WO2007057574A1 - Method and device for counting bodies in a liquid - Google Patents

Method and device for counting bodies in a liquid Download PDF

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Publication number
WO2007057574A1
WO2007057574A1 PCT/FR2006/002538 FR2006002538W WO2007057574A1 WO 2007057574 A1 WO2007057574 A1 WO 2007057574A1 FR 2006002538 W FR2006002538 W FR 2006002538W WO 2007057574 A1 WO2007057574 A1 WO 2007057574A1
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WO
WIPO (PCT)
Prior art keywords
channel
body
light
characterized
device according
Prior art date
Application number
PCT/FR2006/002538
Other languages
French (fr)
Inventor
Philippe Daurenjou
Original Assignee
Ph Diagnostics Sas
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Filing date
Publication date
Priority to FR0511670A priority Critical patent/FR2893414A1/en
Priority to FR0511670 priority
Application filed by Ph Diagnostics Sas filed Critical Ph Diagnostics Sas
Publication of WO2007057574A1 publication Critical patent/WO2007057574A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Electro-optical investigation, e.g. flow cytometers
    • G01N15/1434Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/01Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03Cuvette constructions
    • G01N21/05Flow-through cuvettes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/84Systems specially adapted for particular applications
    • G01N21/85Investigating moving fluids or granular solids
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Electro-optical investigation, e.g. flow cytometers
    • G01N15/1456Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Electro-optical investigation, e.g. flow cytometers
    • G01N15/1484Electro-optical investigation, e.g. flow cytometers microstructural devices
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/01Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03Cuvette constructions
    • G01N2021/0346Capillary cells; Microcells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/01Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03Cuvette constructions
    • G01N21/05Flow-through cuvettes
    • G01N2021/056Laminated construction

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 COUNTING BODIES IN LIQUID

The present invention relates to a method and a device body counting in a liquid. It applies, in particular, enumeration and, optionally, differentiation of particles suspended in a liquid, particularly the cells contained in the blood, optionally diluted, such as erythroid cells, leukocyte and / or thrombocyte. Are known particle counting systems in a liquid, which enable qualitative and quantitative analysis of blood cells. These systems work on the principle of detecting the change in impedance or conductance, known as the "Coulter principle." This change in impedance or conductance does not provide morphological information on the cells. Document US 6,438,279 presents a unitary structure of microcapillary and waveguide and a method for its manufacture. The teaching of this document applies to the fluorescence, that is to say to excitation by a powerful light beam, from a laser, of a substance to emit light for be analyzed. This technology does not apply to the enumeration or classification of living cells, such as different blood cells.

The present invention aims to overcome these drawbacks.

For this purpose, in a first aspect, the present invention provides a body counting device in a liquid, which comprises: a light emitting means having a plurality of wavelengths, said body each having at least one effect optics for at least one said wavelength, a shift-up means on said liquid containing said body with respect to said light emitting means, light receiving means adapted to provide a plurality of signals corresponding to the light reaching it to said wavelengths, means of classification of different body in different classes, depending on the signals supplied by the receiving means and a body of the counting means each of said classes of bodies. Thanks to these provisions, each body can be identified within a body class and a count of the number of bodies in each class can be performed.

According to particular features, the device as briefly described above comprises an optical and fluidic component comprising, on one hand, a liquid flow channel containing said body and, on the other hand, at least one guide of wave connected to said transmission means input, said channel and each input waveguide occurring in an observation area of ​​the body through the channel.

Through these provisions, the respective positions of the channel and the waveguide can be specific and final, without risk adjustment.

According to particular features, the optical and fluidic component further comprises at least one output waveguide connected to said receiving means, said channel and each output waveguide occurring in the observation area of ​​the body through the channel. Through these provisions, the respective positions of the channel and the waveguide can be specific and final, without risk adjustment.

According to particular features, the optical and fluidic component comprises a layer within which are made the channel and each waveguide sandwiched between a substrate layer and a coating layer. Thanks to these provisions, even if very small, less than 0.1 millimeter, and each channel waveguide, their respective positions can be specific.

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

With these arrangements, manufacture of the channel is easy and accurate.

According to particular features, at least one waveguide is formed by local increase in the refractive index of the layer intended to include at least one waveguide. Thanks to these provisions avoids mechanical actions such as drilling in the optical component and fluid.

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

According to particular features, the layer intended to include the channel and at least one waveguide is photosensitive polymeric material.

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

According to particular features, the channel has a cross-sectional dimension smaller than or equal to the largest dimension of the body flowing through said channel. According to particular features, the channel has, at the input and / or output, a funnel shape. This limits the risk of clogging and allows natural body cladding to the center channel and thus the flow.

Thanks to these provisions, it avoids the risk of clogging of the canal by the body and avoids distorting the larger body flowing through this channel.

According to particular features, the device as briefly described above comprises an injection means in the input waveguide, light components of said plurality of wavelengths corresponding to different absorption powers for said body. Thanks to these provisions, classification of body is easy because it can be performed based on the absorption wavelengths of the body.

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

Thanks to these features can be measured synchronously or very slightly shifted, the impedance of each body of which we analyze the optical effect. additional information is thus obtained on each of the body flowing in the channel, which allows, for example, to confirm a classification or constitute subclasses in which one can classify the different bodies.

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

Thanks to these features, one can treat the optical effects of reflection bodies flowing in the channel and thus confirm their classification or store them in subclasses.

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

Thanks to these provisions, it increases the proportion of the light emitted by each light source that reaches the body flowing in the channel.

According to particular characteristics, the light emitting means includes a plurality of light sources emitting in different spectral ranges in succession, the receiving means comprising a light sensor adapted to provide a signal representative of light from each of light sources . Thanks to these arrangements, the light receiving means is simple and the time used to separate the various signals representing the different wavelengths emitted by the light source and thus to treat the optical effects for different lengths wave, different body flowing in the channel. According to particular characteristics, the light receiving means includes a plurality of photosensitive sensors sensitive in different spectral ranges and a divider adapted to divide the light from the emitting means on the different wave sensor according to the length of this light.

Thanks to these provisions, it increases the proportion of the light emitted by each light source that reaches the receiving means.

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

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

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

Thanks to these provisions, discrimination bodies between different classes is more accurate or safer.

According to particular features, the processing means is adapted to process the signals of pulse form and to obtain, according to this form, additional information on the body for storing into subclasses.

Thanks to these features, different body of a class can be stored 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 provides a body counting method in a liquid, characterized in that it comprises: a light emitting step comprising a plurality of wavelengths, said body each having at least one optical effect to said at least one wavelength,

- a moving step on said liquid containing said body with respect to said light emitting means, a light receiving step of providing a plurality of signals corresponding to the light reaching it for said wavelength, a different body classification step in different classes, depending on the signals supplied by the receiving means and - a counting step of the body of each of said classes of bodies.

The advantages, specific objects and features of this method are similar to those of the device object of the present invention as briefly described above, they are not repeated here.

Other advantages, specific objects and features of the present invention appear on reading the description which follows, given, for explanatory purposes and in no way limiting to the appended drawings in which Figure 1 shows schematically in perspective, a first embodiment of the device of the present invention and a means for transmitting representation and reception of optical signals, - Figure 2 shows, schematically, in perspective, the first embodiment of the object of the device present invention, Figure 3 represents, schematically, a particular embodiments of component of the device object of the present invention, Figure 4 is a schematic view, in section, of the component shown in Figure 3, - Figure 5 is a view schematic, sectional, of the component shown in figures 3 and

4, 6A, 6B and 6C show an electrical signal representative of an optical signal in implementing the first embodiment of the present invention and this signal after treatment, - Figure 7 shows schematically a second embodiment of embodiment of the device, Figure 8 shows, diagrammatically and in section, an embodiment of an optical component and microfluidic incorporated in the device illustrated in Figure 7, Figure 9 shows, schematically and in perspective, one embodiment an optical component and microfluidic incorporated in the device illustrated in Figure 7, Figure 10 shows, diagrammatically and in section, an embodiment of an optical component and microfluidic incorporated in the device illustrated in Figure 7,

- Figure 11 shows, schematically and in perspective, an embodiment of an optical component and microfluidic incorporated in the device illustrated in Figure 7,

- Figure 12 represents, schematically, an embodiment of the optical portion of the device illustrated in Figure 7,

- Figure 13 represents, schematically, an embodiment of the optical portion of the device illustrated in Figure 7, - Figure 14 shows schematically one embodiment of the optical portion of the device illustrated in Figure 7, Figure 15 shows a signal representative of the transparency of a sample in a spectral range, being processed, Figure 16 illustrates the classification of samples in a volume and - Figure 17 shows in the form of a flowchart, steps utilized in deals with a particular embodiment of the method of the present invention. It is observed that the components illustrated in the drawings are not to scale. The present invention applies in particular to food areas (analysis of milk, fruit juice or wine, for example), pharmaceutical, medical and biological (fluid analysis such as blood) and to areas where it can disperse particles or cells in a fluid, for example water, without a solvent or a gas, such as air.

The device illustrated in Figs applies, in particular, the enumeration and, optionally, differentiation of particles suspended in a liquid, particularly the cells contained in the blood, optionally diluted, such as erythroid cells, and leukocyte / or thrombocyte.

Blood cells such as red blood cells, white blood cells and platelets in the blood, suspended in plasma. It may be helpful, especially to determine certain conditions and developments of treatment, to know the quantities and the morphologies of these blood cells.

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

Referring to Figure 2, it is found that the mean A essentially comprises a parallelepiped general shape of housing 104 and, inside the housing 104, an optical component 106 provided with an optical network and a microfluidic network integrated optical component 106. the optical component 106, also of parallelepiped shape, is traversed by a channel 107 of the diluted blood flow to be analyzed, calibrated according to the size of blood cells so as not to impede their flow and a guide for wave 108. the channel 107 extends perpendicularly between two opposite faces of the optical component 106 in the middle of these sides. Preferably, the channel 107 has, at the input and / or output, a funnel shape. This limits the risk of clogging and allows natural body cladding to the center channel and thus the flow.

The waveguide 108 extends between two opposite faces of the optical component 106 in the middle of these sides. The waveguide 108 is thus interrupted by the channel 107. The channel 107 thus divides the waveguide 108 in a portion of waveguide 109 and a transmitter portion of waveguide receiver 110. Each guide portion waveform, 109 and 110, has a cross section, for example square, and is coupled to an optical fiber cross-section, e.g., circular, respectively of inlet 111 and outlet 112, via modules groove "V" 113, shown in Figure 5. Figure 5 shows, also, the coupling waveguide square section of the optical fibers of circular cross section, for example using an adhesive 115 to adaptation optical index.

As seen in Ia Figure 2 and in Figure 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 profile funnel, although not shown, may 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 preferably the same height as the fluidic channel 107, or slightly lower.

The assembly formed by the optical component 106 and the groove modules "V" 113 is encapsulated in the 104 light-tight enclosure that protects it from this set and the optical isolation of the waveguide portions 109 and 110. The housing 104 has a hole 119 opening into the outer front face of the housing 104 and a hole 120 opening into the rear outer side of the housing 104. the holes 119 and 120 are both, preferably of 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 the channel 107, passes between the waveguide portions 109 and 110 and exits through the exit hole 120. the housing 104 is made of any suitable material, such as resin or other polymeric material.

For optical component 106, comprising, sandwiched between a lower substrate layer 122 and a coating layer 123, a layer 125 of a photosensitive material, for example an organo-mineral hybrid material or polymeric. This layer 125 may be of composite structure and composed of a stack of a plurality of unit layers.

It is in the layer 125 is brought about the waveguide 108, with its emitting portions 109 and receiver 110. The material which constitutes the layer 125 has the property of becoming insoluble after irradiation while non-irradiated portions remain soluble , for example in solvents alcohol or acetone. the channel 107 is thus achieved by disposing a mask on the region of the future channel 107 and irradiating the rest of the layer 125. Thus, only the material of the layer which has been protected from the radiation by the mask remains soluble and is then solubilized by a suitable solvent.

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

By integrating the component 106 of the waveguide 108 and the channel 107, there is obtained an alignment between the waveguide 108 and the channel 107 during the manufacture of the component.

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

Concerning the functioning of the device object of the present invention, it allows the detection of different types of cells, such as red blood cells, white blood cells and platelets, since these cells differentiate from one another by their power of absorption, deviation or reflection of light traversing the input waveguide portion 109, this absorption, deviation or reflection depending on the wavelength of the light used. For differentiating blood cells which is passed through the channel 107 and through the light beam carried by the waveguide 108, is implemented a plurality of wavelengths or wavelength ranges which correspond to the power of absorption, deflection and / or reflection, preferably maximum, different blood cells. The injection and separation of different components of light is the transmitter side and the receiver side by using wavelength multiplexers, splitters, known under the name of "WDM". By comparing the ratio of light intensities received and transmitted in different wavelengths, indicating the amount of light absorbed, identifying the nature of each cell passing through the light beam.

In general, the passage of a cell through the beam of light carried by the waveguide is detectable by a pulse 127 in at least one of the light beams received for different wavelengths used, as illustrated in Figure 6A, representing the received electric signal to a wavelength in the receiving means C, after conversion of received optical signal to the wavelength, into an electric signal. For this purpose, use is made of, for example, a photodiode or a phototransistor at the output of wavelength splitter. Ia on Figure 6A, the individual pulses each indicate the passage of a cell of one type through the light beam due to the fact that the cell has absorbed, reflected or deflected a portion of the incident light for Ia length of wave considered. It should be noted that the pulse width is information indicating a size, for example a length or volume of the cell. More momentum is more broad size or the volume is important. The height of the pulses provides a further information for differentiating cell types. In fact, by comparing the heights of the pulses for the different wavelengths implemented, can perform the classification of the detected cells.

Figures 6B and 6C illustrate the electrical signal received after an appropriate digital processing. It should be noted that Ie signal can, alternatively, be subjected to an analog processing by electronic components involving electrical filtering operations. The particular embodiment of the device of the present invention just described has many advantages over the known analyzers. Due to the use of a component combining an optical circuit and a microfluidic circuit in a common plane, the alignment tolerances 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 precise alignment excludes any need for further adjustment and is not likely to go awry, allowing, in particular, to provide a device as a portable instrument and carry Ie device without risk adjustment. Moreover, the integrated optical component ensures compatibility with the principle of reading by change in impedance or conductance. The optical reader module does not disrupt the reading change in impedance or conductance since the analyzed cells are not destroyed or altered by the components it through. Using a microprocessor, one can carry out the treatment more than ten samples of optical signals through blood cell passing through the device. The present invention thus provides a very accurate analysis device that is used to count and classify the living cells within the animal or plant biology, but also allows studying photometry specific solutions to multiple sectors activity, including food processing, pharmaceutical and chemical.

Is observed in Figure 7, a reservoir 200 of fluid containing body cells or particles to be counted, a pump 205, a supply line 210, an optical component and microfluidic 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 body is of known type. According to the variants, it comprises or not a stirrer, magnetic example, in order to homogenize its contents. The pump 205 is of known type and injects the liquid contained in the tank 200 into the pipe 210, itself of known type. Various embodiments of the optical component and microfluidic 215 are described and shown in Figures 8, 9, 10 and 11. The liquid outlet 220 leads, according to the variants, either a discharge or a lysis making means , that is to say, destruction of certain body, such as red blood cells, and an impedance sensor to count the body remaining after lysis. Several examples of light sources 225 and several examples of photosensitive sensors 235 are described with reference to Figures 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 from the photosensor 235, to associate the information contained in these signals for different spectral ranges, positioning local extreme values ​​of these signals in a multidimensional space, to deduce 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 to obtain additional information on these bodies and to count, in each class, the objects that are stored there. For example, the width of the pulses is indicative of a dimension of the body and the shoulder present in the pulses are indicative of the presence of separate parts in the body. The information output means 245 is of known type, for example of the computer connector, display screen or printer. It allows the provision of the results obtained by the signal processing means 240. Various treatments applied by the signal processing means are shown in Figures 15 and 16.

Is observed in Figure 8, an optical component and microfluidic 315 having a channel 305, passages 310 and 311 of the optical fibers 230 and 231, collinear, and partitions 320 and 321.

The component 315 is formed, at least a layer comprising the channel 305 and passages 310 and 311, by a transparent material in the spectral ranges implemented. The channel 305 and passages 310 and 311 are formed in the component 315, in known manner, for example by mechanical drilling, by etching processes, by a method similar to that described in US Patent 6,438,279 or as disclosed in first embodiment, with reference to figures 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, the partitions 320 and 321. It is noted that this embodiment has, with respect to the first embodiment embodiment described with reference to figures 1 to 6C, a simpler manufacturing since it is not necessary to dope a material to change the refractive index. However, in variants, partitions or their complement in the material layer that contains are doped to improve the passage of light rays from the optical fiber 230 to the optical fiber 231, through the walls and the channel 305.

It is observed in Figure 9, an optical component and microfluidic 415 having 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 its proximity, instead of crossing the channel 405 and wave guides 410 and 411.

The channel 405, the input waveguides 410 and outlet 1 41 may be made according to any of the techniques described above. Insertion of the conductive surfaces 412 and

413, preferably of a surface similar to the cross section of channel 405 is effected by the addition of channels perpendicular to the plane of channel 405 and waveguides 410 and 411, for example by drilling, by mounting Ia layer with the plane of the channel 405 and wave guides 410 and 411 between two printed circuits carrying these conductive surfaces 412 and 413, or by addition to the time of assembly of the component constituting the layers 415. with the component 415, the sensor 235 is completed by an impedance sensor which measures the change in impedance between the conductive surfaces 412 and 413, in the area between waveguides 410 and 411. the signals representative of the impedance and the signals from the photosensitive sensor 235 are synchronized, that is to say, a body, a particle or cell modulating light transmitted from the waveguide 410 to the waveguide 411 acts simultaneously on the measuring signal 'Impe dance. These signals can be correlated and provide more information on this body, this particle or that cell.

Alternatively, the conductive surfaces are positioned on the same side of the plane of the channel 405 and waveguides 410 and 411 or are in pairs on each side of this plane, the inductance is measured between the upstream surfaces lying on both sides and swallow surfaces lying on both sides. It is observed in Figure 10, an optical component and microfluidic 515 having 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 outlet of the channel of the intersection area 505 and waveguides 510 and 511, of my same manner as in an analyzer embodying the impedance. The channel 505, the input waveguide 510 and output 511 may be made according to any of the techniques described above. However, Ie channel 505 opens, right out of the region of intersection on an opening in a conductive diaphragm 512 and opposite electrode 513 with a.

With the component 515, the sensor 235 is completed by an impedance sensor which measures the change in impedance between the conductive surfaces 512 and 513, in immediate release from the area between the waveguides 510 and 511. The signals representative of the impedance and the signals from the photosensor 235 are almost synchronized, that is to say, a body, a particle or cell modulating light transmitted from the waveguide 510 to the waveguide 511 acts with a very short delay time, which depends on the velocity of the liquid in the channel 505, on the impedance measurement signal. These signals can be correlated and provide more information on this body, this particle or that cell.

It is observed that, to limit the vortices could disturb the impedance measurement, there is advantageously provided an auxiliary channel of additional liquid inlet (not shown because it is above the section plane) 514 and a liquid channel outputs from the channel 505 and the auxiliary channel 540, for example in a plane perpendicular to the plane of channel 505 and waveguides 510 and 511 and forming, between themselves and with the channel 505, a 120 degree angle.

It is observed in Figure 11, an optical component and microfluidic 615 having a channel 605, an input waveguide 610, an output waveguide 611 collinear with the input waveguide 610, and a waveguide 650 whose axis passes through the intersection of the channel area 605 and wave guides 610 and 611 but outside the plane of the channel 605 and waveguides 610 and 611. Preferably, the axis of the waveguide 650 is perpendicular to the plane of the channel 605 and wave guides 610 and 611. the wave guide 650 is connected, for example by an optical fiber, the photosensitive sensor 235 and provides information on the portion of the light reflected by a body, a particle or a cell located in the intersection area.

The channel 605, the input waveguide 610 and output 611 may be made according to any of the techniques described above. The waveguide 650 can be performed by drilling, for example, leaving a septum (not shown) to the channel 605.

It is observed that the various embodiments discussed above, as examples, may be combined to form other micro-fluidic and optical components, for example with both the waveguide 650 and surfaces conductor for measuring the impedance of the intersection zone, as described with reference to one of figures 9 or10.

For the realization of optical and microfluidic component, the reader will also learn from the manufacturing method described in document US 6,438,279.

It is observed that the implementation of the present invention does not require the implementation of an optical and fluidic integrated component. In contrast, in embodiments, one implements opto-electronic circuit production techniques implemented in the field of telecommunications, such as laser and / or photodiodes. The precise alignment of the components can then be used, for example, the technique called "flip-chip".

Preferably, the channel of each embodiment illustrated in Figures 7-1 1 shows, at the input and / or output, a funnel shape. This limits the risk of clogging and allows natural body cladding to the center channel and thus the flow. It is observed in Figure 12, that in embodiments, the optical pattern may comprise a plurality of light sources emitting in different spectral ranges, here three sources 701, 702 and 703 are shown and a plurality of photosensitive sensors same number 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 light sources. For example, the light sources are light emitting diodes or lasers and photosensitive sensors are photodiodes or phototransistors. Between the sources and the sensors are located, a multiplexer / divider 705, an input waveguide 706, the intersection area 707 of a channel 708 carrying the liquid which was characterized and waveguides , the output waveguide 709 and a multiplexer / divider 710.

Thus, the light rays of different spectral ranges are emitted by different light sources, Ie combined by multiplexer / divider 705, injected into the input waveguide 706, through Ia intersection area 707 and the guide of waves 709 and are distributed by the multiplexer / divider 710, based on their spectral ranges, on 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 Ie waveguide 706. Likewise, the photosensitive sensor and the multiplexer / divider

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

It is observed in Figure 13, that in embodiments, the optical pattern may comprise a white light source 801 and a plurality of photosensitive sensors, here three sensors 821, 822 and 823 are shown. 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 laser, a source of light by incandescence. Between the light source and the sensors are an input waveguide 806, the intersection area 807 of a channel 808 carrying Ie liquid which was characterized and waveguides, the guide of output waves 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, through the intersection area 807 and the waveguide 809 and are divided by the multiplexer / divider 810, based on their spectral ranges, on different photosensitive sensors.

Of course, the photosensitive sensors Ie and multiplexer / divider 810 are assembled so that the light rays reaching a photosensor have wavelengths for which the sensor in question is sensitive. It is observed in Figure 14, that in embodiments, the optical pattern may comprise a plurality of light sources emitting in different spectral ranges, here three sources 901, 902 and 903 are shown and a photosensitive sensor 921. The sensor 921 is responsive in each of the spectral ranges transmitted 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 source and the sensor are an input waveguide 906, the intersection area 907 of a channel 908 carrying the liquid which was characterized and the waveguides and the waveguide outlet 909.

In these embodiments, the light sources are sequentially lit to achieve a time multiplexing. Thus, the light beams of different spectral ranges are successively emitted by the various sources of light injected into the input waveguide 906, through the intersection area 907 and the waveguide 909 and reach the photosensitive sensor .

It is observed in Figure 15, the signal corresponding to a wavelength or in a limited spectral range, after high-pass filtering, this pulse or negative peaks (down) corresponding to the light absorption 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 has an extreme value, here Ie minimum and an amplitude corresponding to the difference between an average signal value and the extreme value of the pulse. It is observed in Figure 16, wherein only three spectral wavelengths or ranges or two wavelengths and an impedance are considered in a simplification of the description, that by positioning the amplitudes of the pulses measured simultaneously the signals corresponding to different wavelengths and possibly to the impedance, the body are represented by points having three coordinates.

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

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

The inventors have determined that these different treatments are used to classify and count four types of blood cells: platelets, red blood cells - white blood cells and red blood cells with inclusion of RNA (reticulocytes).

In embodiments, to detect red blood cells with inclusion RNA (reticulocytes), stained RNA, according to techniques known per se, preliminarily to the passage of these cells in the optical observation channel. In embodiments, the one or more sensor (s) photosensitive (s) are based on indium gallium arsenide InGaAs, whose sensitivity extends over the wavelength range of 800 nanometers A1650 nanometers. In embodiments, an acquisition card is used National Instruments (RTM) 8 analog inputs, femax = 1.25 MHz.

It is noted that each optical fiber implementation may be an optical fiber stranded, especially in the case where plural light sources are used, the strands being divided into light of different light sources (see, in particular, Figure 14)

As seen in reference to FIG 17, in the embodiments of the method, during a step 1205, diluted liquid containing the bodies to be counted.

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

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

During a step 1225, the signal is filtered to eliminate low frequency components, e.g., frequencies below 500 Hertz. During a step 1230, optional, one temporally shifts the signal representative of the potential difference, depending on the flow velocity in the channel and the distance between the center of Ia optical viewing area and the center of the interval between the conductive surfaces.

During a step 1235, it performs a thresholding of the signals, that is to say removal of small amplitude signal changes.

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

In a step 1245, it combines these measures tuples, for example, when there are only three signals implemented to form amplitude triplets or 6- tuples and amplitudes widths.

During a step 1250, it distributes the tuples in classes and / or subclasses, based on 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 preliminarily defined by the reference body for observation. These volumes can be automatically generated by learning techniques generating polygons, for example. In a step 1255, there are, for each class and / or sub-class, the number of bodies.

During a step 1260, there is provided the number field in each class and / or subclass. Then, the process returns to step 1220.

Claims

1 - body counting device in a liquid, characterized in that it comprises: a light emitting means having a plurality of wavelengths, said body each having at least one optical effect for at least one said length wave, a movement setting means on said liquid containing said body with respect to said light emitting means, light receiving means adapted to provide a plurality of signals corresponding to the light reaching it to said lengths of wave, classification means different body in different classes, depending on the signals supplied by the receiving means and a body of the counting means each of said classes of bodies.
2 - Device according to claim 1, characterized in that it comprises an optical and fluidic component comprising, on one hand, a flow channel of the liquid containing said bodies and, secondly, at least one guide of wave connected to said transmission means input, said channel and each input waveguide occurring in an observation area of ​​the body through the channel.
3 - Device according to claim 2, characterized in that the optical component and fluid further comprises at least one output waveguide connected to said receiving means, said channel and each output waveguide occurring in the observation of the body through the channel region.
4 - Device according to any one of claims 2 or 3, characterized in that the optical component and fluid comprises a layer within which are made 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 include 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 formed by local increase in the refractive index of the layer intended to include at least one waveguide.
7 - Device according to any one of claims 4 to 6, characterized in that the layer intended to include the channel and at least one waveguide is organo-mineral hybrid material photosensitive. 8 - Device according to any one of claims 4 to 6, characterized in that the layer intended to include the channel and at least one waveguide is photosensitive polymeric material.
9 - Device according to any one of claims 2 to 8, characterized in that the channel has a cross-sectional dimension smaller than or equal to the largest dimension of the body flowing through said channel.
10 - Device according to any one of claims 2 to 9, characterized in that the channel has, at the input and / or output, a funnel shape.
11 - Device according to any one of claims 2 to 10, characterized in that it comprises an injection means in the input waveguide, light components of said plurality of wavelengths corresponding to different absorption powers for said body.
12 - Device according to any one of claims 2-1 1, characterized in that the optical component and fluid (415, 515) includes conductive surfaces (412 and 413, 512 and 513) placed on the surface of the channel (405 , 505) or its proximity, instead of the crossing of the channel and at least one waveguide (410 and 411, 510 and 511) and an impedance sensor which measures the change in impedance between the conductive surfaces .
13 - Device according to any one of claims 2 to 12, characterized in that the optical component and fluid (615) comprises at least one output waveguide (650) adapted to convey light reflected by body in said liquid 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 the light from each of light sources to the light receiving means.
15 - Device according to any one of claims 1 to 14, characterized in that Ie light emitting means includes a plurality of light sources emitting in different spectral ranges in succession, the receiving means comprising a light 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 includes a plurality of photosensitive sensors sensitive in different spectral ranges and a divider adapted to divide the light from the transmitting means on different sensor depending on 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 local extreme values ​​of the signals from the photosensitive sensor in a multidimensional space, and deduce the class of each body represented by these extreme values, each body class being represented by a volume within this multidimensional space. 18 - Device according to claim 17, characterized in that the processing means is further adapted to treat 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 in deducing the class of each body represented by these extreme values, each body class being represented by a volume within 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 signals of the pulse shape and to obtain, according to this form, additional information on these bodies for put them into 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 - body counting method in a liquid, characterized in that it comprises: a light emitting step comprising a plurality of wavelengths, said body each having at least one optical effect for at least one said length wave, a moving step on said liquid containing said body with respect to said light emitting means, a light receiving step of providing a plurality of signals corresponding to the light reaching it to said lengths of oven, a different body classification step in different classes, depending on the signals supplied by the receiving means and a counting step of the body of each of said classes of bodies.
PCT/FR2006/002538 2005-11-17 2006-11-17 Method and device for counting bodies in a liquid WO2007057574A1 (en)

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FR0511670A FR2893414A1 (en) 2005-11-17 2005-11-17 system of differentiation and enumeration of particles suspended in a liquid.
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