WO2017076800A1 - Procédé et dispositif de mesure pour la détermination de globules sanguins - Google Patents

Procédé et dispositif de mesure pour la détermination de globules sanguins Download PDF

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Publication number
WO2017076800A1
WO2017076800A1 PCT/EP2016/076217 EP2016076217W WO2017076800A1 WO 2017076800 A1 WO2017076800 A1 WO 2017076800A1 EP 2016076217 W EP2016076217 W EP 2016076217W WO 2017076800 A1 WO2017076800 A1 WO 2017076800A1
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WO
WIPO (PCT)
Prior art keywords
impedance
frequency
sensor
blood cells
blood
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PCT/EP2016/076217
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German (de)
English (en)
Inventor
Peter Simon
Dr. Marcin FRANKOWSKI
Dr. Jörg NEUKAMMER
Original Assignee
Bundesrepublik Deutschland, Vertreten Durch Das Bundesministerium Für Wirtschaft Und Energie, Dieses Vertreten Durch Den Präsidenten Der Physikalischen Bundesanstalt
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Application filed by Bundesrepublik Deutschland, Vertreten Durch Das Bundesministerium Für Wirtschaft Und Energie, Dieses Vertreten Durch Den Präsidenten Der Physikalischen Bundesanstalt filed Critical Bundesrepublik Deutschland, Vertreten Durch Das Bundesministerium Für Wirtschaft Und Energie, Dieses Vertreten Durch Den Präsidenten Der Physikalischen Bundesanstalt
Publication of WO2017076800A1 publication Critical patent/WO2017076800A1/fr

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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
    • G01N15/10—Investigating individual particles
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • 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
    • G01N15/1023—Microstructural devices for non-optical measurement
    • 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

Definitions

  • the invention relates to a method for the determination of blood cells in a blood sample in the flow with a sensor for measuring the electrical impedance.
  • the invention further relates to a measuring device for the determination of blood cells with such a method.
  • the determination of blood cells in a blood sample with the determination of the types of blood cells contained in the blood sample comprising red and white blood cells and their subpopulations plays a major role in laboratory medicine.
  • optical and / or electrical flow cytometry is known. M. Frankowski, P. Simon, N. Bock A. El Hasni, U. Schnakenberg, J.
  • Neuclotaneous Optical and Impedance Analysis of Single Cells A Comparison of Two Microfluidic Sensors with Sheath Flow Focusing, in: Engineering Life Sciences 2015, 15, pages 286 to 296 describes a method with a combined analysis of blood samples by flow cytometry as a function of the measured electrical impedance and optical properties of microparticles and blood cells.
  • a microstructured sensor has a capacitive sensor sor Hoch, in which an AC field is built through which the blood sample is passed. Another sensor or sensor area is provided for measuring the electrical impedance of the blood sample or a reference medium without blood cells to determine from the difference signal of the two sensors caused by the blood cells impedance change.
  • the subpopulations of granulocytes, monocytes and white blood cell lymphocytes (leucocytes) can be determined from impedance events with characteristic absolute impedance magnitudes at two different frequencies.
  • the object of the present invention is therefore an improved method for the determination of blood corpuscles in a blood sample in the flow with a sensor. to create sensor for measuring the electrical impedance and a corresponding measuring device.
  • the procedure for the determination of blood corpuscles has the steps:
  • Differentiation of the different types of blood cells in a blood sample and determination of the concentration of the individual types of blood cells in the blood sample is possible by means of an electrical flow cytometry in an alternating voltage field using two different frequencies of the alternating voltage field.
  • the blood sample is not subjected to any previous hemolysis.
  • the different types of blood cells can be much better differentiated by the ratio of imaginary part to real part of the impedance of the same volume part of the blood sample at different frequencies. This is especially true for the granulocytes and monocytes, which otherwise can not be differentiated sufficiently without hemolysis using electrical flow cytometry.
  • Z11 at the first frequency is dominated by the real part, ie that the imaginary part lm (Z1) at the first frequency is substantially smaller (eg smaller than 80%) than the real part Re (Z1), then, for the real part Re (Z1), the amount of impedance at the first frequency
  • the impedance at the second frequency can be used when the imaginary part lm (Z2) dominates the real part Re (Z2) at the second frequency (e.g., greater than 80%). Then the ratio lm (Z2) / Re (Z1) can be determined by the ratio
  • a count of values can be carried out, for example, for the individual types Blood cells are characteristic value ranges. This can be used to determine the number of different types of blood cells and their concentration in the blood sample.
  • the object is also achieved by the method for the determination of blood cells in a blood sample in flow with an electrical impedance sensor in that the electrical impedance of the same volume part of the blood sample with a first frequency in the range of 1 MHz to 6 MHz and with a second frequency is carried out in the range of 6 MHz to 15 MHz.
  • the smaller first frequency range improves the selectivity for detecting the small-volume blood bodies, in particular the platelets, while the larger second frequency range is optimized for differentiating the large-volume blood cells, ie the white blood cells and their subpopulations. If the electrical impedances in these two measurement areas are correlated with each other, there is an improved differentiability of the different types of blood cells and their number or concentration in the blood sample.
  • impedance change events are counted in which the impedance change or the ratio of the imaginary part of the impedance at the second frequency to the real part of the impedance at the first frequency exceeds a predetermined threshold value, and the types of blood corpuscles and their number or concentration in the blood sample is determined as a function of the number of impedance change events.
  • the threshold may be a single limit or may include a range of values having a lower and upper limit.
  • simultaneous passage of the hemolysis-free blood sample with blood cells contained therein is effected by an AC voltage field of a first sensor and passing a comparison medium through an AC voltage field of a second sensor. Then, the impedance difference between the electrical impedance simultaneously measured with the first and second sensors is determined.
  • the types of blood cells in the AC field of the first sensor may be determined depending on the ratio of the imaginary part of the impedance difference at the second frequency to the real part of the impedance difference at the first frequency.
  • the at least one sensor Before the blood sample is passed through the at least one sensor, it is preferable to dilute the hemolysis-free blood sample with an aqueous solution.
  • the blood sample is free of hemolysis by no hemolysis is performed before passing through the at least one sensor.
  • the second frequency for measuring the electrical impedance is preferably greater than the first frequency.
  • the first frequency can advantageously be in the range of 1 MHz to 6 MHz, and particularly preferably 2.3 MHz.
  • the second frequency may advantageously be in the range of 6 MHz to 15 MHz and preferably 10.1 MHz.
  • Particularly advantageous is a differentiation of the types of blood cells, which are passed through the sensor, further depending on the amount of electrical impedance or impedance change, which was measured at a frequency.
  • the amount of the electrical impedance change is in this case proportional to the difference between the amount of the electrical impedance of the blood sample and blood cells to the amount of the electrical impedance of the reference medium or the blood sample without blood cells, which are measured in alternating voltage fields with the same frequency. It is advantageous if the impedance of the comparison medium in a second sensor is determined simultaneously or immediately thereafter with the measurement of the electrical impedance of the blood sample.
  • a comparison medium is passed through this sensor with the same sensor provided for measuring the electrical impedance of the blood sample prior to the measurement of the electrical impedance of the blood sample or after this measurement in order to determine the electrical impedance of the comparison medium ,
  • the hemolysis-free blood sample itself is advantageously suitable as a comparison medium, if it is adjusted by dilution in such a way that the distance between two blood statistically larger than the distance between two successive sensor areas. If, in a first sensor region, the impedance changes due to the presence of a blood cell, the difference to the impedance value can be formed in the second sensor, which statistically reproduces the impedance of the same blood sample without blood corpuscles.
  • microstructured flow cytometric sensors may have planar electrodes formed in platinum, which are mounted perpendicular to the direction of flow and typically have a capacitor plate spacing of in the range of about 30 ⁇ to 10 ⁇ and preferably of about 15 ⁇ +/- 5 ⁇ .
  • a diluted standard blood serum or the aqueous solution used to dilute the blood sample can also be used as the comparison medium. Since the comparison medium generally has a constant characteristic, the electrical impedance value measured with the comparison medium and its imaginary and real part do not change over the time at the at least two frequencies or can be taken into account as a constant comparison value. This constant comparison value can be determined by averaging comprising the option of determining the median.
  • a determination of the amount of small-volume blood cells which include the type of platelets, succeeds particularly advantageous if as a characteristic measure the ratio of the imaginary part of the impedance or impedance change at the first frequency to the real part of the impedance or relative to the relative electrical impedance at the second frequency Impedance change at the first frequency is used.
  • the ratio of the imaginary part of the impedance to the real part of the impedance is thus plotted against the relative electrical impedance at the first frequency, ie the amount of the impedance or impedance change.
  • a determination of the amount of large-volume blood cells which include the type of red blood cells and leukocytes with their subpopulation of granulocytes, monocytes and lymphocytes succeeds particularly advantageous if the ratio of the imaginary part of the impedance or impedance change at the second frequency to the real part of the impedance or Impedance change at the first frequency is related to the relative electrical impedance at the second frequency.
  • the ratio of the imaginary part to the real part of the impedance or impedance change at the different frequencies is thus plotted against the relative electrical impedance at the second frequency.
  • the measuring device is set up to carry out the method described above.
  • the evaluation unit may be, for example, a programmable arithmetic unit which is suitably programmed.
  • FIG. 1 shows a sketch of a measuring device 1 for determining blood corpuscles 2 in a flow cytometric sensor 3.
  • FIG. 1 shows a sketch of a diagram of the opacity over the relative impedance amount at the second frequency with the area of the lymphocytes, monocytes and granulocytes. It can be seen that the blood sample is passed in the direction of the arrow through a microstructured channel 4.
  • the microstructured channel 4 are two sensors 5a, 5b introduced at a distance from each other, the two at a distance in a range of about 30 ⁇ to 10 ⁇ and preferably 15 ⁇ +/- 5 ⁇ in the Have channel 4 spaced electrodes 6, between which by means of an AC generator / AC generator 7, an AC field 8 is established.
  • One of the electrodes 6 of a sensor 5a, 5b is connected to the alternator 7 and the opposite electrode 6 is connected to a respective amplifier 9a, 9b.
  • the outputs of the two amplifiers 9a, 9b are supplied to the input of a differential amplifier 10, in whose output an evaluation unit 1 1 is connected.
  • the evaluation unit 11 may, for example, be a programmable arithmetic unit.
  • the evaluation unit 1 1 may have an analog-to-digital converter. It is also conceivable that between the output of the differential amplifier 10 and the evaluation unit 1 1, a separate analog-to-digital converter is turned on.
  • the evaluation unit 1 1 can also have a further multi-frequency two-phase lock-in amplifier, the ac in the form of a sine wave signal applied directly to the electrode plates 6 or generates a synchronization signal S for synchronization of the alternator 7 for generating an AC field 8 ,
  • the hemolysis-free blood sample is adjusted by dilution to the distance between the two sensors 5a, 5b, that statistically only one blood cell 2 in the blood sample in only one of the two sensors 5a, 5b connected in series.
  • the impedance change caused by the blood corpuscles 2 in the sensor 5a / 5b is detected and output at the output of the differential amplifier 10.
  • the evaluation unit 1 1 is now set up in hardware or by suitable programming as an FPGA, ASIC or microcontroller so that it detects not only the amount of electrical impedance of the sensors 5 a, 5 b but also the real part of the impedance and the imaginary part of the impedance.
  • the measuring device 1 is furthermore set up such that the sensors 5a, 5b determine the impedance of the same volume part of a blood sample with at least two different frequencies.
  • the first frequency preferably a frequency in the range of 1 MHz to 6 MHz is selected.
  • the first frequency is particularly preferably 2.3 MHz.
  • a frequency in the range of 6 MHz to 15 MHz is selected. It is preferably at 10.1 MHz.
  • FIG. 2 shows a sketch of a section of the sensor region of a sensor 5a / 5b.
  • the electrodes 6 of the sensor are preferably applied from platinum to a microstructured substrate.
  • the channel 4 is introduced, through which the blood sample is passed in the direction of the arrow.
  • the electrodes 6 cross the channel and are aligned as shown as planar electrodes perpendicular to the flow direction. You have a distance of about 30 m to 10 ⁇ and preferably at about 15 ⁇ +/- 5 ⁇ each other.
  • FIG. 3 shows a diagram of an exemplary measurement result with the measuring device 1 from FIG. 1 after passage of a hemolysis-free blood sample.
  • the first frequency is 2.3 MHz and the second frequency is 10.1 MHz.
  • the ratio of the impedance Lm (Z2) of the electric impedance at the second frequency to the real part Re (Zi) of the electric impedance at the first frequency is the amount of the relative electrical impedance
  • the relative impedance is understood to mean that this is not calibrated in ohms. The same also applies to the imaginary part and the real part of the electrical impedance, where due to the quotient calibration in a Si measuring unit is even less significant.
  • thrombocytes Plt occupy a relatively large field in addition to the noise region, but they are relatively sharp in this noise component as well as in the noise adjacent red blood cells RBC and leukocytes with their subpo- pulations of lymphocytes Ly, monocytes M and granulocytes Gn separated.
  • the platelets Plt are no longer so sharply separated from the noise signal Noise.
  • the red blood cells i. Erythrocytes RBC and sub-population of leukocytes sharply separated.
  • FIG. 5 shows a detail from the diagram of FIG. 4 in the region of the subpopulation of the leukocytes. It can be seen that the statistical distribution of the cell events for the lymphocytes Ly, the monocytes M and the granulocytes Gn are sufficiently differentiated from one another in characteristic value ranges.
  • FIG. 6 shows, for the same measurement results, an evaluation of the opacity over the amount of the relative impedance at the second frequency.
  • the opacity is the ratio of the magnitude of the impedance at the second frequency to the magnitude of the impedance at the first frequency. It becomes clear that the opacity in particular causes the monocytes M and granulocytes Gn to merge into one another and also that the distance to the lymphocytes Ly is no longer so sharp. This makes it clear that by determining the types of blood corpuscles for determining the respective concentration in the blood sample or for differentiation for another purpose, an evaluation as a function of the ratio of the imaginary part of the electrical impedance at the second frequency to the real part of the electrical impedance at the first frequency leads to better results.
  • the blood cells or cells of the blood sample when passing through the electrode pairs, produce impedance changes that are proportional to the volume of the cells, the capacity of their membrane and the conductivity of the cytoplasm. Their respective amounts depend on the selected frequency.
  • the ratio lm (Z2) / Re (Zi) from the imaginary part of the impedance (Z2) at a suitable second frequency h and the real part of the impedance Z1 at the first frequency fi is proportional to the capacity of the cell of the corpuscle per unit volume.
  • two pairs of electrodes 6 are implemented for the purpose of differential measurement.
  • the isotonic solution in which the blood cells are located is measured once with and once without a cell.
  • the difference between the two signals thus contains only the amount of impedance change caused by the cell, i. the blood cell is caused.
  • erythrocytes represent the vast majority of blood cells and with a diameter of 7.5 ⁇ are much larger than thrombocytes with a diameter in the range 1 to 3 ⁇ , a differentiation only with the electrical impedance measurement itself is very difficult.
  • the leucocytes have a size of about 8 to 15 ⁇ a comparable size to the erythrocytes.
  • the impedance change caused by the capacity of the respective blood body is related to the change in impedance caused by the volume of the respective blood body.
  • a pulse height analysis is carried out in which the maximum values or, depending on the polar direction, the minimum values are used as relevant impedance change events in order to calculate the impedances and impedance ratios for these events.
  • the blood sample To prepare the blood sample, it is diluted appropriately with an aqueous solution to match the counts of the cells to the electronic assemblies and the data acquisition. Counting rates of 1 to 10 kHz are typical.
  • a calibration of the identification of blood particles by means of an AC frequency scan in the range of 300 kHz to 100 MHz for the blood of one species and a further, independent method can be performed.
  • Such a calibration can be carried out with the aid of the two-dimensional representation corresponding to FIGS. 3 to 5.

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  • Chemical & Material Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Physics & Mathematics (AREA)
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  • Life Sciences & Earth Sciences (AREA)
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Abstract

L'invention concerne un procédé de détermination de globules sanguins (2) dans un échantillon sanguin pendant l'écoulement à l'aide d'un capteur (5a, 5b) pour la mesure de l'impédance électrique. Le procédé comporte les étapes consistant à : faire passer l'échantillon sanguin sans hémolyse contenant les globules sanguins (2) à travers un champ de tension alternative (8) du capteur (5a, 5b), mesurer l'impédance électrique des globules sanguins (2) individuels dans l'échantillon sanguin à l'aide du capteur (5a, 5b), l'impédance électrique étant mesurée à au moins deux fréquences de mesure différentes du champ de tension alternative (8), et déterminer les types de globules sanguins (2) se trouvant dans le champ de tension alternative (8) du capteur (5a, 5b) en fonction d'une corrélation de l'impédance électrique de la même région volumique de l'échantillon sanguin mesurée à une première fréquence (Re(Z1)) dans la plage de 1 MHz à 6 MHz et à une deuxième fréquence (Im(Z2)) dans la plage de 6 MHz à 15 MHz.
PCT/EP2016/076217 2015-11-05 2016-10-31 Procédé et dispositif de mesure pour la détermination de globules sanguins WO2017076800A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102015119027.3A DE102015119027B4 (de) 2015-11-05 2015-11-05 Verfahren und Messeinrichtung zur Bestimmung von Blutkörperchen
DE102015119027.3 2015-11-05

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CN111370075B (zh) * 2020-03-04 2023-02-03 广东博智林机器人有限公司 糖分含量的检测方法
CN116429837B (zh) * 2023-06-15 2023-09-08 可孚医疗科技股份有限公司 红细胞压积校正方法、系统及电化学测量系统

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HYWEL MORGAN AND TAO SUN AND DAVID HOLMES AND SHADY GAWAD AND NICOLAS G GREEN: "Single cell dielectric spectroscopy", JOURNAL OF PHYSICS D: APPLIED PHYSICS, vol. 40, no. 1, 15 December 2006 (2006-12-15), pages 61 - 70, XP002765654, Retrieved from the Internet <URL:http://iopscience.iop.org/article/10.1088/0022-3727/40/1/S10/pdf> [retrieved on 20170103] *
VYKOUKAL, DAYNENE M. AND GASCOYNE, PETER R. C. AND VYKOUKAL, JODY: "Dielectric characterization of complete mononuclear and polymorphonuclear blood cell subpopulations for label-free discrimination", INTEGR. BIOL., vol. 1, 2 June 2009 (2009-06-02), pages 477 - 484, XP002765653, Retrieved from the Internet <URL:http://pubs.rsc.org/en/content/articlepdf/2009/ib/b906137a> [retrieved on 20170102] *

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