality of blood cells pass through the aperture while being close to each other, and the pulse voltage waveform in such a case is greatly increased from the normal waveform.
  • a deformed waveform hereinafter referred to as “illegal waveform” is obtained.
  • the electric field formed between the electrodes is locally strong near the edges around the opening at the entrance and exit of the aperture. Since it is distributed, the detection sensitivity is higher than the central portion. Therefore, as shown in the passage state shown in the left diagram of FIG. 13A, when a blood cell travels along a path deviating from the center and approaching the edge around the opening, the graph in the right graph of FIG. Thus, the voltage waveform tends to be an incorrect waveform having two protruding peaks.
  • the phenomenon that the above-mentioned incorrect waveform is generated occurs not only in the electric resistance method but also in the optical method shown in FIG.
  • two pulse waveforms overlap like one waveform, and have two peaks as in FIG. Waveform and a wide waveform with a bump on the slope, it is counted as one blood cell that is larger than the true size of each blood cell has passed.
  • the volume-frequency distribution graph that includes the incorrect waveform in the count is a distribution graph in which the frequency is added on the larger volume side compared to the graph when only the normal waveform is counted, which hinders correct diagnosis.
  • Such a problem exists not only in blood cells but also in general particle counting using an electrical resistance method or an optical method.
  • An object of the present invention is to provide a new method for eliminating an irregular waveform from a pulse waveform obtained by an electrical resistance method or an optical technique, and adopting only a pulse waveform that is a normal waveform for counting, and the method It is an object of the present invention to provide a particle counter configured to be able to implement the following.
  • the pulse waveform to be determined is associated with a Gaussian curve defined by a Gaussian function V (t) of the following equation (I) that approximates the pulse waveform;
  • V (t) Vp * exp ( ⁇ t 2 / (2 * c 2 ))
  • Formula (I) (In the above formula (I), t is a time variable, Vp is the maximum value of the pulse waveform to be determined, and c 2 is determined to have a width equal to the pulse width W of the pulse waveform associated with the Gaussian curve.
  • the method further includes a second determination step, and the second determination step is positioned before or after or in parallel with the first determination step.
  • the second determination step calculates a difference between the time Tm at the center of the width W of the pulse waveform to be determined and the time Tp at the maximum value Vp of the pulse waveform, and the difference is a predetermined threshold value.
  • a step of determining that the pulse waveform can be used for counting For the same pulse waveform, the first determination step and the second determination step are both configured to employ the pulse waveform for counting only when it is determined that both can be employed for counting.
  • the particle counting method according to [1] above. [3]
  • the method further includes a first preliminary selection step of pre-selecting the pulse waveform with respect to the pulse width W,
  • the first preliminary selection step is a step for excluding a pulse waveform having a pulse width W outside a predetermined allowable range, which is located before all the determination steps.
  • the particle counting method as described.
  • the method further includes a second pre-selection step of pre-selecting the pulse waveform with respect to the maximum value Vp,
  • the second preliminary selection step is positioned before all the determination steps and is positioned after the first preliminary selection step, and a pulse waveform having a maximum value Vp equal to or higher than a predetermined lower limit value is described above.
  • a step to send to the decision step A pulse waveform having a maximum value Vp less than the lower limit value is adopted for counting.
  • the particle counting method according to [3] above.
  • a particle counter configured to obtain a pulse waveform corresponding to each particle in the sample liquid according to the particle counting method of (A) or (B) below and to count the particles based on the pulse waveform.
  • the particle counter has a calculation unit for counting particles,
  • the calculation unit has at least a first determination unit that determines whether or not the pulse waveform of each particle can be used for counting,
  • the calculation unit further includes a second determination unit, The second determination unit is positioned before or after the first determination unit or in parallel, If the difference between the center time Tm of the width W of the pulse waveform to be determined and the time Tp at the maximum value Vp of the pulse waveform is calculated and the difference is within a predetermined threshold range, It is configured to determine that the pulse waveform can be used for counting, The calculation unit is configured to adopt the pulse waveform for counting only when the first determination unit and the second determination unit determine that both can be used for counting with respect to the same pulse waveform. Being The particle counter according to [5] above.
  • the calculation unit further includes a first preliminary selection unit that pre-selects the pulse waveform with respect to the pulse width W,
  • the first preliminary selection unit is positioned so as to perform data processing before all the determination units, and is configured to exclude a pulse waveform having a pulse width W outside a predetermined allowable range.
  • the particle counter according to [5] or [6] above.
  • the calculation unit further includes a second preliminary selection unit that pre-selects the pulse waveform with respect to the maximum value Vp,
  • the second preliminary sorting unit is positioned so as to perform the sorting process before all the determination units, and is positioned so as to sort the pulse waveforms that are not excluded by the first preliminary sorting unit,
  • a pulse waveform having a maximum value Vp equal to or greater than a predetermined lower limit value is configured to be sent to the determination unit.
  • the calculation unit is configured to employ a pulse waveform having a maximum value Vp less than the lower limit value for counting, The particle counter according to [7] above.
  • an upper limit and a lower limit are set as threshold values for determination in the dispersion value that determines the width of the Gaussian curve, and the dispersion value of the Gaussian curve associated with the pulse waveform of each particle. Is within the range of the upper and lower thresholds.
  • the upper and lower thresholds are fixedly set for the pulse waveform height (pulse height) and the pulse waveform width (pulse width), respectively, and the fixed upper and lower limits are fixed. Whether the pulse waveform is good or bad is determined only by whether or not it is within the threshold range.
  • a method of discriminating an incorrect waveform by checking the symmetry of a voltage waveform, or (ii) a voltage value at an intermediate point in time (t1, t2) at half the maximum peak value of one waveform has also been proposed.
  • the pulse waveform obtained for each particle is converted into a Gaussian curve based on the pulse height and pulse width, and the upper and lower thresholds of each pulse width are set to each waveform. It fluctuates according to the pulse height. That is, in the present invention, the threshold value for waveform determination is not a fixed value.
  • the manner in which the upper and lower thresholds of the pulse width of the Gaussian curve change according to the change in the pulse height of the Gaussian curve is as shown in the graph of FIG.
  • the present invention replaces the determination of the pulse waveform with the determination of a Gaussian curve, and uses the high processing capability of a digital arithmetic device such as a computer or an FPGA (Field Programmable Gate Array), so that the pulse height and pulse width input as the determination conditions are used. It is determined whether it falls within the range of the two-dimensional threshold (that is, the upper and lower thresholds of the pulse width that varies according to the change in the pulse height). Therefore, the determination method of the present invention is a determination method based on a new technical idea that has not existed in the past, and eliminates incorrect waveforms without reducing the processing speed while suppressing erroneous determination. Is possible. The present invention is also a method that can eliminate the drawbacks of the conventional methods (i) and (ii).
  • FIG. 1 is a graph illustrating the principle of determination in the first determination step (or the first determination unit in the apparatus of the present invention) in the method of the present invention.
  • FIG. 1A shows a Gaussian curve (thick solid line) associated with a pulse waveform (pulse voltage) to be determined, a Gaussian curve having an upper limit dispersion value (dashed line), and a lower limit dispersion value. It is the graph which superimposed the Gaussian curve (dashed line).
  • the horizontal axis represents time t, and the vertical axis represents a voltage value as an example.
  • FIG. 1B is a graph showing how the threshold value of the width W of the Gaussian curve changes in correspondence with the height Vp of the Gaussian curve associated with the maximum value of the pulse waveform.
  • FIG. 2 is a graph in which the pulse waveform of each particle actually measured according to the first determination step in the method of the present invention (or the first determination unit in the apparatus of the present invention) is associated with a Gaussian curve and plotted. It is. In the graph, the vertical axis represents the pulse width, and the horizontal axis represents the pulse height.
  • FIG. 3 is a graph illustrating the principle of determination in the second determination step (or the second determination unit in the apparatus of the present invention) in the method of the present invention.
  • FIG. 4 is a flowchart showing a preferred example of the method of the present invention, and shows an example of the flow from step s1 for acquiring the pulse waveform to step s7 for performing the counting process.
  • FIG. 5 is a block diagram schematically showing an example of the configuration of the apparatus of the present invention.
  • FIG. 6 is a scattergram showing the relationship between the maximum value (maximum voltage value) Vp and the pulse width W of the pulse waveform (pulse voltage) of each particle actually measured in the example of the present invention. .
  • FIG. 7 shows the relationship between the maximum value (maximum voltage value) Vp and the pulse width W of the pulse waveform (pulse voltage) determined to be employable for counting in the second determination step in the embodiment of the present invention.
  • FIG. 8 shows the maximum value (maximum voltage value) Vp of the pulse waveform (pulse voltage) determined to be employable for counting in both the first determination step and the second determination step in the embodiment of the present invention.
  • 6 is a scattergram showing a relationship with a pulse width W.
  • FIG. 9 shows an example of a data set of a pulse waveform (pulse voltage) as it is measured and a data set of a pulse waveform (pulse voltage) determined to be employable for counting in the embodiment of the present invention.
  • FIG. 10 is a graph showing the determination performance in the method of the present invention.
  • FIG. 11 is a diagram for explaining the basic principle and apparatus configuration of the electrical resistance method.
  • FIG. 11A shows an arrangement example of apertures and electrodes
  • FIG. 11B schematically shows voltage fluctuations between electrodes that appear when blood cells as particles pass through the apertures.
  • FIG. 12 is a diagram for explaining the basic principle of the particle coefficient and the device configuration based on the optical method.
  • FIG. 12A shows an arrangement example of the flow path and the photoelectric sensor
  • FIG. 12B shows a pulse-like signal (in the example shown in the figure) obtained when blood cells as particles cross the optical axis of the sensor. (Pulse voltage) is schematically shown.
  • FIG. 13 is a diagram illustrating an illegal waveform appearing in voltage measurement by the electric resistance method and its cause.
  • the particle counting method of the present invention is a method of obtaining a pulse waveform corresponding to each particle in a sample solution according to an electric resistance method or an optical technique, and counting particles based on the waveform.
  • the electrical resistance method is taken as an example of the measurement method for obtaining the pulse waveform, and the case where the pulse waveform is obtained as the pulse voltage is taken as an example, but the measurement method for obtaining the pulse waveform is appropriately replaced with an optical method.
  • the obtained pulse voltage may be any signal that changes in a pulse shape.
  • various processing such as pulse waveform determination, preliminary selection, and subsequent counting are automatically performed by an arithmetic unit described later.
  • the pulse waveform to be determined is associated with a Gaussian curve defined by a Gaussian function V (t) of the following equation (I) that approximates the pulse waveform.
  • V (t) Vp * exp ( ⁇ t 2 / (2 * c 2 ))
  • Formula (I) the maximum value (maximum voltage value) of t is the time variable, Vp is to be determined pulse waveform (pulse voltage), c 2 is a variance value, a base of the Gaussian curve of the spread This parameter determines the degree.
  • the c 2 is determined so that the Gaussian curve of the above formula (I) has a width corresponding to the pulse width W of the pulse waveform of the original signal.
  • “*” means an operator that means multiplication
  • “/” means an operator that means division
  • “exp ()” means an exponential function e () in the base e of natural logarithm. Operator.
  • a Gaussian curve (thick solid line graph) to be determined is a Gaussian curve having an upper limit variance (dashed line) and a Gaussian curve having a lower limit variance (dashed line). Between the two, it is determined that the original pulse waveform corresponding to the Gaussian curve can be used for counting.
  • the threshold voltage Vs for the pulse waveform and the Gaussian curve of each particle it is preferable to set the threshold voltage Vs for the pulse waveform and the Gaussian curve of each particle and handle only the portion above the threshold voltage Vs. Then, by defining the width of each of the pulse waveform and the Gaussian curve associated therewith as the width W at the threshold voltage Vs as shown in the graph of FIG. 1A, the width value becomes clear. More accurate calculation processing can be performed. In the following description, the width of each of the pulse waveform and the Gaussian curve is the width W at the threshold voltage Vs.
  • the Gaussian curve defined by the above formula (I) has the same height Vp as the maximum value (maximum voltage value) Vp of the pulse waveform corresponding to the particle to be determined.
  • the maximum value Vp of the pulse waveform is a maximum value of the entire pulse including a voltage value of an irregular protrusion due to a disturbance or the like.
  • the height Vp of the Gaussian curve is the height of the central peak.
  • the Gaussian curve defined by the above formula (I) has a width (for example, the same width) W corresponding to the pulse width W of the pulse voltage of the particle to be determined.
  • a Gaussian function with x as a variable is generally expressed as in the following equation (a).
  • f (x) a * exp ( ⁇ (x ⁇ b) 2 / (2 * c 2 ))
  • Formula (a) In the above equation (a), a is a coefficient that determines the height of the waveform, and b is the peak position (time).
  • the waveform of the pulse voltage that appears when the particle passes through the aperture in the optical method, the waveform of the pulsed signal obtained when the particle crosses the optical axis
  • c 2 W 2 / ( ⁇ 8 * ln (Vs / Vp))
  • Formula (d) “ln ()” is an operator that means a natural logarithm.
  • the variance value c 2 of the Gaussian function associated with the pulse waveform of each particle is the maximum value of the pulse waveform (maximum voltage value of the pulse voltage) Vp, pulse width W, threshold voltage. Determined by Vs.
  • the total range that the dispersion value c 2 can take with respect to each height Vp (the range including the irregular waveform) will correspond to the type of particles and the flow rate of the sample liquid.
  • the dispersion value c 2 is about 20 to 60.
  • the dispersion value c from the above equation (d) depends on the average diameter of the target particles, the voltage between electrodes by the electric resistance method, the beam diameter by the optical method, the respective flow velocity, etc.
  • the upper and lower thresholds of 2 may be determined based on actual measurement values using beads having a single particle size. As an example, in the red blood cell count, the range from the upper limit threshold value to the lower limit threshold value to be set as the variance value c 2 is about 30-50.
  • FIG. 1B is a graph showing the principle of determination in the first determination step more easily. Gauss to be determined within an area between an upper limit curve (indicated by a one-dot chain line) indicating a change in the upper threshold value of the dispersion value and a lower limit curve (indicated by a broken line) indicating a change in the lower threshold value of the dispersion value If the variance value c 2 of the curve is included, it can be determined that the pulse waveform can be used for counting.
  • FIG. 2 is a graph in which the pulse waveform of each particle actually measured is associated with a Gaussian curve and the (dispersion value, height) of each Gaussian curve is plotted. In the graph of FIG.
  • the pulse width is determined by the time that the particle passes through the aperture at a constant flow rate, but the aperture thickness (electric field strength) is more dominant than the particle size. It is.
  • the particles to be determined by the present invention are not particularly limited, but the present invention is useful for counting blood cells such as red blood cells, white blood cells, and platelets (blood cell counting).
  • blood cells such as red blood cells, white blood cells, and platelets (blood cell counting).
  • erythrocytes have a higher concentration than other blood cells, and double passage tends to occur during counting. Therefore, the usefulness of the present invention becomes more remarkable in counting red blood cells.
  • erythrocytes are important particles to be judged for the present invention because the mean red blood cell volume (MCV) is clinically important.
  • MCV mean red blood cell volume
  • the aperture shape of the aperture for carrying out the electrical resistance method and the cross-sectional shape of the flow path for carrying out the optical method are not particularly limited, but a circular shape is usually preferred.
  • the diameter may vary depending on the particles to be counted, but may be appropriately selected from the range of about 0.01 mm to 1 mm for erythrocytes.
  • the maximum value (maximum voltage value) Vp of the pulse waveform is the volume of the particle to be measured, the cross-sectional area of the aperture corresponding thereto, the voltage applied between the electrodes, the passage of the particle It depends on the route.
  • the maximum voltage value Vp of the waveform of the pulse voltage of red blood cells is about 0.1 to 10 V including an incorrect waveform.
  • the threshold voltage Vs may be appropriately determined according to the maximum value (maximum voltage value) Vp of the pulse waveform so that only noise around the voltage 0V can be effectively eliminated.
  • the threshold voltage Vs is about 0.1 to 1V. is there.
  • the pulse width W of red blood cells determined by the threshold voltage Vs varies greatly depending on the flow velocity, but is, for example, about 5 to 30 ⁇ sec including an irregular waveform. In this case, for example, by setting the upper limit threshold value of the variance value c 2 to 40 and setting the lower limit threshold value of the variance value to 30, two curves shown in FIG. 1B are obtained.
  • a second determination step described below may be provided.
  • the second determination step may be before or after the first determination step, or may be located in parallel.
  • the time Tm at the center of the width W of the pulse waveform to be determined (the position of the center line indicated by the alternate long and short dash line) Tm and the maximum value Vp of the pulse waveform
  • the difference ⁇ T with respect to the time Tp (that is, the position shift at the maximum value from the center position of the width) is calculated. If the difference ⁇ T is within a predetermined threshold range, it is determined that the pulse waveform of the particle can be used for counting.
  • the difference ⁇ T is a relative value with respect to one pulse width, and may be a value having a positive or negative sign, and a threshold range and a calculation method may be determined according to them.
  • the threshold range of ⁇ T is about ⁇ 50% of each pulse width.
  • both the determination result in the first determination step and the determination result in the second determination step can be used for counting with respect to the same one particle pulse waveform Only the pulse waveform of that particle is adopted for counting.
  • the second determination step s4 is positioned before the first determination step s5, and only the waveform determined by the second determination step s4 as being employable for counting is used. The determination is made in the first determination step s5.
  • the selection and determination processing of a set (data set) of pulse waveform data (pulse voltage data and the like) obtained by measurement is performed in series, but in the data set A procedure may be used in which an identification number is provided for each data, processing is performed separately in parallel, and comprehensive determination is finally performed on each data.
  • first and second preliminary selection steps described below are further provided prior to all determination steps (first and second determination steps).
  • the pulse waveform obtained by the measurement may include an incorrect waveform that does not need to be determined and has a clearly unspecified height or width. It is preferable to preliminarily filter out such an incorrect waveform in the first and second preliminary selection steps so as not to perform useless determination processing or erroneous determination processing. Only one of the first and second preliminary sorting steps may be provided.
  • the first preliminary selection step s2 is located before the first and second determination steps, and has a pulse waveform having a pulse width outside a predetermined allowable range. This is a step to eliminate this with a simple filter.
  • the allowable range related to the pulse width set in the first preliminary sorting step may be set to an appropriate value according to each blood cell counter with reference to a conventionally known blood cell counting method.
  • the pulse waveform data whose pulse width was within the allowable range in the first preliminary sorting step s2 is sent to the second preliminary sorting step s3.
  • the second preliminary sorting step is also located before the first and second determination steps.
  • the second preliminary sorting step s3 is provided after the first preliminary sorting step s2.
  • a lower limit value for the maximum value of the pulse waveform is set, and a pulse waveform having a maximum value Vp equal to or greater than this lower limit value is simply sent to the first and second determination steps. It is done.
  • a pulse waveform having a maximum value Vp less than this lower limit value is used for counting rather than being excluded (in the flow chart example of FIG. Is sent to step s6).
  • the reason is that, for example, even particles having a small pulse waveform less than the lower limit, such as crushed erythrocytes present in a sample of a patient who has been burned, may be counted.
  • a small pulse waveform less than the lower limit value has a shape that differs greatly from a Gaussian curve, such as a trapezoidal shape that is wide with respect to the height, and may be excluded in the first determination step. Such an incorrect waveform to be counted is preferably used for counting according to the result of the preliminary selection step.
  • FIG. 5 is a diagram schematically illustrating an example of the configuration of the apparatus.
  • the device configuration for carrying out the electric resistance method is shown, but it may be replaced with an optical method.
  • the apparatus 1 is configured to perform the particle counting method according to the present invention, and generally includes a measurement structure unit 2 and a control unit 3.
  • the measurement structure unit 2 includes a flow path 20 for flowing a sample solution, an aperture 21 provided in the flow path, and a pair of electrodes 22 and 23 disposed on both sides (upstream side and downstream side) of the aperture. It is comprised so that the pulse voltage (pulse waveform) of particle
  • the measurement structure unit 2 such as a tank, a pump, and a detailed form of the flow path and ancillary devices, the related art may be referred to.
  • the control unit 3 is a control device for causing the measurement structure unit 2 to perform the electrical resistance method (or measurement by an optical technique), and controls the flow of the sample solution and controls the voltage application to the electrode pair. (In the case of an optical method, drive control of a photoelectric sensor), measurement data of a pulse waveform of each particle is obtained, and the data is processed and counted.
  • the control unit 3 includes at least a constant current power supply unit 4 and a calculation unit 5.
  • the constant current power supply unit is configured to apply a voltage to the electrode pairs 22 and 23 and output the waveform data of the pulse voltage when each particle passes through the aperture to the calculation unit 5.
  • the calculation unit 5 is a calculation device configured to execute the above-described method of the present invention and count the pulse waveform finally determined to be employable for counting.
  • the arithmetic device include a computer that executes a program created so as to implement the method of the present invention. Not only all arithmetic processing is performed by a program, but arithmetic processing such as the above-described FPGA is performed by an electronic circuit. A mode in which a digital arithmetic element constructed so as to be able to be partially used is preferable in that the circuit can be easily adjusted after speeding up the processing and checking the data. For the data communication technology between the computer and the digital arithmetic element, the conventional technology may be referred to. Further, in the aspect of FIG. 5, the configuration for converting the obtained pulse waveform into digital data may be included in either the constant current power supply unit 4 or the calculation unit 5.
  • the calculation unit 5 has at least a first determination unit 6 configured to execute the first determination step in the method of the present invention described above.
  • the determination principle in the first determination unit, the set value for the determination, and the detailed processing content are as described above.
  • the calculating part 5 further has the 2nd determination part 7 comprised so that the 2nd determination step in the method of this invention mentioned above might be performed.
  • the determination principle in the second determination unit, the set value for the determination, and the detailed processing content are as described above.
  • the calculation unit counts the pulse waveform only when the first determination unit 6 and the second determination unit 7 determine that both can be employed for counting with respect to the same pulse waveform. Configured to be adopted.
  • the calculation unit 5 further includes a first preliminary sorting unit configured to execute the first preliminary sorting step in the above-described method of the present invention (not shown in FIG. 5).
  • the first preliminary sorting unit is positioned so as to perform data processing before the first and second determination units.
  • the selection contents in the first preliminary selection section, the set values for the selection contents, and the detailed processing contents are as described above.
  • the calculation unit 5 further includes a second preliminary sorting unit configured to perform the second preliminary sorting step in the method of the present invention (not shown in FIG. 5).
  • the second preliminary sorting unit is positioned so as to perform data processing prior to the first and second determination units, and sorts the pulse waveforms that are not excluded by the first preliminary sorting unit. To position.
  • the selection contents in the second preliminary selection section, the set values for the selection contents, and the detailed processing contents are as described above.
  • the calculation unit 5 is configured to employ, in the counting, the pulse waveform determined by the second preliminary selection unit as having a maximum value (maximum voltage value) Vp less than the lower limit value. ing. The reason is as described above.
  • the computer that is the main device of the calculation unit 5 performs all steps from the acquisition of the pulse waveform in step s1 to the counting process step in step s7 shown in the flowchart of FIG. FPGA).
  • the computer that is the main device of the calculation unit 5 performs all steps from the acquisition of the pulse waveform in step s1 to the counting process step in step s7 shown in the flowchart of FIG. FPGA).
  • FIG. 6 is a scattergram (dispersion diagram) showing the relationship between the maximum pulse voltage Vp and the pulse width W of individual particles obtained by measurement using blood as the sample liquid.
  • 7 to 10 are also scattergrams or graphs showing measurement results when blood is used as the sample solution.
  • a scattergram was determined using an experimental sample solution containing polystyrene beads having three kinds of average particle diameters.
  • the test sample solution is a solution in which three types of monodisperse polystyrene monodisperse particles (average particle diameters of 5 ⁇ m, 6 ⁇ m, and 7 ⁇ m) are mixed in substantially the same ratio in physiological saline. The performance of was verified. As a result, since the distribution of beads entered between the upper and lower thresholds, the threshold was judged to be appropriate.
  • the determination processing in the first determination step was performed by the computer on the pulse waveform data set determined to be employable in the counting in the second determination step.
  • the pulse waveform whose variance value c 2 exceeds the predetermined threshold range is excluded.
  • Upper and lower limits of the threshold variance value c 2 at this time is the pulse shape of the time of the blood or polystyrene monodisperse particles as a sample, the upper limit 30 and the lower limit is set to 43.
  • FIG. 8 is a scattergram showing the relationship between the maximum voltage value Vp of the pulse voltage determined to be employable for counting in the first determination step and the pulse width W. Compared with FIG. 6 and FIG. 7, it can be seen that a pulse waveform having a wide pulse width is effectively eliminated and only a pulse waveform having a normal waveform remains.
  • FIG. 9 also shows the volume frequency distribution of the first data set before processing. As is clear from the graph of FIG. 9, it can be seen that only the frequency on the side with the larger volume is effectively scraped off by the determination processing in the second determination step to the first determination step.
  • FIG. 10 is a graph plotted with the hematocrit value of each sample solution obtained in each of the above experiments as the horizontal axis and the hematocrit value obtained by calculation of each sample solution as the vertical axis.
  • the value of the decision count R 2 representing the degree of correlation between the two is 0.97, indicating a strong correlation, indicating that the determination method according to the present invention is correct.
  • the actual hematocrit value of each sample liquid obtained in each experiment is all higher than the hematocrit value of each sample liquid obtained by calculation. It is estimated that this is because a small amount of gaps are included.
  • the present invention it is possible to effectively exclude an irregular waveform from a pulse waveform obtained by an electric resistance method or an optical technique, and to adopt only a normal pulse waveform for counting. Thereby, in the blood cell count, a volume frequency distribution graph that does not include an incorrect waveform in the count is obtained, and a more accurate diagnosis can be performed.

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Abstract

　L'invention concerne un procédé et un dispositif de comptage de particules pour obtenir une forme d'onde d'impulsion de particules dans un liquide d'échantillon, selon un procédé de résistance électrique ou une procédure optique, et compter les particules sur la base de la forme d'onde. Une forme d'onde d'impulsion à déterminer est associée à une courbe de Gauss se rapprochant de la forme d'onde et, si la variation de la courbe de Gauss se trouve dans la plage de valeurs de seuil de limite supérieure et de limite inférieure prédéterminées en fonction de la hauteur de la courbe de Gauss, il est déterminé qu'il est possible d'adopter cette forme d'onde de tension d'impulsion en vue d'un comptage. Selon la présente invention, des formes d'onde inappropriées peuvent être éliminées de façon appropriée de formes d'onde d'impulsion mesurées.
PCT/JP2014/084488 2013-12-27 2014-12-26 Procédé de comptage de particules et appareil de comptage de particules WO2015099116A1 (fr)

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JPWO2017110753A1 (ja) * 2015-12-25 2018-10-25 国立大学法人大阪大学 個数分析方法、個数分析装置および個数分析用記憶媒体
CN110243729A (zh) * 2018-03-09 2019-09-17 理音株式会社 粒子计数器
US20200070167A1 (en) * 2018-08-31 2020-03-05 Vortex Biosciences, Inc. Processing systems for isolating and enumerating cells or particles
CN111060443A (zh) * 2019-12-24 2020-04-24 深圳开立生物医疗科技股份有限公司 一种干扰脉冲识别方法、装置、存储介质及细胞计数设备
CN112444621A (zh) * 2019-08-30 2021-03-05 深圳迈瑞生物医疗电子股份有限公司 血液细胞分析仪及其计数方法
CN116481982A (zh) * 2023-04-20 2023-07-25 瑞芯智造(深圳)科技有限公司 一种基于库尔特粒度检测仪的颗粒检测方法及检测仪
US11781099B2 (en) 2015-12-25 2023-10-10 Aipore Inc. Number analyzing method, number analyzing device, and storage medium for number analysis

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JPWO2017110753A1 (ja) * 2015-12-25 2018-10-25 国立大学法人大阪大学 個数分析方法、個数分析装置および個数分析用記憶媒体
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CN110243729A (zh) * 2018-03-09 2019-09-17 理音株式会社 粒子计数器
US20200070167A1 (en) * 2018-08-31 2020-03-05 Vortex Biosciences, Inc. Processing systems for isolating and enumerating cells or particles
CN112444621A (zh) * 2019-08-30 2021-03-05 深圳迈瑞生物医疗电子股份有限公司 血液细胞分析仪及其计数方法
CN111060443A (zh) * 2019-12-24 2020-04-24 深圳开立生物医疗科技股份有限公司 一种干扰脉冲识别方法、装置、存储介质及细胞计数设备
CN111060443B (zh) * 2019-12-24 2022-09-30 深圳开立生物医疗科技股份有限公司 一种干扰脉冲识别方法、装置、存储介质及细胞计数设备
CN116481982A (zh) * 2023-04-20 2023-07-25 瑞芯智造(深圳)科技有限公司 一种基于库尔特粒度检测仪的颗粒检测方法及检测仪

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