Device for counting and differentiating particles in a sample stream

Abstract

The invention relates to a device (1) for counting and differentiating particles comprising: - a heating base (3) within which are provided an injection channel (9) for a sample stream and an injection channel (11) for a sleeved stream, and - a measuring cell (5) comprising an optical cuvette (17) arranged to be traversed by a sleeved sample stream, a light source (33) arranged to illuminate the sleeved sample stream, a detector (41) arranged to receive a fluorescence signal emitted by a particle after light absorption, and an outlet channel (23) for the sleeved sample stream. The detector (41) is arranged to measure fluorescence in order to differentiate the particles. The heating base (3) and the outlet channel (23) form measuring electrodes arranged to measure resistivity in order to count the particles. [Fig. 1]

Description

Title of the invention: Device for counting and differentiating particles in a sample stream

[0001] The present invention relates to the counting and differentiation of particles in a sample stream respectively by electrical and optical measurements.

[0002] Flow cytometry (often referred to by the acronym FCM) is a technique for automatically counting particles suspended in a liquid. This technique also allows for the measurement of the characteristics of each particle, such as size, shape, and complexity. The liquid to be analyzed is flowed through a device—called a cytometer—so that the particles pass one by one through a beam of light emitted, for example, by a laser source, a halogen light source, or light-emitting diodes. Pressure injection is generally used to set the liquid in motion.

[0003] The fluid to be analyzed is generally a biological or even organic fluid, for example cerebrospinal fluid, pleural fluid, fluid taken during a bone marrow puncture or synovial fluid.

[0004] Hematology is one of the most remarkable applications of flow cytometry. The liquid to be analyzed is then a blood sample - which may be whole blood or not - and the particles are blood cells, namely leukocytes (or white blood cells), erythrocytes (or red blood cells) or thrombocytes (or platelets).

[0005] Detectors are used to receive the optical signals emitted by the particles by scattering, reflection or refraction of light. These optical signals are then used to determine the characteristics of each detected particle.

[0006] Among optical signals, small-angle scatter (known by the English acronym FSC for "Forward Scatter") allows the particle size to be determined. 90° scatter (known by the English acronym SSC for "Side Scatter") provides information on the internal structure and granularity of the particles. Finally, the fluorescence emitted by the particles is particularly useful for differentiating them.

[0007] In hematology, fluorescence measurement makes it possible to detect fluorescent dyes used as blood cell markers. For example, it is known to couple a fluorochrome—also called a fluorophore—to a nucleic acid probe, a ligand, or an antibody specific to a certain cell class in order to identify blood cells of that class.

[0008] It is also known to label intracytoplasmic nucleic acids - RNA or DNA - blood cells using fluorescent dyes, for example asymmetric cyanines and in particular Thiazole Orange.

[0009] Regarding particle counting, most flow cytometers use electrical measurements, specifically impedance or resistivity measurements. As explained in European patent EP 0 425 381 B1, the flow cytometer comprises measuring electrodes located on either side of an orifice. The electrodes are electrically powered, thus generating an electric current flowing from one electrode to the other. When a particle passes through the orifice, it causes a point change in the resistivity of the medium between the electrodes, resulting in a voltage pulse proportional to the particle's volume. Detecting these pulses allows the particles to be counted.

[0010] It has been observed that temperature has a direct impact on the measurements performed by the cytometer, whether fluorescence or resistivity. In particular, it is preferable for the liquid to be analyzed with as little temperature variation as possible to compensate for the natural instability of the fluorochrome. Thus, the fluorescence intensity can vary by approximately 5% per degree Celsius.

[0011] To avoid these temperature variations, it is known in the prior art to control the temperature by placing the entire cytometer, including the measuring electrodes and the components necessary for optical measurements, namely the light source, detectors, and any optics, in a thermostatically controlled enclosure or box. Maintaining a stable temperature is also possible using a heat exchanger based on the Peltier effect. Unfortunately, such solutions significantly increase the size and cost of the installation including the cytometer.

[0012] The present invention improves the situation.

[0013] In this respect, the invention relates to a device for counting and differentiating particles in a sample stream comprising:

[0014] - a heating base within which an injection conduit of a sample flow and a sleeve flow injection conduit, and

[0015] - a measuring cell comprising an optical cuvette arranged to be traversed along a flow direction by a sample flow from the heating base and sleeved at least by the sleeve flow, a light source arranged to illuminate, along an optical direction substantially orthogonal to the flow direction, the sleeved sample flow passing through the optical tank, a detector arranged to receive a fluorescence signal emitted by a particle of the sleeved sample flow after absorption of the light emitted by the light source, and an evacuation conduit for the sleeved sample flow communicating with the optical tank.

[0016] The device is arranged to differentiate particles from the sample stream at least by a fluorescence measurement carried out by the detector from each fluorescence signal received and to count the particles by a resistivity measurement carried out by a pair of measuring electrodes formed by the heating base and the evacuation conduit between which an electric current flows to which the particles of the sample stream are able to oppose a resistance.

[0017] Advantageously, the device further includes an electrical insulator connected to the exhaust duct for isolating the measuring cell.

[0018] The electrical insulator is, for example, a Faraday cage delimiting an interior space within which the evacuation conduit is received.

[0019] The electrical insulator can be connected to ground or to a guard electrode.

[0020] In one or more embodiments, the heating base is formed from a piece made from a stainless metal alloy.

[0021] In one or more embodiments, the heating base includes an electrical resistance arranged to convert electrical energy into thermal energy by Joule effect.

[0022] Advantageously, the measuring cell further includes a thermal control circuit arranged to control the temperature of the heating base to a setpoint temperature in order to maintain a substantially constant temperature within the measuring cell.

[0023] In one or more embodiments, the measuring cell further comprises an intermediate piece interposed between the heating base and the optical cell. The intermediate piece has a cavity into which the sample flow injection channel and the sleeving flow injection channel open, and within which the sample flow is intended to be sleeved by the sleeving flow injected into the injection channel. The cavity communicates with the optical cell.

[0024] An additional sleeving flow injection channel can be provided in the intermediate piece for additional sleeving of the sample flow.

[0025] Advantageously, a portion of the additional duct for injecting a sleeving flux is housed within the electrical insulator.

[0026] In one or more embodiments, the light source is a laser source.

[0027] In one or more embodiments, the detector is a photomultiplier.

[0028] In one or more embodiments, the exhaust duct is manufactured at starting from a material containing platinum.

[0029] Typically, the heating base forms a cathode and the exhaust conduit forms an anode.

[0030] In one or more embodiments, an opening is provided to allow The inlet of the sleeved sample stream into the optical cuvette. The orifice is sized so that the passage of a particle generates an increase in the resistivity of the medium located between the measuring electrodes, resulting in a variation of the voltage at the measuring electrodes.

[0031] Other features, details and advantages will become apparent upon reading the detailed description below, and upon analysis of the accompanying drawings on which:

[0032] [Fig-1] illustrates a cross-sectional view of a device for counting and differentiation particle depletion from a sample stream according to the invention, and

[0033] [Fig.2] schematically illustrates associated preparation and processing circuits to the device of [Fig.1].

[0034] Fig. 1 illustrates a device 1 for counting and differentiating particles from a sample stream.

[0035] According to the principle of flow cytometry, the device 1 is arranged to receive a sample flow in which particles are suspended.

[0036] In the following description, we consider the case in which device 1 is intended for a particular field of application: hematology. However, those skilled in the art understand that device 1 can also be used to analyze other biological fluids.

[0037] The sample stream can thus be considered a blood sample comprising blood cells such as leukocytes (or white blood cells), erythrocytes (or red blood cells), or thrombocytes (or platelets). In particular, the sample stream can be described as whole blood when it includes all the blood cells listed above. The analysis of the blood cell lines is called a complete blood count (CBC). Any abnormalities detected can allow a healthcare professional to diagnose certain conditions such as anemia or cancer.

[0038] All these blood cells originate from the same stem cell located in the bone marrow and called a hemocytoblast. These stem cells then differentiate into several subpopulations.

[0039] In the case of hematopoietic cells, those skilled in the art know that cell analysis allows for the discrimination of the main cell lineages, including leukocytes, erythrocytes, and thrombocytes. The leukocyte lineage is itself subdivided into several categories, such as, for example, lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Normally, the blood sample consists of mature cells, that is, cells that are no longer dividing.

[0040] For each of the cell types, the different levels of maturation are known. Thus, the first cells of the erythrocytic lineage are called proerythroblasts. Through successive cell divisions, this lineage continues with basophilic erythroblasts, then type I polychromatophilic erythroblasts, and finally type II polychromatophilic erythroblasts. The latter develop into acidophilic erythroblasts, and then into reticulocytes after the expulsion of the nucleus.

[0041] It is these reticulocytes which, after total loss of residual RNA, differentiate in the blood into erythrocytes.

[0042] Leukocytes originate from the bone marrow in the form of myeloblasts. The leukocyte lineage continues with progranulocytes, which then develop into basophilic, eosinophilic, or neutrophilic granulocytes. Initially unsegmented, the nuclei undergo segmentation as the cells mature.

[0043] Myeloblasts are also the origin of the monocytic lineage which leads to monoblasts, promonocytes and then monocytes which pass into the peripheral blood.

[0044] The pluripotent stem cell from which the myeloblast originates also gives rise to the lymphocyte lineage by differentiation into lymphoid stem cells, part of which – the T lymphocyte lineage – continues its maturation in the thymus and lymph nodes, while the other part remains in the bone marrow to generate the B lymphocyte lineage. These B lymphocytes, once activated in the form of plasma cells, produce antibodies to fight pathogenic antigens.

[0045] Finally, thrombocytes originate from megakaryoblasts, themselves derived from the myeloid progenitor cell from which the myeloblast originates. Once they reach the final stage of their maturation, namely thrombocytogenic megakaryocytes, they produce platelets by the breakdown of their cytoplasm. Young platelets—reticular platelets—contain a load of RNA that is the remnant of their cell of origin.

[0046] Device 1 is arranged to count blood cells from this sample stream from resistivity measurements and to differentiate these blood cells from optical measurements, and in particular from fluorescence measurements.

[0047] The device 1 includes a heating base 3, a measuring cell 5 and optionally an electrical insulator 7.

[0048] The heating base 3 generally forms a support for the device 1. Thus, during its use, the device 1 can rest on the heating base 3 which has, for example, a flat surface to ensure the stability of the device 1.

[0049] In the context of the invention, the heating base 3 is arranged to perform at least the three functions detailed below.

[0050] The first function of the heating base 3 is to ensure the injection, into the device 1, of a sample flow and a sleeving flow.

[0051] To this end, and as illustrated in [Fig.1], an injection channel 9 for a sample flow and an injection channel 11 for a sleeve flow are provided in the heating base 3.

[0052] The injection conduit 9 is arranged to receive a sample stream and guide its flow within the device 1. The injection conduit 9 has at least one opening outside the device 1 to allow the sample stream to be injected. Such injection is typically performed under pressure to set the sample stream in motion. Injection under pressure is advantageous when the sample stream flows, in at least a portion of the injection conduit 9, in a direction opposite to gravity.

[0053] The injection conduit 11 is arranged to receive a sleeving flow and guide its flow within the device 1. Like the injection conduit 9, the injection conduit 11 has at least one opening outside the device 1 to allow the sleeving flow to be injected. Here again, the sleeving flow can be injected under pressure.

[0054] The sleeving flow – sometimes called sleeving fluid – enables hydrodynamic sleeving of the sample flow. Hydrodynamic sleeving has a dual purpose: first, the sleeving flow surrounds the sample flow and guides it out of the injection conduit 9; second, the sleeving flow constricts the sample flow to stretch it and reduce its diameter.

[0055] The second function of the heating base 3 is to increase the temperature of the measuring cell 5, in particular by heating the sample flow and the sleeve flow.

[0056] To achieve this, the heating base 3 includes, for example, an electrical resistance 13 – or resistor – arranged to convert electrical energy into thermal energy by the Joule effect. It is understood that the heating base 3 is then connected to an electrical power supply (not shown in [Fig. 1]) to power the electrical resistance 13 and thus increase the temperature of the heating base 3 until the desired temperature is reached. By way of example, the electrical resistance 13 has a resistance of approximately 20 ohms (Q).

[0057] Other known heating means can obviously be used to increase the temperature of the heating base 3. In general, the heating base 3 is arranged to increase its temperature and, by heat transfer, that of the measuring cell 5.

[0058] The inventors have observed that temperature and its variations have an impact on the measurements performed by device 1, whether resistivity or fluorescence measurements. The intensity of the fluorescence can thus vary by nearly 5% per degree Celsius (°C) while resistivity increases, respectively decreases, when temperature increases, respectively decreases.

[0059] As illustrated in [Fig. 1], the heating base 3 is positioned in contact with the measuring cell 5. This compact design allows the heating base 3 to be as close as possible to the measurements being taken. The heating base 3 thus directly affects the temperature at which the resistivity and fluorescence measurements are recorded, not only by heating the sample flow and the sleeve flow, but also by the local increase in temperature in the immediate vicinity of the measuring cell 5.

[0060] The heating base 3 is thus arranged to provide targeted heating, unlike, for example, the enclosure or thermostatically controlled chamber used in the prior art, which imposes a temperature on the entire cytometer indiscriminately. Furthermore, the heating base 3 drastically reduces the overall size compared to the enclosure or thermostatically controlled chamber.

[0061] By way of example, the heating base 3 can reach and maintain a temperature substantially equal to 35°C.

[0062] It should be noted that, generally speaking, the important thing is to have a sufficiently stable temperature to limit the variation of the resistivity and fluorescence measurements. However, the inventors have found that such stability is more easily achieved by raising the temperature of the measuring cell 5, particularly compared to the temperature of the device 1.

[0063] Consequently, the heating base 3 is arranged to raise the temperature of the measuring cell 5 to a temperature strictly higher than that—that is, the average temperature—of the device 1, and to maintain substantially constant the temperature thus reached by the measuring cell 5. By "substantially constant," it is meant here that, ideally, the temperature reached by the measuring cell 5 by means of the heating base 3 is kept constant. However, in practice, it is not always possible to maintain a perfectly constant temperature, so the temperature actually maintained may deviate by nearly 5% from the target temperature.

[0064] Finally, the third function of the heating base 3 is to form an electrode for measuring resistivity. More precisely, the heating base 3 constitutes a cathode.

[0065] For this purpose, the heating base 3 can be formed from a part made from a stainless metal alloy, for example steel. As an example, 316 stainless steel, which has the particularity of containing molybdenum, can be used to give the heating base 3 the properties of a cathode.

[0066] The general principle of measuring resistivity and base contribution heating cell 3 to this measurement will be detailed below with reference to the description of measuring cell 5.

[0067] The measuring cell 5 is arranged to perform resistivity and fluorescence measurements within the device 1. As mentioned previously, resistivity measurements generally allow blood cells to be counted, while fluorescence measurements, possibly coupled with other optical measurements, allow them to be differentiated.

[0068] It should be noted that resistivity measurements can also be used to differentiate blood cells. In particular, resistivity measurements make it possible to distinguish, within the leukocyte lineage, granulocytes, monocytes, and lymphocytes. The combination of resistivity measurements and optical measurements allows for a more reliable differentiation of the different cell populations.

[0069] The measuring cell 5 includes an optional intermediate piece 15, an optical tank 17, a light emission module 19, a light reception module 21 and an evacuation conduit 23.

[0070] The intermediate piece 15 is arranged to be inserted between the heating base 3 and the optical tank 17. Advantageously, for the sake of compactness, the intermediate piece 15 is, on the one hand, in contact with the heating base 3 – and even in line with it – and, on the other hand, in contact with the optical tank 17. For the sake of simplifying the design of the device 1, the heating base 3 and the intermediate piece 15 can form a single unit. Furthermore, the intermediate piece 15 and the optical tank 17 can be manufactured so as to be fixed to each other.

[0071] The intermediate piece 15 has a cavity 25 into which the injection conduit 9 and the injection conduit 11 open. Consequently, the sample flow is intended to be sleeved within the cavity 25 by the sleeve flow injected into the injection conduit 11.

[0072] The cavity 25 advantageously has the general shape of a cone to facilitate the sheathing of the sample flow. In other words, and as illustrated in [Fig. 1], the dimensions of the cavity 25 decrease as the sheathed sample flow approaches the optical cuvette 17.

[0073] Furthermore, cavity 25 communicates with the optical cuvette 17 via orifice 27. Orifice 27 is provided in device 1 to allow the entry of the sleeved sample stream into the optical cuvette 17. Orifice 27 is therefore sized to allow the passage of blood cells from the sleeved sample stream. It is understood here that the purpose of the sleeved stream injected into the injection conduit 11 is to center, sleeve, and stretch the sample stream relative to orifice 27 and to facilitate the passage of blood cells one after another into orifice 27 at a satisfactory flow rate.

[0074] It is at the level of the orifice 27 - which can be described as a counting orifice - that the blood cells of the sample flow are counted by a resistivity measurement.

[0075] As illustrated in [Fig.1], an additional conduit 29 for injecting a sleeving flow can be provided in the intermediate part 15 to achieve additional hydrodynamic sleeving of the sample flow.

[0076] The additional conduit 29 is comparable to the injection conduit 11. Thus, the additional conduit 29 is arranged to receive a sleeving flow and guide its flow within the device 1. The additional conduit 29 has at least one opening outside the device 1 to be able to inject, for example under pressure, the sleeving flow into it.

[0077] More precisely, the additional conduit 29 opens into an annular chamber 31 formed in the intermediate piece 15 and communicating with the optical cuvette 17. While the first sleeving, obtained by injecting a sleeving flow into the injection conduit 11, allows the sample flow to be centered and refined for counting blood cells at the orifice 27, the second sleeving allows the guidance of the sleeved sample flow within the optical cuvette 17 to be reinforced. The second sleeving also prevents the phenomenon of recirculation, i.e. a new passage of a blood cell through the optical measurement point, i.e. the intersection of the circulation direction X and the optical direction Y on the [Fig.1].

[0078] It should be noted that the intermediate piece 15 is accessory. Indeed, the outlet of the injection conduit 9 can coincide with the orifice 27 so that the injected sample flow is conducted directly to the optical cell 27. In such a case, the sleeving flow injected into the injection conduit 11 provides hydrodynamic sleeving of the sample flow in the optical cell 17 for the purposes of fluorescence measurement. It is then understood that additional sleeving is not necessary. It is also possible to provide the intermediate piece 15 without the injection conduit 29. In this particular case, the sleeving flow injected into the injection conduit 11 provides hydrodynamic sleeving of the sample flow in the cavity 25. Such sleeving contributes both to the resistivity measurement at the orifice 27 and to the fluorescence measurement within the optical cell 17.

[0079] The optical tank 17 is arranged to be traversed in a flow direction X by the sample flow from the heating base 3 either indirectly, in the presence of the intermediate piece 15, or directly, in the absence of the intermediate piece 15.

[0080] The sleeving of the sample flow carried out at the level of the optical cuvette 17 allows the sample flow to be centered, stretched and guided so that the blood cells flow one after another along the flow direction X from the orifice 27 to the drain duct 23.

[0081] The optical tank 17 is further arranged to allow the passage of light. Indeed, the optical tank 17 is positioned, within the measuring cell 5, between the light-emitting module 19 and the light-receiving module 21. The optical tank 17 is thus delimited by walls made of a transparent material, for example glass.

[0082] The light emission module 19 is arranged to emit light in the direction of the optical tank 17, and in particular of the sleeved sample flow circulating in the optical tank 17.

[0083] In the embodiment illustrated in [Fig.1], the light emission module 19 comprises a light source 33 and a shaping optic 35.

[0084] The light source 33 is arranged to illuminate, along an optical direction Y substantially orthogonal to the flow direction X, the flow of sleeved sample passing through the optical tank 17. By "substantially orthogonal," it is meant here that, ideally, the flow direction X and the optical direction Y form an angle of 90°. However, in practice, it is not always possible to obtain exactly an angle of 90°, so that the angle actually obtained may deviate by nearly 5% from 90°.

[0085] Typically, the light source 33 is arranged to emit a light beam along the optical direction Y.

[0086] Advantageously, the light source 33 is a laser source. A laser source typically comprises a laser diode arranged to emit coherent monochromatic light. For example, the light source 33 is a blue laser source, in which case the laser diode emits light with a wavelength between 380 and 500 nanometers (nm). In particular, it is possible to use a laser diode from the OSRAM brand (registered trademark) emitting at 488 nm. Such a laser diode has a power output of approximately 50 milliwatts (mW).

[0087] The shaping optics 35 are arranged to converge the rays of the light beam emitted by the light source 33 at a point of passage of the blood cells in the sleeved sample flow. Such a point of passage can be identified in [Fig. 1] by the intersection of the flow direction X and the optical direction Y.

[0088] The shaping optics 35 are particularly useful when the light beam is wide and the rays composing it are emitted by the light source 33 in different directions. The shaping optics 35 are of less interest when the light source 33 is a laser source and the emitted light beam is narrow with light rays that are substantially parallel to each other. The shaping optics 35 is optional.

[0089] The shaping optics 35 may include a spherical lens with a focal length of approximately 75 millimeters (mm). For example, the shaping optics 35 is formed by an achromatic doublet from the brand THORLABS (registered trademark) corresponding to the reference AC127-075-A.

[0090] Advantageously, the shaping optics 35 is positioned so that the circulation direction X is included in the object focal plane of the shaping optics 35.

[0091] The blood cells of the sleeved sample flow circulate in the optical tank 17 from the orifice 27 to the discharge conduit 23 along the flow direction X. The blood cells are illuminated by the light source 33 at a point corresponding to the intersection of the flow direction X and the optical direction Y. Due to the sleeve, the blood cells are illuminated one after the other.

[0092] Each blood cell illuminated by the light source 33 then emits a fluorescence signal. The light emission of a blood cell, or more precisely, of a fluorochrome coupled to a nucleic acid probe, a ligand, or an antibody to bind specifically to the blood cell, is called a "fluorescence signal." This light emission results from the excitation of the fluorochrome's electrons caused by the absorption of photons from the light emitted by the light source 33.

[0093] Advantageously, several fluorochromes can be used to label different types of blood cells and thus differentiate them. It is also possible to use fluorescent dyes.

[0094] Among the known fluorescent markers, it is possible to use, for example, 4',6-diamidino-2-phenylindole (better known by the acronym DAPI), Thiazole Orange, phycoerythrin, the Alexa Fluor range of fluorochromes produced by the company Molecular Probes (registered trademark) or propidium iodide.

[0095] The light receiving module 21 is arranged to receive a fluorescence signal emitted by a blood cell from the sleeved sample stream after absorption of the light emitted by the light source 33.

[0096] In the example illustrated in [Fig. 1], the light receiving module 21 comprises a focusing optic 37, a diaphragm 39 and a detector 4L

[0097] The focusing optics 37 are arranged to focus the fluorescence signal emitted by each blood cell onto the detector 4L

[0098] The focusing optics 37 includes, for example, a doublet of lenses.

[0099] The diaphragm 39 is arranged to perform spatial filtering of the fluorescent signal fluorescence. The diaphragm 39 is more precisely an aperture diaphragm allowing the passage of light rays other than the fluorescence signal to the detector 41.

[0100] The detector 41 is arranged to perform a fluorescence measurement from each received fluorescence signal. In particular, the detector 41 is arranged to convert each received fluorescence signal into an electrical signal. The electrical signal is representative of the fluorochrome emitting the fluorescence signal and therefore of the type of blood cell to which the fluorochrome is bound.

[0101] It is understood here that the detector 41 performs a fluorescence measurement for each blood cell in the sleeved sample flow which, in response to the absorption of the light emitted by the light source 33, emits a fluorescence signal towards the light receiving module 21.

[0102] Advantageously, the detector 41 is a photomultiplier tube (PMT). A photomultiplier tube has the advantage of a high gain, on the order of 10⁶, which is particularly suitable for detecting a fluorescence signal, which is generally quite weak. For example, a silicon photomultiplier tube (SiPM) can be used.

[0103] The description of device 1, and more specifically of measuring cell 5, focuses on the measurement of fluorescence. However, it should be noted that other optical signals can be analyzed. For example, small-angle scattering (FSC) allows the size of blood cells to be determined, while 90° scattering (SSC) provides information on the shape, internal structure, and granularity of blood cells.

[0104] The measurement cell 5 can thus include detectors dedicated to the analysis of optical signals other than the fluorescence signal. It is known, in particular, to use dichroic mirrors to decompose optical signals and redirect them to the appropriate detectors. It is understood that, contrary to what [Fig. 1] suggests, the light-receiving module 21 is not necessarily located on the optical Y-axis.

[0105] Therefore, those skilled in the art understand that the embodiment illustrated in [Fig. 1] is not limiting and that the measuring cell 5 may include one or more additional light-emitting modules and one or more additional light-receiving modules for performing optical measurements other than fluorescence measurement. Such measurements include, for example, small-angle scattering measurements, 90° scattering measurements, extinction measurements, and large-angle scattering measurements. These optical measurements are complemented Comments on fluorescence measurement allow the collection of information regarding the characteristics of blood cells. Such information notably helps to improve blood cell differentiation.

[0106] From a structural point of view, it is also possible, rather than adding one or more light emission modules and one or more light reception modules, to equip the light emission module 19 with one or more additional light sources - which can also be laser sources - and the necessary optics; and to equip the light reception module 21 with one or more additional detectors - which can also be photomultipliers - and the necessary optics.

[0107] It should therefore be noted here that the device 1 is arranged to differentiate blood cells at least by a fluorescence measurement, but that this can be supplemented by optical measurements such as those mentioned above, and that, where appropriate, the measuring cell 5 includes the means necessary for this purpose. By way of example, international patent application WO 2021 / 144545 A1 describes emission guns and reception guns for carrying out small-angle scattering measurements, 90° scattering measurements, extinction measurements, and large-angle scattering measurements.

[0108] Finally, the evacuation conduit 23 communicates with the optical tank 17 and is arranged to evacuate the sleeved sample stream.

[0109] Furthermore, the discharge conduit 23 is further arranged to form an electrode for measuring resistivity. More specifically, the discharge conduit 23 constitutes an anode. For this purpose, the discharge conduit 23 can be made from a platinum-containing material.

[0110] It is therefore understood that the heating base 3 and the discharge conduit 23 form a pair of measuring electrodes arranged to perform a resistivity measurement. To do this, an electric current is applied across the terminals of the measuring electrodes, namely the cathode formed by the heating base 3 and the anode formed by the discharge conduit 23. An electric current then flows between the heating base 3 and the discharge conduit 23, and passes through the orifice 27. Consequently, the passage of a blood cell from the sample flow through the orifice 27 generates a momentary increase in the resistivity of the medium located between the measuring electrodes, resulting in a voltage pulse proportional to the volume of the blood cell.

[0111] Equivalently, the quantity measured by the pair of measuring electrodes can be an impedance.

[0112] Furthermore, the measuring cell 5 can also include a thermal control circuit (not shown in [Fig.1]) coupled to the heating base 3 and arranged to control the temperature of the heating base 3 to a setpoint temperature.

[0113] Indeed, as explained previously, temperature has an impact on resistivity and fluorescence. It is therefore particularly advantageous to maintain the measuring cell 5 at a substantially constant temperature to obtain reliable values ​​for resistivity and fluorescence intensity.

[0114] Such a thermal control circuit takes for example the form of a control loop with, as inputs, a setpoint temperature and the temperature measured at the level of the measuring cell 5. The thermal control circuit may include one or more temperature sensors to measure the temperature.

[0115] In addition to the temperature measured at the measuring cell 5, the thermal control circuit can also receive as input the temperature of device 1, and not just that of the measuring cell 5. The temperature of device 1 does indeed have an impact on the temperature of the measuring cell, and it can therefore be advantageous to take this into account. The temperature of device 1, and thus of the measuring cell 5, can also depend on the temperature of the laboratory where device 1 is installed. Typically, the thermal control circuit can therefore include a temperature sensor for the measuring cell 5, but also a temperature sensor for device 1, or even a temperature sensor for the laboratory.

[0116] Finally, the electrical insulator 7 is arranged to protect the measuring cell 5 from electromagnetic fields. To this end, the electrical insulator 7 is connected to the exhaust conduit 23. Such isolation of the measuring cell 5 makes it possible to limit disturbances in the resistivity measurement and to obtain a better signal-to-noise ratio (SNR).

[0117] The electrical insulator 7 is for example a Faraday cage delimiting an interior space within which the evacuation conduit 23 is received.

[0118] In the example illustrated in [Fig. 1], the electrical insulator 7 is connected to ground. Furthermore, electrical connections of the electrical insulator 7 can be connected either to ground or to a guard electrode.

[0119] Indeed, the exhaust duct 23 can behave like an electrical wire during the use of the device 1 and thus interfere with the resistivity measurements. The electrical insulator 7 prevents such interference.

[0120] Furthermore, in the particular embodiment in which the measuring cell 5 includes the intermediate piece 15 and in which an additional conduit 29 for injecting a sleeve flux is provided in the intermediate piece 15, a portion of the additional conduit 29 may be housed within the electrical insulator 7. It is understood here that the additional conduit 29 comprises a part, visible on [Fig.1], provided within the intermediate piece 15 and a part, not shown on [Fig.1], outside the intermediate piece 15 and of which at least a portion is housed within the electrical insulator 7.

[0121] Just like the exhaust duct 23, the additional duct 29 can indeed behave like an electrical wire and thus interfere with resistivity measurements. Here again, the electrical insulator 7 prevents such interference.

[0122] Fig. 2 schematically illustrates preparation and processing circuits associated with device 1.

[0123] A hydraulic distribution assembly 43 is arranged to supply the device 1 with sample and sleeve flow.

[0124] In particular, the hydraulic distribution assembly 43 may include a blood sample storage unit in which several preparation operations may be carried out before the injection of a sample stream into the injection conduit 9 of the device 1. For example, one or more fluorochromes respectively coupled to a nucleic probe, a ligand or an antibody may be mixed with blood samples to promote the binding of each fluorochrome with a blood cell according to the type thereof.

[0125] As explained previously, the use of fluorochromes makes it possible to differentiate blood cells from a fluorescence signal emitted by each blood cell to the light receiving module 21 after absorption of the light emitted by the light source 33.

[0126] Furthermore, it should be noted here that the device 1 can be used to analyze biological or even organic fluids other than blood samples. Consequently, the hydraulic distribution assembly may include a storage unit for cerebrospinal fluid, pleural fluid, fluid obtained during a bone marrow aspiration, or synovial fluid. A "storage unit" here generally refers to any container or receptacle capable of storing a biological fluid in order to supply the device 1 with a sample flow of that fluid. The storage unit may, for example, be a sample tube or a blood collection tube.

[0127] A resistivity measurement processing circuit 45 is arranged to receive the voltage impulse generated by the passage of each blood cell through the orifice 27 of the device 1.

[0128] Indeed, as explained previously, the passage of a blood cell through the orifice 27 causes a punctual increase in the resistivity of the medium located between the measuring electrodes, namely the heating base 3 and the evacuation conduit 23.

[0129] The processing circuit 45 is therefore arranged to detect each voltage pulse and count the blood cells in the sample flow. To this end, the processing circuit 45 includes, for example, a voltmeter connected to the terminals of the measuring electrodes to measure the voltage between the cathode – i.e., the heating base 3 – and the anode - therefore the exhaust duct 23.

[0130] The processing circuit 45 may also include a voltage amplification card - also called a voltage amplifier - to raise the voltage measured across the terminals of the measuring electrodes and thus facilitate the detection of a pulse.

[0131] The output of the processing circuit 45 is typically a number of blood cells detected and counted.

[0132] A processing circuit 47 for optical measurements is arranged to receive at least the fluorescence measurements performed by detector 4L

[0133] More specifically, the processing circuit 47 is arranged to receive each electrical signal generated by the detector 41 from a fluorescence signal received from a blood cell. The electrical signal received by the processing circuit 47 is characteristic of the fluorescence measurement and therefore of the type of blood cell.

[0134] The fluorescence measurements of detector 41 thus enable the processing circuit 47 to differentiate several types of blood cells, for example: lymphocytes, monocytes, neutrophils, eosinophils, basophils, or erythroblasts. In general, the processing circuit 47 can be configured to differentiate the various cells of the leukocyte, erythrocyte, and thrombocytic lineages detailed previously.

[0135] The output of the processing circuit 47 is typically the type of blood cell detected, or even the distribution of blood cells according to their type.

[0136] As explained previously, optical measurements other than fluorescence measurement can be performed to identify and differentiate the characteristics of blood cells, including small-angle scattering measurements, 90° scattering measurements, extinction measurements, and large-angle scattering measurements. The processing circuit 47 can, where appropriate, be arranged to receive additional optical measurements performed by detectors other than detector 41, whether such detectors are included in the light-receiving module 21 or in other light-receiving modules not shown in [Fig. 1].

[0137] Finally, a screen 49 is arranged to receive the outputs of the processing circuit 45 and the processing circuit 47 and to make the results of the counting and differentiation accessible to a user, for example a healthcare professional.

[0138] The screen 49 is thus arranged to display the number of blood cells counted and the distribution of these blood cells by type. The screen 49 can also be coupled to means for printing the results on paper.

[0139] In the example illustrated in [Fig.2], the hydraulic distribution assembly 43, the treatment circuit 45, the treatment circuit 47 and the screen 49 are separate from the device 1. However, all or part of these elements may be integrated into device 1. In particular, the processing circuit 45 and the processing circuit 47 may possibly be considered as part of the measuring cell 5.

Claims

Demands

1. Device (1) for counting and differentiating particles in a sample stream comprising: - a heating base (3) in which are provided an injection channel (9) for a sample stream and an injection channel (11) for a sleeved stream, and - a measuring cell (5) comprising an optical cuvette (17) arranged to be traversed in a direction of flow (X) by a sample stream from the heating base (3) and sleeved at least by said sleeved stream, a light source (33) arranged to illuminate, in an optical direction (Y) substantially orthogonal to the direction of flow (X), the sleeved sample stream passing through the optical cuvette (17), a detector (41) arranged to receive a fluorescence signal emitted by a particle of the sleeved sample stream after absorption of the light emitted by the light source (33),and a sleeved sample flow discharge conduit (23) communicating with the optical cell (17), said device (1) being arranged to differentiate particles from the sample flow at least by a fluorescence measurement performed by the detector (41) from each received fluorescence signal and to count said particles by a resistivity measurement performed by a pair of measuring electrodes formed by the heating base (3) and the discharge conduit (23) between which an electric current flows to which the particles of the sample flow are able to oppose a resistance.

2. Device (1) according to claim 1, further comprising an electrical insulator (7) connected to the exhaust conduit (23) for the isolation of the measuring cell (5).

3. Device (1) according to claim 2, wherein the electrical insulator (7) is a Faraday cage delimiting an interior space within which the evacuation conduit (23) is received.

4. Device (1) according to claim 2 or 3, wherein the electrical insulator (7) is connected to ground or to a guard electrode.

5. Device (1) according to any one of the preceding claims, wherein the heating base (3) is formed from a piece made from a stainless metal alloy.

6. Device (1) according to any one of the preceding claims, in which the heating base (3) includes an electrical resistance (13) arranged to convert electrical energy into thermal energy by Joule effect.

7. Device (1) according to any one of the preceding claims, wherein the measuring cell (5) further comprises a thermal control circuit arranged to control the temperature of the heating base (3) to a setpoint temperature for the purpose of maintaining a substantially constant temperature within the measuring cell (5).

8. Device (1) according to any one of the preceding claims, wherein the measuring cell (5) further comprises an intermediate piece (15) interposed between the heating base (3) and the optical tank (17), said intermediate piece (15) having a cavity (25) into which open the injection conduit (9) of the sample flow and the injection conduit (11) of the sleeving flow and within which the sample flow is intended to be sleeved by the sleeving flow injected into the injection conduit (11), said cavity (25) communicating with the optical tank (17).

9. Device (1) according to claim 8, wherein an additional sleeving flow injection conduit (29) is provided in the intermediate piece (15) for additional sleeving of the sample flow.

10. Device (1) according to claims 2 and 9 taken in combination, wherein a portion of the additional conduit (29) for injecting a sleeving flux is housed within the electrical insulator (7).

11. Device (1) according to any one of the preceding claims, wherein the light source (33) is a laser source.

12. Device (1) according to any one of the preceding claims, wherein the detector (41) is a photomultiplier.

13. Device (1) according to any one of the preceding claims, wherein the discharge conduit (23) is made from a material comprising platinum.

14. Device (1) according to any one of the preceding claims, wherein the heating base (3) forms a cathode and the discharge conduit (23) forms an anode.

15. A device (1) according to any one of the preceding claims, wherein an orifice (27) is provided to allow the entry of the sleeved sample stream into the optical cuvette (17), said orifice (27) being dimensioned such that the passage of a particle generates an aug- Change in the resistivity of the medium located between the measuring electrodes resulting in a variation of the voltage at said measuring electrodes.

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