WO2007057574A1

WO2007057574A1 - Method and device for counting bodies in a liquid

Info

Publication number: WO2007057574A1
Application number: PCT/FR2006/002538
Authority: WO
Inventors: Philippe Daurenjou
Original assignee: Ph Diagnostics Sas
Priority date: 2005-11-17
Filing date: 2006-11-17
Publication date: 2007-05-24
Also published as: FR2893414A1

Abstract

A device for counting bodies in a liquid comprises a light emitting device exhibiting a plurality of wavelengths, wherein each said body produces at least one optical effect for at least one wavelength, means for relatively displacing the liquid containing said bodies with respect to the light emitting device, receiving means used for delivering a plurality of signals corresponding to the light arriving thereto for said wavelength, means for classifying different bodies into different categories according to the signals transmitted by the receiving means and means for counting the bodies of each category. In the certain embodiments, the inventive device comprises optical and fluid components, a channel for draining the liquid containing said bodies, at least one input waveguide connected to said emission means, wherein said channel and each input wave guide meet each other in an area for observing bodies running through the channel.

Description

METHOD AND DEVICE FOR DENOMBRETING BODIES IN A LIQUID

The present invention relates to a method and a device for counting bodies in a liquid. It applies, in particular, to the enumeration and, optionally, the differentiation, of particles suspended in a liquid, in particular cells contained in the blood, possibly diluted, such as erythrocyte, leukocyte and / or thrombocyte cells. Particle counting systems in a liquid are known, which allow qualitative and quantitative analyzes of blood cells. These systems operate on the basis of the principle of detecting impedance or conductance variation, known as the Coulter Principle. This variation of impedance or conductance does not make it possible to obtain morphological information on the cells. US 6,438,279 discloses a unitary structure of microcapillary and waveguide and a method for its manufacture. The teaching of this document applies to fluorescence, that is to say to the excitation by a powerful ray of light, resulting from a laser, a substance so that it emits light intended for to be analyzed. This technology does not apply to the enumeration or classification of living cells, for example different blood cells.

The present invention aims to remedy these disadvantages.

For this purpose, according to a first aspect, the present invention provides a device for counting bodies in a liquid, which comprises: light emitting means comprising a plurality of wavelengths, said bodies each having at least one effect optical system for at least one said wavelength, a means for moving relative to said liquid containing said bodies, with respect to said light-emitting means, an adapted light receiving means providing a plurality of signals corresponding to the light reaching for said wavelengths, a means for classifying different bodies in different classes, according to the signals provided by the receiving means and a means for counting the bodies of each of said body classes. With these features, each body can be identified in a body class and a count of the number of bodies of each class can be made.

According to particular features, the device as briefly described above comprises an optical and fluidic component comprising, on the one hand, a channel flow of the liquid containing said bodies and, secondly, at least one input waveguide connected to said transmitting means, said channel and each input waveguide meeting in an area of observation of bodies crossing the canal.

Thanks to these provisions, the respective positions of the channel and the waveguide can be precise and definitive, without risk of maladjustment.

According to particular features, the optical and fluidic component further comprises at least one output waveguide connected to said reception means, said channel and each output waveguide meeting in the observation zone of the bodies crossing the canal. Thanks to these provisions, the respective positions of the channel and the waveguide can be precise and definitive, without risk of maladjustment.

According to particular features, the optical and fluidic component comprises a layer inside which the channel and each waveguide are made, sandwiched between a substrate layer and a coating layer. Thanks to these arrangements, even in the case of very small dimensions, less than 0.1 millimeters, of the channel and of each waveguide, their respective positions can be precise.

According to particular features, the layer intended to comprise the channel and each waveguide is, initially, soluble material become insoluble, outside the channel, after irradiation.

Thanks to these provisions, the manufacture of the channel is easy and precise.

According to particular characteristics, at least one waveguide is constituted by locally increasing the optical index of the layer intended to comprise at least one waveguide. Thanks to these provisions, mechanical actions, for example drilling, are avoided in the optical and fluidic component.

According to particular features, the layer intended to comprise the channel and at least one waveguide is made of an organo-mineral hybrid photosensitive material.

According to particular features, the layer intended to comprise the channel and at least one waveguide is of polymeric light-sensitive material.

Thanks to each of these arrangements, the manufacture of the optical and fluidic component is easy and the service life of the component is increased.

According to particular features, the channel has a cross section smaller in size greater than or equal to the largest dimension of the bodies flowing in said channel. According to particular characteristics, the channel has, at the inlet and / or at the outlet, a funnel shape. This limits the risk of clogging and allows a natural cladding bodies in the center of the channel and therefore the flow.

Thanks to these provisions, it avoids the risk of clogging of this channel by bodies and avoids deforming the largest bodies circulating in this channel.

According to particular features, the device as briefly described above comprises means for injecting into the input waveguide, light components of said plurality of wavelengths corresponding to different absorption powers. for said bodies. Thanks to these provisions, the classification of the bodies is easy because it can be performed according to the absorption wavelengths of the bodies.

According to particular features, the optical and fluidic component comprises conductive surfaces placed on the surface of the channel or in its proximity, instead of crossing the channel and at least one waveguide and an impedance sensor that measures the impedance variation between the conductive surfaces.

Thanks to these arrangements, it is possible to measure in a synchronized or slightly offset way, the impedance of each body whose optical effect is analyzed. Thus, complementary information is obtained on each of the bodies circulating in the channel, which makes it possible, for example, to confirm a classification or to constitute subclasses in which the different bodies can be classified.

According to particular features, the optical and fluidic component comprises at least one output waveguide adapted to convey light reflected by bodies in said liquid and flowing in the flow channel.

Thanks to these arrangements, it is possible to treat the optical effects of reflection of the bodies circulating in the channel and, thus, to confirm their classification or to arrange them in subclasses.

According to particular features, the light emitting means comprises a plurality of light sources emitting in different spectral ranges and a multiplexer adapted to transmit light from each of the light sources to the light receiving means.

Thanks to these provisions, the proportion of the light emitted by each light source which reaches the bodies circulating in the channel is increased.

According to particular features, the light emitting means comprises a plurality of light sources emitting successively in different spectral ranges, the receiving means comprising a photosensitive sensor adapted to provide a signal representative of the light coming from each of the light sources. . Thanks to these arrangements, the light receiving means is simple and time is used to separate the different signals representing the different wavelengths emitted by the light source and hence to treat the optical effects, for the different lengths. different bodies circulating in the channel. According to particular features, the light receiving means comprises a plurality of sensitive photosensitive sensors in different spectral ranges and a divider adapted to distribute light from the transmission means on the different sensors as a function of the wavelength of the light. this light.

By virtue of these arrangements, the proportion of the light emitted by each light source which reaches the reception means is increased.

According to particular features, the device as briefly described above comprises signal processing means adapted to position the local extreme values of the signals coming from the photosensitive sensor in a multidimensional space, and to deduce therefrom the class of each body represented by these extreme values, each body class being represented by a volume in this multidimensional space.

Thanks to these arrangements, the bodies are classified according to their optical effects for the different wavelengths and two bodies having the same optical effect for one or more wavelengths are discriminated according to their optical effect for the other wavelength (s).

According to particular features, the processing means is further adapted to process an impedance measurement signal and to position the local extreme values of the signals from the photosensitive sensor and the impedance measurement signal, in a multidimensional space. and to deduce from them the class of each body represented by these extreme values, each class of body being represented by a volume in this multidimensional space.

Thanks to these provisions, the discrimination of the bodies between the different classes is more precise or safer.

According to particular features, the processing means is adapted to process the pulse shape of the signals and to obtain, as a function of this form, complementary information on these bodies to classify them in subclasses.

Thanks to these provisions, different bodies of a class can be arranged in different subclasses.

According to particular features, the signal processing means is adapted to count the number of bodies in each class and / or subclass.

According to a second aspect, the present invention relates to a method for counting bodies in a liquid, characterized in that it comprises: a light emission step comprising a plurality of wavelengths, said bodies each having at least one optical effect for at least one said wavelength,

a step of moving relative to said liquid containing said bodies, with respect to said light-emitting means, a step of receiving light to provide a plurality of signals corresponding to the light reaching it for said wavelengths; step of classifying different bodies in different classes, according to the signals provided by the receiving means and - a step of counting the bodies of each of said body classes.

Since the advantages, aims and particular characteristics of this process are similar to those of the device that is the subject of the present invention as succinctly set forth above, they are not recalled here.

Other advantages, aims and particular characteristics of the present invention will emerge on reading the following description, given for an explanatory and in no way limiting purpose with reference to the appended drawings, in which: FIG. 1 is a diagrammatic representation of perspective, a first embodiment of the device object of the present invention and a representation of means for transmitting and receiving optical signals, - Figure 2 shows, schematically, in perspective, the first embodiment of the device object of the present invention. FIG. 3 schematically represents a component of particular embodiments of the device that is the subject of the present invention; FIG. 4 is a diagrammatic sectional view of the component illustrated in FIG. 3; FIG. schematic, in section, of the component illustrated in FIGS.

4, FIGS. 6A, 6B and 6C show an electrical signal representative of an optical signal implemented in the first embodiment of the present invention and this signal after processing; FIG. 7 is a schematic representation of a second embodiment of FIG. embodiment of the device, FIG. 8 is a diagrammatic sectional representation of an embodiment of an optical and microfluidic component incorporated in the device illustrated in FIG. 7. FIG. 9 is a diagrammatic perspective view of an embodiment of the device. an optical and microfluidic component incorporated in the device illustrated in FIG. 7, FIG. 10 represents, schematically and in section, an embodiment of an optical and microfluidic component incorporated in the device illustrated in FIG. 7,

FIG. 11 represents, schematically and in perspective, an embodiment of an optical and microfluidic component incorporated in the device illustrated in FIG. 7,

FIG. 12 schematically represents an embodiment of the optical part of the device illustrated in FIG. 7,

FIG. 13 represents, schematically, an embodiment of the optical part of the device illustrated in FIG. 7; FIG. 14 is a schematic representation of an embodiment of the optical part of the device illustrated in FIG. 7, FIG. a signal representative of the transparency of a sample in a spectral range, being processed, FIG. 16 illustrates the classification of samples in a volume and FIG. 17 represents, in the form of a logic diagram, the steps implemented in FIG. in a particular embodiment of the method that is the subject of the present invention. It is observed that the components illustrated in the figures are not to scale. The present invention applies, in particular, to the food fields (analysis of milk, fruit juice or wine, for example), pharmaceutical, medical and biological (analyzes of fluids such as blood) and to the areas in which dispersing particles or cells in a fluid, for example water, without, a solvent or a gas, such as air.

The device illustrated in the figures applies, in particular, to the enumeration and, optionally, the differentiation, of particles suspended in a liquid, in particular cells contained in the blood, possibly diluted, such as erythrocyte, leukocyte and / or or thrombocytic.

Blood cells, such as red blood cells, white blood cells and platelets are suspended in plasma in the blood. It can be useful, in particular, to determine certain pathologies and evolutions of treatment, to know the quantities and the morphologies of these blood cells.

Preferably, the blood to be analyzed is diluted with water or a physiological liquid to reduce the concentration of the cells in the liquid. This diluted blood is then passed through a channel whose internal dimensions allow the passage of the cells. This channel is crossed by one or more light beams. At each passage of a blood cell, the incident light is modulated and possibly at least partially deviated and / or absorbed. FIG. 1 represents a first embodiment of such a device, which comprises a means A for differentiating and counting blood cells suspended in the blood entering the device through an inlet port 101 and leaving the device by a device outlet port 102 and transmitter means B and receiver C of the light signal of differentiation and enumeration of blood cells.

Referring to FIG. 2, it can be seen that the means A essentially comprises a casing of parallelepipedal general shape 104 and, inside this casing 104, an optical component 106 provided with an optical network and a microfluidic network. integrated in the optical component 106. The optical component 106, also of parallelepipedal shape, is traversed by a diluted blood flow channel 107 to be analyzed, calibrated according to the dimensions of the blood cells to avoid impeding their flow and a guide of 108. The channel 107 extends perpendicularly between two opposite faces of the optical component 106, in the middle of these faces. Preferably, the channel 107 has, in input and / or output, a funnel shape. This limits the risk of clogging and allows a natural cladding bodies in the center of the channel and therefore the flow.

The waveguide 108 extends between two other opposite faces of the optical component 106, in the middle of these faces. The waveguide 108 is thus interrupted by the channel 107. The channel 107 thus divides the waveguide 108 into a transmitting waveguide portion 109 and a receiving waveguide portion 110. Each guide portion 109 and 110, has a cross section, for example square, and is coupled to a cross-section optical fiber, for example, circular, respectively input 111 and output 112, through modules to V-groove 113, shown in FIG. 5. FIG. 5 also illustrates the coupling of square-section waveguides to optical fibers of circular cross-section, for example using an adaptive glue 115. of optical index.

As seen in FIG. 2 and FIG. 3, the microfluidic channel 107 has a square cross-section which widens towards the inlet in the manner of a funnel 117, while maintaining the same height over the entire length. Such a funnel profile, although not shown, can also be provided on the output side of the channel 107. It should be noted that the two waveguide portions 109 and 110 also have a square section advantageously of the same height as the fluidic channel 107, or slightly lower.

The assembly formed by the optical component 106 and the "V" groove modules 113 is encapsulated in the opaque casing 104 which ensures the protection of this assembly and the optical isolation of the waveguide portions 109 and 110. housing 104 has a hole 119 opening into the outer front face of the housing 104 and a hole 120 opening into the outer rear face of the housing 104. The holes 119 and 120 are both of preferably circular cross-section and aligned with the fluid channel 107. The diluted blood to be analyzed enters the housing 104 through the inlet hole 119, flows along channel 107, passes between waveguide portions 109 and 110 and exits through exit hole 120. Housing 104 is made of any suitable material, such as resin or other polymeric material.

Regarding the optical component 106, it comprises, sandwiched between a lower substrate layer 122 and a coating layer 123, a layer 125 of a photosensitive material, for example an organomineral or polymeric hybrid material. This layer 125 may be of composite structure and composed of a stack of a plurality of elementary layers.

It is in the layer 125 that the waveguide 108 is produced, with its emitting portions 109 and receiving 110. The material constituting the layer 125 has the property of becoming insoluble after irradiation while the non-irradiated parts remain soluble. for example in alcohol or acetone type solvents. The channel 107 is thus produced by placing a mask on the zone of the future channel 107 and by irradiating the remainder of the layer 125. Thus, only the material of the layer which has been protected from irradiation by the mask remains soluble and is then solubilized with a suitable solvent.

The waveguide 108 is made in the hybrid layer 125 by appropriate doping of the material to constitute the waveguide, so that the material in the waveguide has a higher optical index than outside. of the waveguide. The reflection of light on the walls of the waveguide is thus total.

Thanks to integration with the component 106 of the waveguide 108 and the channel 107, an alignment is obtained between the waveguide 108 and the channel 107 during the manufacture of the component.

For example, the cross section of the channel 107, essentially square, may have a side of 80 microns, the cross section of the waveguide 108, essentially square, may have a 60 micron side, the substrate may have a thickness of the order of 525 microns plus or minus 25 microns and the coating 123 a thickness of about 150 microns. With regard to the case 104 containing the optical component 106, it may have a length of 30 millimeters, a width of 10 millimeters and a height of 10 millimeters. The length of the optical component 106 may be 6 to 10 millimeters.

As regards the operation of the device which is the subject of the present invention, it allows the detection of different types of cells, such as red blood cells, white blood cells and platelets, because these cells are differentiated from each other by their power of absorption, deflection or reflection of the light traveling through the inlet waveguide portion 109, this absorption, deflection or reflection depending on the length wave of light used. To differentiate the blood cells that are passed through the channel 107 and through the light beam carried by the waveguide 108, a plurality of wavelengths or ranges of wavelengths are implemented. , which correspond to the absorption, deflection and / or reflection, preferably maximum, of the different blood cells. The injection and separation of the different light components is done from the transmitter side and the receiver side using wavelength division multiplexers, known as "WDM". By comparing the ratio of the light intensities received and emitted in the different wavelengths, revealing the amount of light absorbed, the nature of each cell passing through the light beam is identified.

In general, the passage of a cell through the light beam conveyed by the waveguide is detectable by a pulse 127 in at least one of the received light beams for the different wavelengths used, as illustrated in FIG. 6A, which represents the electrical signal received, for a wavelength in the reception means C, after conversion of the optical signal received for this wavelength, into an electrical signal. For this purpose, it implements, for example, a photodiode or a phototransistor output of the wavelength divider. In FIG. 6A, the different pulses each indicate the passage of a cell of a certain type through the light beam due to the fact that the cell has absorbed, reflected or deflected a portion of the incident light for the length of the light. considered wave. It should be noted that the width of the pulse is information indicating a dimension, for example a length or a volume of the cell in question. The larger the impulse, the larger the size or volume. The pulse height provides other information to differentiate cell types. Indeed, by comparing the heights of the pulses for the different wavelengths implemented, it is possible to classify the detected cells.

Figures 6B and 6C illustrate the received electrical signal after appropriate digital processing. It should be noted that the signal may, alternatively, be subjected to analog processing by electronic components involving electrical filtering operations. The particular embodiment of the device object of the present invention which has just been described offers many advantages over known analyzers. Due to the use of a component combining an optical circuit and a microfluidic circuit, in a common plane, the tolerances on the alignments are reduced and the alignment is stable between the portions 109 and 110 of the waveguide 108 and between these portions and the channel 104. This very precise alignment excludes any need for a subsequent adjustment and does not run the risk of being out of adjustment, which makes it possible, in particular, to produce a device in the form of a portable instrument and to carry Ie. device without risk of maladjustment. On the other hand, the integrated optical component makes it possible to ensure compatibility with the principle of reading by variation of impedance or conductance. The optical reading module does not disturb the reading by impedance variation or conductance since the cells analyzed are not destroyed or impaired by the components it passes through. By using a microprocessor, it is possible to process more than ten optical signal samples per blood cell passing through the device. The present invention therefore makes it possible to obtain a very precise analysis device that is usable for counting and classifying living cells, which are part of animal or plant biology, but also makes it possible to photometrically study solutions specific to multiple sectors of the animal. activity, especially agri-food, pharmaceutical and chemical.

FIG. 7 shows a reservoir 200 of liquid containing bodies, cells or particles to be counted, a pump 205, an inlet pipe 210, an optical and microfluidic component 215, a liquid outlet 220, a light source 225, two optical fibers 230 and 231, a photosensitive sensor 235, a signal processing means 240 and an information output means 245.

The reservoir 200 of liquid containing bodies is of known type. According to the variants, it comprises, or not, an agitator, for example magnetic, in order to homogenize its contents. The pump 205 is of known type and injects the liquid contained in the reservoir 200 in the pipe 210, itself of known type. Various embodiments of the optical and microfluidic component 215 are described and shown in FIGS. 8, 9, 10 and 11. The liquid outlet 220 leads, according to the variants, either to an evacuation or to a means of performing lysis. that is to say, destruction of certain bodies, for example red blood cells, then an impedance sensor to count the bodies remaining after lysis. Several examples of light sources 225 and several examples of photosensitive sensors 235 are described with reference to FIGS. 12, 13 and 14.

The optical fibers 230 and 231 are of known type and are transparent and form waveguides, in the spectral ranges used. The signal processing means 240 is adapted to filter each signal coming from the photosensitive sensor 235, to associate the information contained in these signals for different spectral ranges, to position the local extreme values of these signals in a multidimensional space, to deduce from it the class of each body represented by these extreme values. Preferably, the signal processing means 240 is further adapted to process the shape of the pulses of these signals in order to obtain complementary information on these bodies and to count, in each class, the bodies stored therein. For example, the width of the pulses is indicative of a body size and the shoulders present in the pulses are indicative of the presence of distinct parts in the bodies. The information output means 245 is of known type, for example computer connector type, display screen or printer. It enables the results obtained by the signal processing means 240 to be made available. Various treatments applied by the signal processing means are represented in FIGS. 15 and 16.

FIG. 8 shows an optical and microfluidic component 315 comprising a channel 305, passages 310 and 311 of collinear optical fibers 230 and 231, and partitions 320 and 321.

The component 315 is formed, at least for a layer comprising the channel 305 and the passages 310 and 311, with a transparent material in the spectral ranges used. The channel 305 and the passages 310 and 311 are formed in the component 315 in a known manner, for example by mechanical drilling, by etching processes, by a method similar to that described in US Pat. No. 6,438,279 or as disclosed in US Pat. first embodiment, with reference to FIGS. 1 to 6C. The optical fibers 230 and 231 are mechanically introduced into the passages 310 and 311, respectively and, optionally, assembled by gluing, with an optical adhesive, to the partitions 320 and 321. It is observed that this embodiment has, compared to the first embodiment embodiment shown with reference to Figures 1 to 6C, a simpler manufacture since it is not necessary to dope a material to change the optical index. However, in variants, the partitions or their complement in the layer of material that contains them are doped to improve the passage of light rays from the optical fiber 230 to the optical fiber 231, through the partitions and the channel 305.

FIG. 9 shows an optical and microfluidic component 415 comprising a channel 405, an input waveguide 410, an output waveguide 411 collinear with the input waveguide 410, and conductive surfaces 412 and 413 placed on the surface of the channel

405 or near it, instead of the crossing of channel 405 and waveguides 410 and 411.

The channel 405, input waveguides 410 and output 41 1 can be made according to one of the techniques described above. The insertion of conductive surfaces 412 and

413, preferably of a surface similar to the cross section of the channel 405 is made by the addition of channels perpendicular to the plane of the channel 405 and the waveguides 410 and 411, for example by drilling, by the mounting of the layer comprising the plane of the channel 405 and the waveguides 410 and 411 between two printed circuits bearing these conductive surfaces 412 and 413, or by adding at the time of assembly the layers constituting the component 415. Thanks to the component 415, the sensor 235 is completed by an impedance sensor which measures the impedance variation between the conductive surfaces 412 and 413, in the area between the waveguides 410 and 411. The signals representative of the impedance and the signals from the photosensitive sensor 235 are therefore synchronized, i.e. a body, particle or cell modulating the light transmitted from the waveguide 410 to the waveguide 411 acts simultaneously on the impedance measurement signal. These signals can thus be correlated and give more information on this body, this particle or this cell.

Alternatively, the conductive surfaces are positioned on the same side of the plane of the channel 405 and the waveguides 410 and 411 or are in pairs on each side of this plane, the inductance being measured between the upstream surfaces lying on both sides and downstream surfaces on both sides. FIG. 10 shows an optical and microfluidic component 515 comprising a channel 505, an input waveguide 510, an output waveguide 51 1 collinear with the input waveguide 510, and conductive surfaces 512 and 513 placed at the exit of the intersection zone of the channel 505 and the waveguides 510 and 511, in the same manner as in an analyzer employing the impedancemetry. Channel 505, input waveguides 510 and output waveguides 511 can be made according to one of the techniques described above. However, the channel 505 opens, as soon as it leaves the intersection zone, onto an opening made in a conductive diaphragm 512 and facing an electrode 513.

With the component 515, the sensor 235 is completed by an impedance sensor which measures the impedance variation between the conductive surfaces 512 and 513, immediately out of the area between the waveguides 510 and 511. The signals representative of the impedance and the signals from the photosensitive sensor 235 are thus almost synchronized, ie a body, a particle or a cell modulating the light transmitted from the waveguide 510 to the waveguide 511 operates with a very short delay time, which depends on the speed of the liquid in the channel 505, on the impedance measurement signal. These signals can thus be correlated and give more information on this body, this particle or this cell.

It can be seen that, in order to limit the vortices that can disturb the impedance measurement, an auxiliary auxiliary liquid inlet channel (not shown because it is above the sectional plane) and a liquid outlet channel 514 are advantageously provided. from the channel 505 and the auxiliary channel 540, for example in a plane perpendicular to the plane of the channel 505 and the waveguides 510 and 511 and forming, between them and with the channel 505, an angle of 120 degrees.

FIG. 11 shows an optical and microfluidic component 615 comprising a channel 605, an input waveguide 610, an output waveguide 611 co-linear with the input waveguide 610, and a waveguide 650 whose axis passes through the zone of intersection of the channel 605 and the waveguides 610 and 611 but outside the plane of the channel 605 and the waveguides 610 and 611. Preferably, the axis of the waveguide 650 is perpendicular to the plane of the channel 605 and the waveguides 610 and 611. The waveguide 650 is connected, for example by a fiber optical, to the photosensitive sensor 235 and provides information on the portion of the light that is reflected by a body, particle or cell in the intersection area.

Channel 605, input waveguides 610 and output waveguides 611 can be made according to one of the techniques described above. Waveguide 650 may be made by drilling, for example, leaving a bulkhead (not shown) at channel 605.

It is observed that the various embodiments described above, by way of example, can be combined to form other optical and microfluidic components, for example comprising, both the waveguide 650 and surfaces conductors for measuring the impedance of the intersection zone, as set forth with reference to one of FIGS. 9 or 10.

For the production of the optical and microfluidic component, the reader will also be able to draw on the method of manufacture disclosed in the document US Pat. No. 6,438,279.

It is observed that the implementation of the present invention does not require the implementation of an integrated optical and fluidic component. On the contrary, in embodiments, optoelectronic circuit fabrication techniques implemented in the telecommunication field, for example lasers and / or photodiodes, are implemented. The precise alignment of the components can then use, for example, the so-called "flip-chip" technique.

Preferably, the channel of each embodiment illustrated in FIGS. 7 to 11 presents, in input and / or in output, a funnel shape. This limits the risk of clogging and allows a natural cladding bodies in the center of the channel and therefore the flow. FIG. 12 shows that, in embodiments, the optical scheme may comprise a plurality of light sources emitting in different spectral ranges, here three sources 701, 702 and 703 are represented and a plurality of photosensitive sensors in FIG. same as the light sources, 721, 722 and 723. Each of the sensors is sensitive in a spectral range emitted light by at least one of the light sources. For example, the light sources are light-emitting diodes or lasers and the photosensitive sensors are photodiodes or phototransistors. Between the sources and the sensors are a multiplexer / divider 705, an input waveguide 706, the intersection zone 707 of a channel 708 carrying the liquid that is characterized, and waveguides. , the output waveguide 709 and a multiplexer / divider 710.

Thus, the light rays of different spectral ranges are emitted by the different light sources, combined by the multiplexer / divider 705, injected into the input waveguide 706, cross intersection area 707 and waveguide 709 and are distributed by multiplexer / divider 710, according to their spectral ranges, on the different photosensitive sensors.

Of course, the light sources and the multiplexer / divider 705 are assembled so that the light rays emitted by the different light sources are oriented towards the waveguide 706. Similarly, the photosensitive sensors and the multiplexer / divider

710 are assembled so that the light rays that reach a photosensitive sensor have wavelengths for which the sensor in question is sensitive.

FIG. 13 shows that, in embodiments, the optical diagram may comprise a white light source 801 and a plurality of photosensitive sensors, here three sensors 821, 822 and 823 are represented. Each of the sensors is sensitive in a spectral range emitted by the light source 801. For example, the light source 801 is a light emitting diode or lasers, an incandescent light source. Between the light source and the sensors are an input waveguide 806, the intersection zone 807 of a channel 808 carrying the liquid that is characterized and waveguides, the guide of output wave 809 and a multiplexer / divider 810.

Thus, the light rays of different spectral ranges are emitted by the light source 801, injected into the input waveguide 806, pass through the intersection zone 807 and the waveguide 809 and are distributed by the multiplexer / divider 810, according to their spectral ranges, on the different photosensitive sensors.

Naturally, the photosensitive sensors and the multiplexer / divider 810 are assembled so that the light rays that reach a photosensitive sensor have wavelengths for which the sensor in question is sensitive. FIG. 14 shows that, in embodiments, the optical diagram may comprise a plurality of light sources emitting in different spectral ranges, here three sources 901, 902 and 903 are represented and a photosensitive sensor 921. The sensor 921 is sensitive in each of the spectral ranges emitted by the light sources 901, 902 and 903. For example, the light sources are light-emitting diodes or lasers and the photosensitive sensor is a photodiode or a phototransistor. Between the sources and the sensor is an input waveguide 906, the intersection zone 907 of a channel 908 carrying the liquid that is characterized and waveguides, and the waveguide 909 output.

In these embodiments, the light sources are successively turned on in order to perform time division multiplexing. Thus, the light rays of different spectral ranges are successively emitted by the different light sources, injected into the input waveguide 906, pass through the intersection zone 907 and the waveguide 909 and reach the photosensitive sensor.

FIG. 15 shows that the signal corresponding to a wavelength or a limited spectral range, after filtering high, has negative (downward) pulses or peaks corresponding to the light absorption of a body flowing in the channel opposite the waveguides and variations corresponding to noise, variations that can be eliminated by thresholding the signal. These pulses have an extreme value, here the minimum and an amplitude, corresponding to the difference between an average value of the signal and the extreme value of the pulse. FIG. 16, in which only three wavelengths or spectral ranges or two wavelengths and one impedance are considered, with the aim of simplifying the description, by positioning the amplitudes of the pulses measured simultaneously on the signals corresponding to the different wavelengths and possibly to the impedance, the bodies are represented by points having three coordinates.

Based on these coordinates, the bodies are arranged in classes corresponding to volumes in the multidimensional representation of Figure 16.

In Figure 16, six bodies 1101 to 1106 are shown and distributed in three classes or families of bodies 111 1 to 1113, which correspond to three volumes. For each body, its representation is made by three segments whose intersection is at the point whose coordinates are defined by the amplitudes of the pulses of the various processed signals and whose length, on either side of this point of intersection is representative, on the axis corresponding to a wavelength, of the width of the pulse. The bodies in the same class are discriminated according to these pulse widths, or segment lengths, in subclasses.

The inventors have determined that these different treatments make it possible to classify and enumerate four types of blood cells: platelets, red blood cells, white blood cells and red blood cells with inclusion of RNA (reticulocytes).

In embodiments, for detecting red blood cells with inclusion of RNA (reticulocytes), the RNA is stained, according to techniques known per se, prior to the passage of these cells in the optical observation channel. In embodiments, the photosensitive sensor (s) are based on indium arsenide and gallium InGaAs, whose sensitivity extends over the wavelength range from 800 nanometers to 1650 nm. nanometers. In embodiments, a National Instruments (registered trademark) acquisition card with 8 analog inputs, femax = 1.25 MHz, is used.

It can be observed that each optical fiber used may be a multi-strand optical fiber, in particular in the case where several light sources are used, the strands being distributed facing the different light sources (see, in particular, FIG. 14)

As can be seen from FIG. 17, in embodiments of the method, during a step 1205, the liquid containing the bodies to be enumerated is diluted.

Then, in a step 1210, the diluted liquid containing the bodies is set in motion, for example, by the action of a pump, so that they pass between a light emitting means having a plurality of wavelengths and light receiving means capable of providing signals representative of the received light for different wavelengths.

During a step 1215, optional, creates a potential difference between two conductive surfaces. During a step 1220, for each range of wavelengths, or spectral ranges, a signal representative of the light received by the reception means and, optionally, a signal of potential difference between the conductive surfaces is received. .

During a step 1225, the signals are filtered to eliminate the low frequency components, for example frequencies below 500 Hertz. During an optional step 1230, the potential difference signal is shifted temporally as a function of the flow velocity in the channel and the distance between the center of the optical observation zone and the center of the optical observation zone. the gap separating the conductive surfaces.

During a step 1235, a thresholding of the signals is carried out, that is to say an elimination of the variations of low amplitude signal.

During a step 1240, a measurement is made of the width and the amplitude of each pulse of each signal.

During a step 1245, these measurements are associated in n-tuples, for example, when there are only three signals implemented, to form amplitude triplets or amplitudes of 6-uples. widths.

During a step 1250, the n-tuples are distributed in classes and / or subclasses, according to their presence in volumes defining classes and / or subclasses in a multidimensional space corresponding to the n -uples. It is observed that these volumes are preliminary defined by the observation of reference bodies. These volumes can be generated automatically by learning techniques generating polygons, for example. During a step 1255, there is, by each class and / or subclass, the number of bodies.

During a step 1260, the body numbers are provided in each class and / or subclass. Then, we return to step 1220.

Claims

1 - Apparatus for counting bodies in a liquid, characterized in that it comprises: light emitting means comprising a plurality of wavelengths, said bodies each having at least one optical effect for at least one said length wave, a relative displacement means of said liquid containing said body, with respect to said light-emitting means, a light-receiving means adapted to provide a plurality of signals corresponding to the light reaching it for said lengths of light; wave, a means of classifying different bodies in different classes, according to the signals provided by the receiving means and a means for counting the bodies of each of said classes of bodies.

2 - Device according to claim 1, characterized in that it comprises an optical and fluidic component comprising, on the one hand, a liquid flow channel containing said bodies and, on the other hand, at least one guide of an input wave connected to said transmitting means, said channel and each input waveguide meeting in an observation zone of the bodies passing through the channel.

3 - Device according to claim 2, characterized in that the optical and fluidic component further comprises at least one output waveguide connected to said receiving means, said channel and each output waveguide meeting in the area of observation of the bodies crossing the canal.

4 - Device according to any one of claims 2 or 3, characterized in that the optical and fluidic component comprises a layer within which are formed the channel and each waveguide, sandwiched between a substrate layer and a coating layer.

5 - Device according to claim 4, characterized in that the layer intended to comprise the channel and each waveguide is initially soluble material become insoluble, outside the channel, after irradiation. 6 - Device according to any one of claims 4 or 5, characterized in that at least one waveguide is constituted by local increase of the optical index of the layer intended to comprise at least one waveguide.

7 - Device according to any one of claims 4 to 6, characterized in that the layer intended to comprise the channel and at least one waveguide is in organo-mineral hybrid photosensitive material. 8 - Device according to any one of claims 4 to 6, characterized in that the layer intended to comprise the channel and at least one waveguide is polymeric light-sensitive material.

9 - Device according to any one of claims 2 to 8, characterized in that the channel has a cross section of smaller size greater than or equal to the largest dimension of the body flowing in said channel.

10 - Device according to any one of claims 2 to 9, characterized in that the channel has, in input and / or output, a funnel-shaped.

11 - Device according to any one of claims 2 to 10, characterized in that it comprises means for injecting into the input waveguide, light components of said plurality of wavelengths corresponding to different absorption powers for said bodies.

12 - Device according to any one of claims 2 to 1 1, characterized in that the optical and fluidic component (415, 515) comprises conductive surfaces (412 and 413, 512 and 513) placed on the surface of the channel (405 , 505) or in its vicinity, instead of crossing the channel and at least one waveguide (410 and 411, 510 and 511) and an impedance sensor which measures the impedance variation between the conductive surfaces .

13 - Device according to any one of claims 2 to 12, characterized in that the optical and fluidic component (615) comprises at least one output waveguide (650) adapted to convey light reflected by bodies of found in said liquid and flowing in the flow channel.

14 - Device according to any one of claims 1 to 13, characterized in that the light emitting means comprises a plurality of light sources emitting in different spectral ranges and a multiplexer adapted to transmit light from each of the light sources to the light receiving means.

15 - Device according to any one of claims 1 to 14, characterized in that the light emitting means comprises a plurality of light sources emitting successively in different spectral ranges, the receiving means comprising a photosensitive sensor adapted to provide a signal representative of light from each of the light sources.

16 - Device according to any one of claims 1 to 15, characterized in that the light receiving means comprises a plurality of sensitive photosensitive sensors in different spectral ranges and a divider adapted to distribute light from the transmitting means on the different sensors according to the wavelength of this light.

17 - Device according to any one of claims 1 to 16, characterized in that it comprises a signal processing means (240) adapted to position the values Local extremes of the signals from the photosensitive sensor in a multidimensional space, and deduce the class of each body represent by these extreme values, each body class being represented by a volume in this multidimensional space. 18 - Device according to claim 17, characterized in that the processing means is further adapted to process an impedance measurement signal and to position the local extreme values of the signals from the photosensitive sensor and the measurement signal. impedance, in a multidimensional space, and to deduce the class of each body represent by these extreme values, each class of body being represented by a volume in this multidimensional space.

19 - Device according to any one of claims 17 or 18, characterized in that the processing means is adapted to process the pulse shape of the signals and to obtain, depending on this form, additional information on these bodies for put them in subclasses. 20 - Device according to one of claims 17 to 19, characterized in that the signal processing means is adapted to count the number of bodies in each class and / or subclass. 21 - Method for counting bodies in a liquid, characterized in that it comprises: a light emission step comprising a plurality of wavelengths, said bodies each having at least one optical effect for at least one said length wave, a relative displacement step of said liquid containing said body, with respect to said light-emitting means, a light-receiving step for providing a plurality of signals corresponding to light reaching it for said lengths of light; wave, a step of classifying different bodies in different classes, according to the signals provided by the receiving means and a step of counting the bodies of each of said body classes.