Method for analyzing a biological sample containing biological cells and analysis device for carrying out the analysis method

Description

[0001] The present invention relates to a method for analyzing a biological sample containing biological cells and in particular blood cells, and an analysis apparatus suitable for implementing such an analysis method.

[0002] The cells circulating in the blood include non-nucleated cells such as red blood cells or erythrocytes (about 5 million per mm 3 of normal blood), platelets (about 300,000 per mm 3 ), and nucleated cells, leukocytes (about 10,000 per mm 3 ). The blood may contain other nucleated cells, such as erythroblasts, which are immature red blood cells, or other rarer cells. Each type of cell constitutes what is also called a population.

[0003] Other biological fluids contain blood cells, such as cerebrospinal fluid or urine. Subsequently, we speak of a biological sample, but this is not limited to a blood sample but to all biological fluids containing blood cells.

[0004] Conventional hematology analyzers, whether using flow cytometry or not, are intended to perform the Blood Count and to provide qualitative information when quantitative abnormalities are detected by cell type.

[0005] Specialized cytometry equipment in immunohematology allows, thanks to more sophisticated techniques and the use of specific reagents, to target hematological pathologies that can affect all types of cells.

[0006] It should be remembered that flow cytometry consists of performing at least one hydrodynamic focusing and passing the blood cells one by one through a measuring device which, depending on what is implemented, produces a certain number of physical measurements for each cell. For the measurements to be made distinctly, the cells must be separated and pass at speeds that allow the measurements and their acquisition. In addition, the counts must be sufficient to allow correct statistical evaluations of each population. To do this, the blood sample is not analyzed pure but diluted. In addition, given the thousand times higher quantities of red blood cells than leukocytes, the sample, to perform an analysis that is statistically representative of the leukocyte populations, must pass for several minutes in the cytometer.The other solution, to reduce the time taken for the sample to pass through the cytometer by a factor of ten, consists of carrying out, in addition to a dilution of the sample, a lysis which selectively destroys the red blood cells in order to have a sample sufficiently concentrated in leukocytes and thus have statistically correct counts for these populations.

[0007] The flow cytometry parameters produced are of two kinds: either of a physical or morphological nature: measurement of cell volume by impedance (Coulter effect), optical absorption measurements, diffraction at different angles generated by the passage of each cell in a laser beam and fluorescence measurement of nucleic acids made fluorescent by one or more fluorochromes previously placed in the presence of the cells. These data make it possible to characterize each cell by its volume, by its optical absorption which depends on its surface and its content, by its internal complexity, by the nature of its surface, its composition and by its nucleic activity for nucleated cells; or of an immunological nature (immunohematology): the sample is placed in the presence of reagents containing antibodies conjugated to a fluorochrome and specific for receptors expressed on the surface of certain cells.Passing cells through one or more laser beams generates signals proportional to cell surface markers and thus allows the cells to be characterized.

[0008] A method for analyzing a biological sample containing biological cells comprises the following steps in a known manner: passage of the biological cells of the biological sample to be analyzed in a measuring cell of a flow cytometer, measurement of N cytometry parameters, such as cytometry parameters of a morphological or immunological nature, for each biological cell contained in the biological sample to be analyzed, determination, for each biological cell of the biological sample to be analyzed, of a point in an N-dimensional space whose coordinates are defined according to the cytometry parameters measured for the corresponding biological cell, where N is an integer greater than or equal to 2, for equipment whose function is limited to blood count, grouping of the points into different clusters of cells according to the cytometry parameters measured, so as to define a sample cluster file, the grouping step then being carried out using automatic zoning methods,statistical methods or point density methods, for more sophisticated equipment carrying out hemato-immunological measurements, either visual analysis by an operator, such as a hematologist, a cytometry specialist of representations of the points determined on two measurement axes and grouping of the points into different clusters of cells according to the grouping criteria by thresholds or by zones chosen by the operator, or automatic grouping of the points into different clusters of cells according to the cytometry parameters measured, so as to define a sample cluster file, and computer analysis of the sample cluster file and in particular of the different clusters of cells, the analysis step aiming to qualify and quantify the clusters of cells having common characteristics,identification by the operator of one or more anomalies in the sample cluster file compared to normal samples.

[0009] From the anomalies identified, for example by crossing numerical thresholds (abnormally low or abnormally high number of a type of cells), and from his experience in medical cytometry, the operator may be able either to directly determine the pathology of the patient whose biological sample was analyzed and the associated treatment, or to issue a prognosis concerning the possible pathology of the patient whose biological sample was analyzed and to consider additional blood tests in order to confirm his prognosis.

[0010] However, the grouping, analysis, and identification steps are quite subjective and highly dependent on the cytometry operator's experience, so that a diagnosis or prognosis given for a patient by an operator depends on the operator's qualification and experience. Regardless, these steps are laborious and time-consuming. The processing rates of these devices are low compared to those of hematology counters.

[0011] In addition, cytometry in immunohematology uses antibodies to characterize cells in a very specific manner and uses expensive products, making it difficult for cytometry to establish itself as a routine method.

[0012] Finally, this type of analysis aimed at grouping cells with common characteristics aims to count and qualify populations in order to qualify anomalies by population type. This type of analysis is therefore not interested in all the data as a global image of the patient's sample.

[0013] Document US5605805 discloses a method for determining the lineage of acute leukemia cells in a sample by flow cytometry, the method including steps of identifying normal cell populations by comparing an immunological profile and a scatter profile of each cell population to a scatter profile and an immunological profile expected for normal cells; subtracting the scatter profile of the normal cell population from the scatter profile of each group containing cells belonging to the normal cell population; and determining the lineage of the remaining abnormal cell populations by comparing the scatter profiles and immunological profiles of the abnormal cell populations to the scatter profiles and immunological profiles expected for known leukemic cell populations.

[0014] The present invention aims to overcome these drawbacks and to introduce a new paradigm by using the data collected by the flow cytometer to characterize the biological cells, automatically group them by cluster, identify and qualify the populations of the clusters but also to consider all of the data as an image representation of the biological sample and also to process this image in relation to reference images.

[0015] The technical problem underlying the invention consists in particular in providing a method for analyzing a biological sample containing biological cells which optimizes the processing of data from the measurements, which ensures reproducibility of the results obtained, while ensuring that this method is compatible with high-speed equipment and does not require any particular skill or time from the operator.

[0016] To this end, the present invention relates to a method for analyzing a biological sample containing biological cells including blood cells, the analysis method comprising the following steps: passage of the biological cells of the biological sample to be analyzed in a measuring cell of a flow cytometer, measurement of N cytometry parameters of the biological cells contained in a biological sample to be analyzed, determination, for each biological cell of the biological sample to be analyzed, of a point in an N-dimensional space whose coordinates are defined as a function of the cytometry parameters measured for the corresponding biological cell of the biological sample to be analyzed, where N is an integer greater than or equal to 3, automatic grouping of the determined points into different clusters of cells as a function of the cytometry parameters measured for each biological cell of the biological sample to be analyzed, so as to define a sample cluster file,the sample cluster file advantageously being a computer file digitally describing the biological sample to be analyzed and preferably being written according to a standard, identification of the cell populations defined by the different cell clusters of the sample cluster file, counting the points of each cell cluster of the sample cluster file, comparison of the sample cluster file with reference cluster files, each of the reference cluster files being defined from cytometry parameters of a respective pathological or abnormal biological sample.

[0017] Such a configuration of the analysis method according to the present invention makes it possible to automatically identify a possible resemblance between the sample cluster file, which digitally describes the biological sample to be analyzed, and one or more reference cluster files, and therefore to improve the relevance of the indications given to an operator (via, for example, specific indications or alarms added to the measurements given) at the end of the comparison step and thus to contribute to giving the most relevant recommendations possible to the operator in order to speed up the diagnoses.In particular, the analysis method according to the present invention allows the laboratory to save time in the decisions to be taken, to specify the scope of subsequent investigations and to direct, if necessary, hematologists more quickly towards additional analyses which can be blood smears followed by microscopic analyses or immunological analyses and this with simple, very inexpensive and robust means. It is not a question of substituting this method for the image analysis of the blood smear but of extracting the maximum possible information from the digital blood count and this without additional cost or material or time.

[0018] The analysis method according to the present invention consists more particularly in carrying out, on the basis of cytometric measurements, a comparative morphological analysis of the biological sample considered, with reference biological samples, and more precisely a comparative morphological analysis of the sample cluster file, which is established from the analyzed biological sample, with reference cluster files established from pathological or abnormal biological samples for which the clinical data are known.

[0019] The analysis method according to the present invention is therefore based on the creation of reference files obtained from samples whose pathology is known. This is in fact a learning process carried out by compiling clinical trials and which can progress according to new reference files corresponding to new anomalies or pathologies.

[0020] Each learning version is not specific to each machine but is validated and applies to all machines of the same type that operate with reagents of identical compositions for the preparations (dilution, lysis, fluorescent labeling).

[0021] The cells that circulate in the blood result from what is produced by hematopoiesis, by the detachment of endothelial cells and by infections by allogenic agents such as bacteria or parasites like plasmodium. Hematopoiesis produces in the bone marrow, on the one hand leukocytes, that is to say polymunuclear cells or neutrophilic, eosinophilic and basophilic granulocytes, monocytes and lymphocytes, and on the other hand erythrocytes and platelets. Each type of cell can be affected by different types of pathologies that can be at the origin in the blood of immature cells such as erythroblasts which are erythrocytes still without their nucleus or reticulocytes which no longer have a nucleus but still have ribosomal and mitochondrial activity.

[0022] According to one embodiment of the invention, the cytometry parameters measured are physical measurements such as the volume of each biological cell, optical absorption, wide-angle diffraction, small-angle diffraction.

[0023] The analysis method may further have one or more of the following characteristics, taken alone or in combination.

[0024] According to one embodiment of the invention, the biological cells to be analyzed may be blood cells, and more particularly blood cells that have been the subject of a preparation, either of an isotonic dilution to preserve the cells and to be able to space them sufficiently in the cytometer and measure, under the best conditions, each cytometry parameter, or of a selective lysis operation which will eliminate the red blood cells which are approximately a thousand times more numerous than the white blood cells, this lysis operation having the effect of being able to identify and count the leukocytes in a shorter time but this also has the consequence of modifying, depending on the type of lysis, the volume and the optical response of the non-lysed cells observed.

[0025] In fact, the preferred embodiment of the invention uses two cytometers in parallel, a first in which the biological sample is only diluted and the second, which processes the lysed biological sample, more particularly to observe the nucleated cells. This makes it possible to collect data in spaces with dimensions N > 3 for both the lysed biological sample and the non-lysed biological sample. This means that non-nucleated cells such as erythrocytes and platelets are processed by the same types of algorithms as nucleated cells.

[0026] According to one embodiment of the invention, the cluster grouping step is carried out by automatic processing, according to specific algorithms, of the points determined by the N cytometry parameters corresponding to each biological cell of the biological sample to be analyzed. The different points can for example be grouped into different clusters of points according to statistical criteria or criteria of density of these points in the N-dimensional space, so as to define a file digitally describing the biological sample to be analyzed by points representing each cell, grouped into clusters positioned in the N-dimensional space.

[0027] According to one embodiment of the invention, the analysis method comprises a step of sampling and digitizing a set of analog signals generated during the duration of the measurement step so as to define a first raw data file analogous to a digitized oscillogram for each of the N measurement channels, and a step of synchronizing and grouping the N digitized signals for each biological cell of the sample to be analyzed by a first level of computer processing.

[0028] According to one embodiment of the invention, the grouping step consists of grouping the determined points into different clusters of cells using statistical methods or methods making it possible to isolate the clusters by spatial density analysis of the points representing the cells in the N-dimensional space.

[0029] According to one embodiment of the invention, each coordinate axis of the N-dimensional space corresponds to a respective cytometry parameter.

[0030] According to one embodiment of the invention, N is an integer greater than or equal to 4, and may for example be equal to 5.

[0031] According to one embodiment of the invention, the sample cluster file is in FCS (Flow Cytometry Standard) format.

[0032] According to one embodiment of the invention, the analysis method further comprises a step of transmitting an alarm message when the sample cluster file is at least partially identical or similar to a reference cluster file, and for example when predetermined cell clusters of the sample cluster file are identical or similar to predetermined cell clusters of the reference cluster file.

[0033] According to one embodiment of the invention, the alarm message sent contains indications relating to a pathology or an abnormality associated with the reference cluster file to which the sample cluster file is at least partially identical or similar. These provisions make it possible to communicate to the operator a probability of pathology associated with the analyzed biological sample, but in no case constitutes a diagnosis of the pathology from which the patient from whom the analyzed biological sample was taken may be suffering.

[0034] According to one embodiment of the invention, the analysis method further comprises a step of analyzing the sample cluster file so as to detect at least one possible anomaly in the sample cluster file.

[0035] In an example which is not part of the claimed invention, the comparison step is carried out only when at least one anomaly is detected during the analysis step.

[0036] According to one embodiment of the invention, when at least one anomaly is detected during the analysis step, the alarm message sent during the transmission step also contains information relating to the at least one anomaly detected.

[0037] According to one embodiment of the invention, the analysis step comprises a step of analyzing, for each cluster of cells in the sample cluster file, at least one morphological parameter of said cluster of cells.

[0038] According to one embodiment of the invention, the at least one morphological parameter of each cell cluster of the sample cluster file may comprise the positioning of said cell cluster, the distribution of the points of said cell cluster, the number of points of said cell cluster, and / or the presence or absence of said cell cluster. Thus, the detection of an abnormal count of a cell cluster, of an abnormal relative positioning between different cell clusters or of the presence of a cell cluster relating to a population of abnormal cells makes it possible to detect an anomaly in the sample cluster file.

[0039] According to one embodiment of the invention, the analysis step comprises a step of detecting an anomaly if at least one morphological parameter of at least one cluster of cells in the sample cluster file exceeds a respective predetermined threshold value.

[0040] According to one embodiment of the invention, the analysis step comprises the following steps: comparing, for each cell cluster of the sample cluster file, the number of points grouped in said cell cluster with at least one respective predetermined threshold value, detecting an anomaly if the number of points grouped in at least one of the cell clusters is lower and / or higher than the at least one respective predetermined threshold value.

[0041] According to one embodiment of the invention, the analysis step comprises the following steps: comparing, for each cell cluster of the sample cluster file, the number of points clustered in said cell cluster with a respective predetermined lower threshold value or with a respective predetermined upper threshold value, detecting an anomaly if the number of points clustered in at least one of the cell clusters is less than the respective predetermined lower threshold value or greater than the respective predetermined upper threshold value.

[0042] According to one embodiment of the invention, the analysis step comprises the following steps: analysis of the distribution of points in each cell cluster of the sample cluster file, detection of an anomaly if the distribution of points in at least one of the cell clusters is not Gaussian.

[0043] According to one embodiment of the invention, the analysis step comprises the following steps: analysis of the positioning of cell clusters in the sample cluster file, detection of an anomaly if at least two cell clusters in the sample cluster file are at least partially confused.

[0044] According to one embodiment of the invention, the analysis step comprises the following steps: analysis of cell clusters from the sample cluster file, detecting an anomaly if the presence or absence of at least one predetermined cell cluster is detected.

[0045] According to one embodiment of the invention, the analysis step comprises the following steps: analysis of the sample cluster file, detection of an anomaly if the number of cell clusters is higher or lower than a predetermined reference value.

[0046] According to one embodiment of the invention, the analysis step comprises a step of searching for clusters of abnormal or atypical cells, i.e. located outside the clusters of normal cells, the clusters of abnormal or atypical cells being able, for example, to correspond to immature cells or to parasites. The analysis method may further comprise a step of comparing these clusters of abnormal or atypical cells with the reference files.

[0047] According to one embodiment of the invention, the step of measuring cytometry parameters comprises at least one step of measuring cytometry parameters representative of the morphology and / or the structure of the biological cells of the biological sample to be analyzed.

[0048] According to one embodiment of the invention, the step of measuring cytometry parameters comprises at least one step of measuring, for each biological cell of the biological sample to be analyzed, at least one optical property of said biological cell.

[0049] According to one embodiment of the invention, the step of measuring cytometry parameters comprises at least one step of measuring, for each biological cell of the biological sample to be analyzed, at least one electrical and / or electromagnetic property of said biological cell.

[0050] According to one embodiment of the invention, the step of measuring cytometry parameters comprises a step of measuring a quantity of light absorbed or re-emitted by each biological cell of the biological sample to be analyzed.

[0051] According to one embodiment of the invention, the step of measuring cytometry parameters comprises: a step of measuring the intensity of a light beam scattered at small angles by each biological cell, and / or a step of measuring the intensity of a light beam scattered at 90° by each biological cell, and / or a step of measuring the intensity of a light beam scattered along an optical path of the incident light beam by each biological cell.

[0052] The light scattered by each biological cell provides information on the morphology and structure of said biological cell. In particular, the intensity of a light beam scattered at small angles, for example at angles less than 15°, advantageously equal to 4° and / or 9°, by each biological cell is substantially proportional to the size of said biological cell, while the intensity of a light beam scattered at 90° by each biological cell is proportional to the shape, internal structure and granularity of said biological cell. In addition, the intensity of a light beam in the optical axis of the incident light beam by each biological cell is proportional to the size and viability of said biological cell. Such a measurement of scattering along the optical path of the incident light beam corresponds to a measurement of the intensity of the light absorption of each biological cell.

[0053] Thus, the simultaneous use of these two or three aforementioned parameters (impedance measurement, optical absorption measurement, scattering measurements at different angles) makes it possible to distinguish, in a biological sample, for example platelets, erythrocytes, lymphocytes, monocytes and the different populations of polymorphonuclear cells.

[0054] The angle values ​​communicated below are understood to be relative to the optical path of the incident light beam.

[0055] According to one embodiment of the invention, the step of measuring cytometry parameters comprises a step of measuring the intensity of at least one fluorescence beam emitted by each biological cell, for example at 90°.

[0056] According to one embodiment of the invention, the step of measuring cytometry parameters further comprises a step of measuring the variation in electrical impedance generated by the passage of biological cells through a measuring chamber.

[0057] According to one embodiment of the invention, the step of measuring cytometry parameters comprises the following steps: emission of an incident light beam towards the biological cells passing through the measuring chamber such that the incident light beam crosses the path of the biological cells, detection of at least one light beam from each biological cell passing through the measuring chamber.

[0058] According to one embodiment of the invention, the passage step comprises at least one step of hydrodynamic sheathing of the biological cells passing into the measuring chamber.

[0059] According to one embodiment of the invention, the detection step comprises a step of simultaneous detection of at least one light beam diffused by each biological cell passing through the measuring chamber and of at least one fluorescence beam emitted by each biological cell passing through the measuring chamber.

[0060] According to one embodiment of the invention, the detection step comprises a step of simultaneous detection of light beams diffused in at least two different directions by each biological cell passing through the measuring chamber and of at least two fluorescence beams emitted by each biological cell passing through the measuring chamber at at least two different wavelengths.

[0061] According to one embodiment of the invention, the analysis method comprises a step of determining the structure and / or the shape of said biological cells.

[0062] According to one embodiment of the invention, the analysis method comprises a step of determining the concentration of biological cells and / or the distribution of biological cells in the respective cell clusters.

[0063] According to one embodiment of the invention, the analysis method further comprises the following steps: passing biological cells from a reference biological sample through a measurement cell of a flow cytometer, measuring N cytometry parameters for each biological cell contained in the reference biological sample, determining, for each biological cell of the reference biological sample, a point in an N-dimensional space whose coordinates are defined as a function of the cytometry parameters measured for said biological cell of the reference biological sample, where N is an integer greater than or equal to 3, automatically grouping the determined points relating to the reference biological sample into different clusters of cells as a function of the cytometry parameters measured for each biological cell of the reference biological sample, so as to define a reference cluster file, repeating said steps of passing, measuring,determining and grouping for a plurality of reference biological samples so as to define a plurality of reference cluster files.

[0064] According to one embodiment of the invention, the analysis method comprises, prior to the step of passing the biological cells of the biological sample to be analyzed, a step of preparing the biological sample to be analyzed. The preparation step comprises, for example, a step of diluting the biological sample to be analyzed, for example using an isotonic diluent. The preparation step may further comprise, in addition to the dilution step, a step of selective lysis of at least some of the biological cells contained in the biological sample to be analyzed, and for example erythrocytes.

[0065] According to one embodiment of the invention, the preparation step comprises a step of labeling at least some of the biological cells contained in the biological sample to be analyzed, and more particularly the nucleic acids of at least some of the biological cells contained in the biological sample to be analyzed, with a fluorochrome, such as a fluorescent dye.

[0066] According to one embodiment of the invention, the analysis method comprises a step of integrating the sample cluster file as a reference cluster file. Such an integration step is notably carried out after a hematologist has identified the pathology relating to the biological sample to be analyzed and has associated indications relating to such a pathology with the sample cluster file.

[0067] According to one embodiment of the invention, the analysis method, and in particular the analysis step, comprises a step of comparing the sample cluster file with normal cluster files, each of the normal cluster files being defined from cytometry parameters of a respective normal biological sample. In the present description, the term "normal" biological sample means a biological sample which is not pathological and which is not abnormal.

[0068] According to one embodiment of the invention, the preparation step comprises the addition, to the biological sample to be analyzed, of one or more reagents containing antibodies specific for receptors found on the membranes of biological cells. These antibodies are conjugated either to fluorescent tracers or to particles which make it possible to generate one or more specific signals on each cell. Thus, the analysis method according to the invention makes it possible to add, to the basic physical quantities converted digitally into cytometry parameters, immuno-hematological measurements on demand in order to be able to better specify or confirm a diagnosis.

[0069] The present invention further relates to an analysis apparatus comprising: a flow cytometer comprising a measuring cell intended for the passage of biological cells of a biological sample to be analyzed and measuring means configured to measure cytometry parameters of the biological cells of the biological sample to be analyzed, and a processing unit configured to: determine, for each biological cell of the biological sample to be analyzed, a point in an N-dimensional space whose coordinates are defined as a function of the cytometry parameters measured for the corresponding biological cell of the biological sample to be analyzed, where N is an integer greater than or equal to 3, group points into different clusters of cells as a function of the cytometry parameters measured for each biological cell of the biological sample to be analyzed, so as to define a sample cluster file, compare the sample cluster file with reference cluster files,each of the reference cluster files being defined from cytometry parameters of a respective pathological or abnormal biological sample.

[0070] According to one embodiment of the invention, the analysis device is an analysis device for in vitro diagnosis, such as a hematology device.

[0071] According to one embodiment of the invention, the measuring cell of the flow cytometer is inclined relative to the horizontal, for example at an angle of approximately 45°.

[0072] In any case, the invention will be better understood with the aid of the description which follows with reference to the appended schematic drawings representing, by way of non-limiting example, an embodiment of this flow cytometer. Figures 1 et 2 are perspective views of a flow cytometer belonging to a flow cytometer according to the present invention. Figure 3 is a cross-sectional view of the flow cytometer of the figure 1 . Figures 4 is an enlarged scale view of a detail of the figure 3 . Figure 5 is seen in cross-section of the flow cytometer of the figure 1 . Figure 6 is a sectional view along line VI-VI of the figure 2 . Figures 6 And 7 are enlarged scale views of details of the figure 3 . Figure 8 is a top view of an analysis apparatus comprising the flow cytometer according to the present invention. figures 1 à 7 represent a flow cytometer 3, also called a cytometric measuring head, belonging to an analysis device 2 according to the present invention.

[0073] The flow cytometer 3 comprises a single-piece support 4 which may, for example, be metallic. The support 4 is parallelepipedal and delimits an internal receiving housing 5. The support 4 comprises in particular six passage openings arranged respectively on the six external faces of the support 4.

[0074] The flow cytometer 3 further comprises a measuring cell 6 (shown more particularly in the figure 4 ) which at least partly delimits a measuring chamber 7, an injection device 8 arranged to inject a flow of biological cells F into the measuring chamber 7, and an evacuation device 9 arranged to evacuate the flow of biological cells F injected into the measuring chamber 7 outside the flow cytometer 3.

[0075] As shown on the figure 4 , the measuring cell 6 is annular and is interposed in a sealed manner between the injection and evacuation devices 8, 9. The measuring cell 6 is housed in the receiving housing 5 delimited by the support 4, and is fluidically isolated from the receiving housing 5. The measuring cell 6 is advantageously made of an electrically insulating material that is transparent to light, and for example of polymethyl methacrylate, glass or quartz to avoid autofluorescence.

[0076] The injection and evacuation devices 8, 9 are fixed respectively on two opposite external faces of the support 4, and for example on the opposite lateral external faces of the support 4. However, the injection and evacuation devices 8, 9 could also be fixed respectively on the two upper and lower external faces of the support 4.

[0077] As shown more particularly on the figures 3 And 6, the injection device 8 comprises an injection nozzle 11 delimiting an internal chamber 12. The injection nozzle 11 is provided with an injection orifice 13 opening into the measuring chamber 7 and arranged to fluidically connect the internal chamber 12 to the measuring chamber 7.

[0078] The injection device 8 further comprises a first tubular supply conduit 14 intended to supply the internal chamber 12 with a biological sample to be analyzed containing, in suspension, biological cells to be analyzed.

[0079] As shown on the figure 1 , the injection device 8 further comprises a discharge conduit 15 fluidically connected to the internal chamber 12 and intended to discharge the contents of the internal chamber 12 to the outside of the flow cytometer 3. The discharge conduit 15 is more particularly intended to discharge to the outside of the flow cytometer 3 a rinsing fluid introduced into the internal chamber 12 via the first supply conduit 14.

[0080] The injection device 8 further comprises a second supply conduit 16 intended to supply the internal chamber 12 with a sheathing fluid. The injection nozzle 11 and the second supply conduit 16 are configured such that the sheathing fluid introduced into the internal chamber 12 via the second supply conduit 16 is capable of hydrodynamically sheathing the biological sample introduced into the internal chamber 12 before the biological sample passes through the injection orifice 13.

[0081] As shown on the figure 7 , the evacuation device 9 delimits an internal chamber 17 opening into the measuring chamber 7 and further comprises a tubular evacuation conduit 18 fluidically connected to the measuring chamber 7 and intended to evacuate the flow of biological cells F injected into the measuring chamber 7 to the outside of the flow cytometer 3. The evacuation conduit 18 extends partly into an internal chamber 17 and opens into the measuring chamber 7 opposite the injection orifice 13.

[0082] The evacuation device 9 further comprises a third supply conduit 19 fluidically connected to the measuring chamber 7 and intended to supply the measuring chamber 7 with a sheathing fluid. The measuring chamber 7 and the third supply conduit 19 are configured such that the sheathing fluid introduced into the measuring chamber 7 via the third supply conduit 19 is capable of hydrodynamically sheathing the flow of biological cells F flowing through the measuring chamber 7.

[0083] As shown on the figures 1 And 7, the evacuation device 9 further comprises a discharge conduit 21 fluidically connected to the measuring chamber 7 and intended to discharge the contents of the measuring chamber 7 outside the flow cytometer 3. The discharge conduit 21 is more particularly intended to discharge outside the flow cytometer 3 a rinsing fluid introduced into the measuring chamber 7 via the third supply conduit 19.

[0084] The flow cytometer 3 further comprises measuring means configured to measure cytometry parameters of the biological cells to be analyzed, and in particular to measure optical and electrical properties of the biological cells to be analyzed.

[0085] According to the embodiment shown in the figures 1 à 7 , the measuring means comprise an emission device 22 arranged to emit an incident light beam in the direction of the measuring chamber 7 and capable of crossing, i.e. intersecting, the flow of biological cells F introduced into the measuring chamber 7, and several collection devices 23a, 23b, 23c angularly offset relative to the flow of biological cells F and arranged to collect light beams from the biological cells passing through the measuring chamber 7. Nevertheless, the measuring means could comprise, for example, several emission devices angularly offset relative to the flow of biological cells, and also only one or more collection devices.

[0086] The emission and collection devices are mounted on the upper and lower external faces of the support 4 and extend in a plane substantially perpendicular to the flow direction of the flow of biological cells F. The collection device 23a is for example arranged opposite the emission device 22 relative to the measuring cell 6, while the collection devices 23b and 23c are arranged perpendicular to the emission device 22 relative to the measuring cell 6. However, according to an alternative embodiment of the invention, the emission device 22 and the collection device 23a could be mounted on the lateral external faces of the support 4.

[0087] The emission device 22 comprises a light source 24 arranged to generate the incident light beam. The light source 24 may for example be a laser source arranged to generate a laser beam.

[0088] According to the embodiment shown in the figures 1 à 7 and as this results more particularly from the figure 1 , the collection device 23a comprises a plurality of optical collection elements, and more particularly a central collection optical fiber 25a, and one or more peripheral collection optical fibers 25b. For example, the central collection optical fiber 25a is intended to collect the light beams coming from the measuring chamber 7 along the optical path of the incident light beam, i.e. at 0°, and the peripheral collection optical fibers 25b are intended for some to collect the light beams coming from the measuring chamber 7 at an angle of the order of 4° and for others to collect light beams coming from the measuring chamber 7 at an angle of the order of 9°. The collection device 23a could however comprise a single peripheral collection optical fiber 25b.

[0089] The collection device 23b could for example comprise a single collection optical element, such as a central collection optical fiber, and the collection device 23c could for example also comprise a single collection optical element, such as a central collection optical fiber.

[0090] The measuring means further comprise a plurality of detection elements (not shown in the figures) each associated with a respective collection device 23a-23c. Each detection element is arranged to provide at output a measurement signal determined as a function of the light beams collected by the respective collection device. As each biological cell passes through the incident light beam, each measurement signal provided at output by each detection element is for example proportional to the quantity of light absorbed or re-emitted by said biological cell. Each detection element may for example be a photodetector, such as a photodiode or also a photomultiplier.

[0091] The measuring means further advantageously comprise an electrical impedance variation measuring device arranged to measure the electrical impedance variation generated by the passage of the biological cells through the injection orifice 13. The electrical impedance variation measuring device comprises for example a first and a second electrode (not shown in the figures) arranged respectively on either side of the injection orifice 13. The first and second electrodes are intended to be in electrical contact with the flow of biological cells F so as to establish an electric field through the injection orifice 13.According to an alternative embodiment of the electrical impedance variation measuring device, the latter could comprise a single electrode arranged at least partly in the internal chamber 17, and the potential of the internal chamber 12 could be grounded, such that the electrical impedance variation measuring device is configured to measure an electrical impedance variation between the internal chamber 12 and the electrode placed in the internal chamber 17.

[0092] Such an electrical impedance variation measuring device makes it possible to count the number of biological cells passing through the injection orifice 13, and also to determine the size, and more precisely the volume of the biological cells. The operation of such an electrical impedance variation measuring device is known to those skilled in the art and is therefore not described in detail. It should however be noted that the passage of each biological cell through the injection orifice 13 causes an electrical pulse proportional to the size or volume of said biological cell.

[0093] As shown on the figure 8 , the analysis apparatus 2 further comprises a processing unit 32 configured to analyze the cytometry parameters measured by the measuring means of the flow cytometer 3, and in particular to analyze the measurement signals provided by each detection element. The processing unit 32 is more particularly configured to differentiate and identify the biological cells of a biological sample to be analyzed, and in particular to determine the structure and shape of the biological cells from the cytometry parameters measured by the measuring means. The processing unit 32 more particularly comprises at least one electronic processing card equipped with a microprocessor.

[0094] As shown on the figure 8 , the analysis device 2 may comprise two flow cytometers 3, and also a loading module 33 arranged to move at least one rack in a first direction of movement D1, an unloading module 34 arranged to move at least one rack in a second direction of movement D2, a stirring module (not visible on the figure 8 ) arranged to move at least one rack between the loading module and the unloading module, the stirring module and the loading and unloading modules defining a generally U-shaped rack transport path. Advantageously, the analysis apparatus 2 also comprises a sampling module 36 arranged to take samples of biological liquid from containers received in a rack positioned in the stirring module.

[0095] The analysis device 2 may also include: a loading rotor 37 arranged between the loading and unloading modules and with a substantially vertical rotation axis, the loading rotor 37 comprising a plurality of housings 38 capable of receiving containers containing samples of biological liquid to be analyzed or reactive products and in particular capable of receiving cartridges for carrying out configurable tests, therefore immunohematology but also immunology on whole blood, the sampling module 36 being arranged to take samples or reactive products from the containers received in the loading rotor 37, rotational drive means associated with the loading rotor 37 and arranged to rotate the loading rotor 37 around its rotational axis.a preparation rotor 39 with a substantially vertical rotation axis, the preparation rotor 39 comprising a plurality of preparation bowls 41, the sampling module 36 being arranged to supply the preparation bowls 41 with samples of biological liquid or with reactive products previously collected, and rotational drive means associated with the preparation rotor 39 and arranged to rotate the preparation rotor 39 around its rotation axis.

[0096] The presence of cartridges on the loading rotor 37 makes it possible to add additional preparation reagents, and therefore to add, to the measurements of cytometry parameters of a physical or morphological nature, measurements of cytometry parameters of an immuno-hematological nature.

[0097] Furthermore, the analysis apparatus 2 may be provided, at the level of the preparation rotor 39, with a magnetic device which makes it possible to capture magnetic particles in solution. These magnetic particles are coated with antibodies making it possible to selectively capture a certain type of cell, for example all leukocytes. Thus, after resuspension in an isotonic diluent, the prepared biological sample no longer contains anything but leukocytes without having to resort to lysis to destroy the red blood cells which are a thousand times more numerous. Therefore the leukocytes are intact and it is then possible to adapt the dilution rates in order to be able to carry out the measurements of the N cytometry parameters on a significant number of cells with the possibility of identifying few or rare cells. Furthermore, the leukocytes may also be selectively labeled for conventional immunological identification (for example T lymphocytes).

[0098] A method for analyzing a biological sample containing biological cells using a flow cytometer 2 according to the present invention will now be described.

[0099] Such an analysis method includes the following steps: preparation of the biological sample to be analyzed, the preparation step comprising for example a step of dilution of the biological sample to be analyzed, for example using an isotonic diluent, and / or a step of selective lysis of at least some of the biological cells contained in the biological sample to be analyzed, such as erythrocytes, and / or a step of labeling at least some of the biological cells contained in the biological sample to be analyzed with a fluorochrome, passage of the biological cells contained in the biological sample to be analyzed in the measuring chamber 7 of the flow cytometer 3, measurement of N cytometry parameters for each biological cell contained in the biological sample to be analyzed, such as cytometry parameters representative of the morphology and / or the structure of the biological cells of the biological sample to be analyzed, using the flow cytometer 3, determination,for each biological cell of the biological sample to be analyzed, of a point in an N-dimensional space whose coordinates are defined as a function of the cytometry parameters measured for said biological cell of the biological sample to be analyzed, each coordinate axis of the N-dimensional space corresponding to a respective measured cytometry parameter or to a value calculated from said respective measured cytometry parameter, automatic grouping of the points relating to the biological sample to be analyzed into different cell clusters as a function of the cytometry parameters measured for each biological cell of the biological sample to be analyzed, so as to define a sample cluster file, the sample cluster file being for example in FCS (Flow Cytometry Standard) format, automatic identification of the cell populations defined by the different cell clusters of the sample cluster file,automatic counting of points of each cell cluster of the sample cluster file, comparison of the sample cluster file with reference cluster files, each of the reference cluster files being defined from cytometry parameters of a respective pathological or abnormal biological sample, issuance of an alarm message when the sample cluster file is at least partially identical or similar to a reference cluster file, and in particular when predetermined cell clusters of the sample cluster file are identical or similar to predetermined cell clusters of the reference cluster file, the emitted alarm message advantageously containing indications relating to a pathology or an abnormality associated with the reference cluster file to which the sample cluster file is at least partially identical or similar, the steps of determining,grouping, comparison and transmission being carried out by the processing unit 32.,

[0100] Such an automatic grouping step can be carried out in various ways known to those skilled in the art, and is therefore not described in detail in the present description.

[0101] According to one embodiment of the invention, the analysis method comprises a step of sampling and digitizing a set of analog signals generated during the duration of the measurement step so as to define a first digitized raw data file for each of the N measurement channels, and a step of synchronizing and grouping the N digitized signals for each biological cell of the sample to be analyzed by a first level of computer processing. Said sampling and digitizing step is carried out by the processing unit 32 which transmits the files by Ethernet link to a PC-type computer unit (not shown in the figure). figure 8 ) who analyzes it.

[0102] According to one embodiment of the analysis method, the latter further comprises a step of analyzing the sample cluster file so as to detect at least one possible anomaly in the sample cluster file, the analysis step being carried out by the processing unit 32. In an example which is not part of the claimed invention, the comparison step is carried out only when at least one anomaly is detected during the analysis step and the alarm message sent during the sending step then also contains information relating to the at least one detected anomaly.These provisions make it possible, on the one hand, to avoid carrying out the comparison step if the biological sample to be analyzed is normal and not pathological, and therefore to reduce the execution time of the calculations and to provide the results of the analysis more quickly to the operator, and on the other hand to communicate to the operator an alarm message that is as detailed as possible when the biological sample to be analyzed is pathological or abnormal.

[0103] The analysis step advantageously includes the following steps: analyzing, for each cell cluster of the sample cluster file, at least one morphological parameter of said cell cluster, such as the positioning of said cell cluster, the distribution of points of said cell cluster, the number of points of said cell cluster, and / or the presence or absence of said cell cluster, detecting an anomaly if at least one morphological parameter of at least one cell cluster of the sample cluster file exceeds a respective predetermined threshold value.

[0104] According to one embodiment of the invention, the analysis step more particularly comprises the following steps: comparing, for each cell cluster of the sample cluster file, the number of points grouped in said cell cluster with at least one respective predetermined threshold value, analyzing the distribution of the points in each cell cluster of the sample cluster file, analyzing the positioning of the cell clusters of the sample cluster file, analyzing the presence and / or absence of at least some predetermined cell clusters, detecting an anomaly if the distribution of the points in at least one of the cell clusters is not Gaussian, detecting an anomaly if at least two cell clusters of the sample cluster file are at least partially confused, detecting an anomaly if the presence or absence of at least one predetermined cell cluster is detected, detecting an anomaly if the number of cell clusters is greater or less than a predetermined reference value,detecting an anomaly if the number of points grouped in at least one of the cell clusters is lower and / or higher than a respective predetermined threshold value.

[0105] According to one embodiment of the invention, the analysis step comprises a step of comparing the sample cluster file with normal cluster files, each of the normal cluster files being defined from cytometry parameters of a respective normal biological sample. These arrangements make it possible in particular to facilitate the detection of anomaly in the sample cluster file.

[0106] According to one embodiment of the invention, the step of measuring cytometry parameters comprises the following steps: emission, using the emission device 22, of an incident light beam towards the biological cells passing through the measuring chamber 7 such that the incident light beam crosses the path of the biological cells, detection, using the collection devices 23a-23c, of different light beams coming from each biological cell passing through the measuring chamber 7.

[0107] Taking into account the configuration and arrangement of the various collection devices 23a-23c, the step of measuring cytometry parameters includes in particular the following steps: measuring the intensity of the light beams scattered at small angles by each biological cell using the collection optical fibers 25b, 25c of the collection device 23a, measuring the intensity of a light beam scattered along the optical path of the incident light beam by each biological cell using the central collection optical fiber 25a of the collection device 23a, measuring the intensity of a light beam scattered at 90° by each biological cell using the collection device 23b, and measuring the intensity of a fluorescence beam emitted at 90° by each biological cell using the collection device 23c.

[0108] Advantageously, the step of measuring cytometry parameters further comprises a step of measuring the variation in electrical impedance generated by the passage of the biological cells through the measuring chamber 7, using the device for measuring the variation in electrical impedance.

[0109] Advantageously, the analysis method comprises the following initial steps: measurement of cytometry parameters for each biological cell contained in a reference biological sample using the flow cytometer 3, determination, for each biological cell of the reference biological sample, of a point in an N-dimensional space whose coordinates are defined as a function of the cytometry parameters measured for each biological cell of the reference biological sample, each coordinate axis of the N-dimensional space corresponding to a respective measured cytometry parameter, automatic grouping of the points relating to the reference biological sample into different cell clusters according to the cytometry parameters measured for each biological cell of the reference biological sample, so as to define a reference cluster file, each reference cluster file being for example in FCS (Flow Cytometry Standard) format, the determination and grouping steps being carried out by the processing unit 32, repeating said initial measurement, determination and grouping steps for a plurality of reference biological samples so as to define a plurality of reference cluster files.

[0110] It goes without saying that the invention is not limited to the sole embodiment of the flow cytometer and to the sole modes of implementation of the analysis method, described above as examples; on the contrary, it encompasses all the implementation variants.

Claims

1. An analysis method for analyzing a biological sample containing biological cells including blood cells, the analysis method comprising the following steps: - passage of the biological cells of the biological sample to be analyzed into a measuring cell (6) of a flow cytometer (3), - measurement of N cytometry parameters for each biological cell of the biological sample to be analyzed, - determination, for each biological cell of the biological sample to be analyzed, of a point in a N-dimensional space whose coordinates are defined depending on the cytometry parameters measured for the corresponding biological cell, where N is an integer greater than or equal to 3, - automatic clustering of the determined points into different cell clusters depending on the measured cytometry parameters, so as to define a sample cluster file, - identification of the cell populations defined by the different cell clusters of the sample cluster file defined at the clustering step, - counting of the points of each cell cluster of the sample cluster file defined at the clustering step, the method being characterized by the following step: - comparison of the sample cluster file defined at the clustering step with reference cluster files, each of the reference cluster files being defined from cytometry parameters of a respective pathological or abnormal biological sample.

2. The analysis method according to claim 1, which further comprises a step of emitting an alarm message when the sample cluster file defined at the clustering step is at least partially identical or similar to a reference cluster file.

3. The analysis method according to claim 2, wherein the emitted alarm message contains indications related to a pathology or an abnormality associated to the reference cluster file to which the sample cluster file defined at the clustering step is at least partially identical or similar.

4. The analysis method according to any one of claims 1 to 3, which further comprises a step of analyzing the sample cluster file defined at the clustering step so as to detect at least one possible anomaly in the sample cluster file.

5. The analysis method according to claim 4, wherein the analysis step comprises the following steps: - analysis, for each cell cluster of the sample cluster file defined at the clustering step, of at least one morphological parameter of said cell cluster, - detection of an anomaly if at least one morphological parameter of at least one cell cluster of the sample cluster file defined at the clustering step exceeds a respective predetermined threshold value.

6. The analysis method according to claim 4 or 5, wherein the analysis step comprises the following steps: - comparison, for each cell cluster of the sample cluster file defined at the clustering step, of the number of points clustered in said cell cluster with at least one respective predetermined threshold value, - detection of an anomaly if the number of points clustered in at least one of the cell clusters is less and / or greater than the at least one respective predetermined threshold value.

7. The analysis method according to any one of claims 4 to 6, wherein the analysis step comprises the following steps: - analysis of the distribution of the points in each cell cluster of the sample cluster file defined at the clustering step, - detection of an anomaly if the distribution of the points in at least one of the cell clusters is not Gaussian.

8. The analysis method according to any one of claims 4 to 7, wherein the analysis step comprises the following steps: - analysis of the positioning of the cell clusters of the sample cluster file defined at the clustering step, - detection of an anomaly if at least two cell clusters of the sample cluster file defined at the clustering step are at least partially confused.

9. The analysis method according to any one of claims 4 to 8, wherein the analysis step comprises the following steps: - analysis of the cell clusters of the sample cluster file defined at the clustering step, - detection of an anomaly if the presence or absence of at least one predetermined cell cluster is detected.

10. The analysis method according to any one of claims 1 to 9, wherein the step of measuring cytometry parameters comprises at least one step of measuring cytometry parameters representative of the morphology and / or the structure of the biological cells of the biological sample to be analyzed.

11. The analysis method according to claim 10, wherein the step of measuring cytometry parameters comprises at least one step of measuring, for each biological cell of the biological sample to be analyzed, at least one optical property of said biological cell.

12. The analysis method according to claim 11, wherein the step of measuring cytometry parameters comprises: - a step of measuring the intensity of a light beam scattered at small angles by each biological cell, and / or - a step of measuring the intensity of a light beam scattered at 90° by each biological cell, and / or - a step of measuring the intensity of a light beam scattered along an optical path of the incident light beam by each biological cell.

13. The analysis method according to claim 11 or 12, wherein the step of measuring cytometry parameters comprises a step of measuring the intensity of at least one fluorescence beam emitted by each biological cell, for example at 90°.

14. The analysis method according to any one of claims 1 to 13, wherein the step of measuring cytometry parameters includes the following steps: - emission of an incident light beam towards the biological cells passing through the measurement chamber such that the incident light beam crosses the path of the biological cells, - detection of at least one light beam from each biological cell passing through the measurement chamber.

15. The analysis method according to any one of claims 1 to 14, which further comprises the following steps: - passage of the biological cells of a reference biological sample into a measuring cell (6) of a flow cytometer (3), - measurement of N cytometry parameters for each biological cell of the reference biological sample, - determination, for each biological cell of the reference biological sample, of a point in a N-dimensional space whose coordinates are defined depending on the cytometry parameters measured for said biological cell of the reference biological sample, where N is an integer greater than or equal to 3, - automatic clustering of the determined points relating to the reference biological sample into different cell clusters depending on the cytometry parameters measured for each biological cell of the reference biological sample, so as to define a reference cluster file, - repeating said passage, measurement, determination and clustering steps for a plurality of reference biological samples so as to define a plurality of reference cluster files.

16. An analysis apparatus comprising: - a flow cytometer (3) comprising a measurement cell (6) intended for the passage of biological cells of a biological sample to be analyzed and measuring means (22, 23a, 23b, 23c) configured to measure cytometry parameters of the biological cells of the biological sample to be analyzed, and - a processing unit (32) configured to: - determine, for each biological cell of the biological sample to be analyzed, a point in a N-dimensional space whose coordinates are defined depending on the cytometry parameters measured for the corresponding biological cell, where N is an integer greater than or equal to 3, - cluster the points into different cell clusters depending on the cytometry parameters measured for each biological cell of the biological sample to be analyzed, so as to define a sample cluster file, characterized in that the analysis apparatus being further configured to: - compare the sample cluster file defined at the clustering step with reference cluster files, each of the reference cluster files being defined from cytometry parameters of a respective pathological or abnormal biological sample.