ES2255349B1 - PROCEDURE AND DEVICE FOR THE IDENTIFICATION AND SEQUENTIAL CHARACTERIZATION OF DISCRETE UNITS IN SUSPENSION IN A CAPILLARY JET IN LAMINAR FLOW REGIME, THROUGH AN OPTICAL OR ELECTROMAGNETIC SENSOR. - Google Patents

Abstract

Procedure and device for the identification and sequential characterization of discrete units in suspension in a capillary jet in laminar flow regime, by means of an optical or electromagnetic sensor. The object of the present invention is a process for the identification and sequential characterization of discrete units in suspension in a capillary jet in a laminar flow regime, by means of an optical or electromagnetic sensor, which includes the following steps: - impulsion by an overpressure of up to 200 atm through a hole, simultaneously and concentrically, of a liquid L carrying the discrete units, together with another fluid F immiscible with it, which envelops it giving rise to a jet. - adjustment of the flow of the jet to a value between one and three hundred times the square of the surface tension corresponding to the interphase of the liquid L and the enveloping fluid F, divided by the square root of the product of the density of the liquid L and the third power of the increase in pressure p; - adjustment of the diameter and the flow of the jet to the requirements of the frequency of passage and threshold of detection of the sensor; - registration by the sensor of the passage of discrete units and characterization in succession of each of them.

Description

Procedure and device for sequential identification and characterization of discrete units in suspension in a capillary stream in laminar flow regime, by means of an optical or electromagnetic sensor.

Object of the invention

The object of the present invention is a procedure for sequential identification and characterization of discrete units in suspension in a capillary jet in regime laminar flow, by means of an optical or electromagnetic sensor, which includes the following stages:

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drive through a overpressure of up to 200 atm through a hole, simultaneously and concentrically, of a liquid L carrying the units discrete, together with another F fluid immiscible with it, which Wraps it, giving rise to a jet.

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jet flow setting a a value between one and three hundred times the square of the surface tension corresponding to the interphase of the liquid L and the envelope fluid F, divided by the square root of product of the density of the liquid L and the third power of the pressure increase Δp;

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diameter and flow adjustment of the jet to the requirements of step frequency and threshold of sensor detection;

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registration by step sensor of discrete units and succession characterization of each of them.

An object of the present invention is also a procedure for sequential identification and characterization of discrete units in suspension in a hair jet in laminar flow rate, by means of an optical sensor or electromagnetic, according to the above, characterized in that the fluid envelope F is a gas.

Furthermore, in this invention a procedure for sequential identification and characterization of discrete units in suspension in a capillary jet in regime laminar flow, by means of an optical or electromagnetic sensor, according to the above, characterized in that the viscosity of the liquid L is adjusted by making this product of the mixture of at least two miscible liquids of different viscosity and compatible with the discrete units in suspension, such that the length of the capillary jet is greater than 300 micrometers, preferably greater than 1 millimeter, measured from the hole to the point where the hair jet disintegrates in drops.

Such a procedure can be applied to the identification and sequential characterization of various types of discrete units: cells, viruses, microcrystals, particles inert, probe-particles with affinity for substances to be analyzed, mono- or multivesicular microcapsules.

On the other hand, the procedure may include a stage of separation of discrete units.

Likewise, an object of the invention is device for sequential identification and characterization of discrete units suspended in one or more capillary jets in laminar flow rate, by means of an optical sensor or electromagnetic, characterized in that it includes:

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a source of liquid L carrier of discrete units;

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a source of the fluid F envelope

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a waterproof barrier, which separates the two sources cited from the outside environment, and from which one or more jets of carrier liquid L emerge surrounded concentrically by the envelope fluid F through one or more holes correspondingly practiced in said barrier;

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an element that ensures a overpressure of up to 200 atm from sources with respect to outside environment and allows all emerging flows of the o of the holes are in the range described in the claim 1;

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a sensor, located in front of the section stable of the concentric jets, which registers the units discrete in sequential transit.

Optionally, the envelope fluid F can be a gas

The device can be applied to the identification and sequential characterization of various types of discrete units: cells, viruses, microcrystals, particles inert, probe-particles with affinity for substances to be analyzed, mono- or multivesicular microcapsules.

It can also include elements to make the separation of said discrete units from the hair jet.

State of the art

Flow cytometry (CMF) is a technology intended to measure very diverse cellular parameters, such as surface, cytoplasmic and nuclear antigens, acids nucleic, enzymatic activity, calcium flow, potential membrane and pH. This technology is characterized, developed in the last 35 years, for its ability to measure a large number of cells, individually, for very short periods of time. Among its advantages are its accuracy, precision, speed and ability to process a large number of Samples in short time. The CMF allows an analysis multiparameter, extensive to a large number of cells and to multiple functional processes; it's easy to get a good integration of responses and mechanisms, as well as adequate resolution of cellular heterogeneity and a phenotypic selection of subpopulations. However, the equipment, its maintenance and Reagents are still expensive.

In CMF, the cells or particles subject to measurement move dragged by a flow or liquid stream laminar, being the current measuring ranges of 100-25,000 cells per second in peripheral blood or other body fluids. Each cell goes through a point where the jet receives the impact of a laser, which emits light from a adequate wavelength; the characteristics of each cell determine a specific pattern of dispersion and of intensity in emitted radiation. Said radiation is captured and purified by an optical system, which concentrates and transforms the signal turning it into voltage pulses. These are coded and Interpreted by a computer. The data obtained in this way they can be handled very versatile, being very reliable and accuracy. Flow cytometry far exceeds the capacity of other traditional methodologies and allows to guide the diagnosis, classification and prognosis of numerous diseases.

The cells can carry coupled fluorescent molecules ( labelling, tagging or molecular labeling), which, once excited by the laser, emit a fluorescent response in a longer wavelength, which facilitates the identification of specific subgroups within large cell populations . When the cells parade in a row through the measurement zone, where the laser light intersects the jet, each labeled cell produces a brief fluorescent flash, the intensity of which is directly proportional to the number of marked copies present in the cell. The markers ( fluorescent dyes ) are conjugated with the antigens present on the surface of the cell, acting as classification signals. Currently there are different protocols that allow to bind antibodies to fluorescent compounds. Among the fluorochromes used, fluorescein isothiocyanate (FITC), tetramethylrodamine isothiocyanate (TRITC), dichlortriazinylaminofluorescein (DTAF), Texas Red, bimane and others stand out. Certain biological properties in fluorescent markers are desirable, such as their reactivity with chemical groups, their non-covalent binding to structures, their interaction with ions or radicals, their susceptibility to enzymatic reactions and their permeability and retention across the cell membrane.

A unique aspect of flow cytometry is which measures the fluorescence individually associated with each cell or particle. Instead, spectrophotometric measurements are performed in bulk, valuing absorption or light transmission in percentages for the whole sample. In the CMF, for the On the contrary, each cell is interrogated individually by the laser. To achieve this, and this is one of the central aspects of the In the present invention, it is necessary to ensure a predictable trajectory and sequential cells, which are hydrodynamically focused to force its movement in a row inside the axis of a jet liquid.

Next, it is the task of some tubes photomultipliers (photo multiplier tubes, PMTs) and ones solid state detectors collecting photonic emissions from each cell and convert them into analog potential differences. The analog signals are then digitized by converters (analog to digital converters, ADCs) and stored for later analysis.

Flow cytometry (CMF) allows measurement of numerous parameters of interest, both at the molecular level, by fluorescence (extracellular proteins, DNA or RNA sequences free, circulating immune complexes), as at the subcellular level (individual virions, liposomes, chromosomes, organelles or nuclei isolated), at the cellular level (bacteria, unicellular fungi, cells human and animal, plant protoplasts) or supracellular level (hybridomas and cell fusions, spheroids, organisms multicellular). You can also analyze parameters intracellular (intracellular components, intracellular functions  or intracellular environment) and extracellular parameters (interaction with other cells, cell differentiation).

An important aspect of many CMF systems It is your ability to discriminate. In effect, flow cytometry has emerged as a powerful tool in the differentiation of cellular subpopulations. To do this, the cell carrier jet is subjected to oscillation by means of a quartz crystal piezoelectric. The disturbance thus induced causes the rupture of the jet in microdrops, whose generation frequency is so high that each drop has at most a single cell. If the cell in question satisfies the required criteria, the carrier drop receives a electric charge pulse Next, the drops through an electric field that produces the deflection of the flow and the  Subpopulation separation. Such separation processes are currently common in the field of experimental hematology. See, for example, the description in Flow Cytometry and Sorting, second edition, M.R. Melamed, T. Lindmo, M.L. Mendelsohn, Editors. Wiley-Liss, New York, 1991.

In addition to measuring signals from fluorescent flashes of cells, CMF systems allow indicate other aspects of transient cells, particularly the cell size and density. The incidental laser light is dispersed differently depending on the relative size of the cell and the presence of inner organelles. The size and the density are essential parameters in hematology to differentiate types of cells in the blood.

The usual components of a CMF system They are:

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Sample and chamber injection flow

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Fluorescence emission and optical bench

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Signal amplification of fluorescence

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Signal elimination no desired through a discriminator

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Signal quantification: Analog-to-digital converter

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Classification of the signals: Histogram of frequencies

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Data presentation: graphics of points and projections

Regarding the field of applications of the CMF, numerous fields can be cited. In general clinical practice, CMF systems allow diagnosis or prognosis based in cellular analysis, evaluation and monitoring of treatment, or analysis of cell injury and death. They are wide diagnostic applications of flow cytometry, both for characterization of normal cells and for identification and detection of abnormal cells. In therapeutics, it Applies for cell selection in cell therapy (analysis of parents in autologous transplantation, cross-testing in heterologous transplants, detection and quantification of leukocytes residuals in hemopreparations), analysis of the therapeutic action at cellular level (changes in structural or functional parameters), analysis of therapeutic action at patient level (detection of relapses and minimal residual disease, establishment of prognostic patterns of therapeutic success) or detection and analysis of resistance to therapy (analysis of uptake and retention of  drugs, drug metabolism analysis).

The applications of the Flow cytometry in the analysis of cell injury and death: sublethal cell injury detection (changes in parameters structural or functional), determination of cytotoxic effects global (quality control of cell preparations), characterization of cell death mechanisms (identification and analysis of apoptotic cells, identification of necrotic cells). In immuno-hematology it has CMF applied to antigen analysis (immunophenotype of surface, detection of intracellular antigens or antigens circulating), to the analysis of antibodies (circulating or linked to cells), to the analysis of cell function (proliferation, biochemistry, cell death). See for example in O'Connor, J.E., Guasch, R.M., Carretero, F. (1994) "Flow Cytometry: Technical Bases and Analytical Applications ", in" Diagnostic Applications of Cytofluorimetric Methods Using Monoclonal Antibodies ", European School of Transfusion Medicine, Milano, pp.  3-14; It can also be found at Sansonetty F., O'Connor, J.E. (1998) "Brief introduction to Cytometry of Fluxo-Uma das alternatives for "fazer" Cytology and Analytical Cytopathology. "Micron 1: 14-20.

A field in which the CMF begins to result relevant is in the identification and quantification of acids nucleic, both for diagnostic purposes (identification of gene sequences associated with pathogenic organisms or genetic alterations of the patient) as to the quantification of changes in gene expression patterns. See in Wedemeyer N, Potter T (2001) "Flow cytometry: an" old "tool for novel applications in medical genetics ". Clin Genet 60: 1-8.

Finally, numerous applications of CMF in the environmental field are cited, such as the analysis of unicellular populations in their environment (identification and taxonomy, biomass estimation), the detection of contamination, the detection of toxicity in situ (exposure markers, biochemical effect markers, use of natural or laboratory microorganisms as toxicity indicators, genomic analysis in cells from higher organisms).

As for current trends, the sector of the flow cytometers is experiencing an accelerated change, tending to reduce its size and energy consumption. Are reached benefits of up to 100,000 cells per second, with particularized identifications that affect up to one cell between 10 million It is also feasible to measure the light scattering in two or three angles, fluorescence in 12 or more regions Spectral

Fluidomechanical aspects of CMF

The objective of the fluid system in CMF is the transport a current to the laser beam ensuring its Optimal and sequential exposure to the light source. To do this it is it is necessary that only one cell or particle transits through the beam in  every moment This is achieved by injecting the sample into a stream of enveloping fluid inside the flow chamber. The sample flows as a coaxial core with the enveloping fluid, staying guided by a hydrodynamic principle known as focus.

The so-called Flow Focusing or FF technology (Gañán-Calvo 1998, Physical Review Letters 80, 285), applying a special geometry, uses the pneumatic path to generate liquid micro cubes that subsequently, after the exit orifice, are broken into drops of size Very small and substantially homogeneous. The latter technology is also capable of generating micro-jets of liquid by another liquid instead of gas, or it can generate micro-jets of gas within a liquid (the same liquid or a different one used as a forger, that is, with the same role played by the gas in the pneumatic process), with which microbubbles of homogeneous size are generated.

The present invention aims to apply the advantages of a simple and robust technology design FF to identification in CMF.

Brief Description of the Invention

The present invention refers to a procedure and associated device for identification and characterization, as well as the possible separation, of units discrete (eukaryotic cells, bacteria, viruses, particles carriers of biological or genetic information, microcrystals, inert particles, particles-probe with affinity for substances a analyze, mono- or multivesicular microcapsules). Those cited units are suspended in a liquid L that is made circular in laminar jet and stable by extrusion concentric through a hole, together with another fluid envelope F, F being preferably a gas. The compound jet resulting consists of a liquid core in which they travel in suspension of discrete units of interest, surrounded by the current consisting of the surrounding fluid F. A sensor in correspondence with the composite jet takes care of the identification and sequential characterization of the units discreet

The proposed procedure is based on the formation of a jet presenting a laminar and stable section, suitable for sequential circulation of discrete units object of high speed analysis; the conditions for jet formation are regulated by a range of flow rates dictated by fluidomechanical variables of the carrier liquid and the envelope fluid, preferably a gas.

In cytometry the procedures are known analytical based on the optical scanning of a liquid jet carrier of cells to be analyzed; however, the procedures Conventionals face problems of binding in the nozzles and canalicules through which the cell suspension and have little flexibility in terms of size of cells or particles to be analyzed, as well as in terms of to the flow regime. Alternatives based on the use of another auxiliary liquid, which surrounds during formation of the jet to the carrier liquid in order to increase the flow stability, avoid clogging and increase control variables However, the presence of this liquid auxiliary, which is mixed with the carrier, can cause interference or contamination, forces an expensive maintenance, and the volume of waste generated is large.

Essential aspects of the invention proposed in The following claims are: (1) the immiscible character of the two fluids used, which prevents interference or the particle migration between carrier liquid and fluid envelope (2) the possibility of adapting, without modifying the device, the diameter and flow rate in the jet at unit circulation and identification requirements discrete; (3) the possibility of using a gas, in particular air, as a surrounding fluid, with the consequent savings in components, disposal of much of the volume of waste and simplification in the procedure; (4) functional simplicity resulting, which allows multiplying on the same device the number of jets and sensor beams, thus multiplying substantially the productivity of the system, with a volume of limited residue

Description of the figures

Figure 1 shows a device adapted to the procedure described for identification and characterization sequential discrete units suspended in a jet capillary in laminar flow regime, by means of an optical sensor or electromagnetic, showing the following aspects of the invention:

(one)

liquid power supply carrier of discrete units L;

(2)

liquid ejection nozzle carrier L;

(3)

enclosure fluid pressure chamber F;

(4)

liquid outlet carrier L and envelope fluid F;

(5)

waterproof barrier that separates power supplies of both ambient fluids Exterior;

(6)

transmitter;

(7)

optical sensor or electromagnetic.

The liquid L forms a jet in which they travel the discrete units object of identification and characterization sequential, being hydrodynamically focused by a fluid envelope F.

Embodiment of the invention

The invention described herein contemplates a procedure and its corresponding device for sequential identification and characterization of discrete units in suspension in a capillary stream in laminar flow regime, by means of an optical or electromagnetic sensor. The invention includes two immiscible fluids: one, necessarily a liquid, has for mission transport the discrete units under study in a stream organized as a slender stream, with a diameter superior characteristic or of the order of discrete units. The other fluid is the focusing one (F): it can be a gas, but it doesn't It is mandatory. Both fluids come from both sources independent. Its movement is produced by impulse due to a overpressure of up to 200 atm with respect to the outside environment. Under said drive, both fluids go outside through one or more holes (4), with simultaneous and concentric flow. Every hole is practiced in a waterproof barrier (5), which separates to the two sources cited from the outside environment. Through hole emerges a stream of carrier liquid L surrounded concentrically by the enveloping fluid F. During the flow of outflow of both fluids, the enveloping or focusing fluid surrounds the jet of carrier liquid, molded under its simple action aerodynamics to said carrier liquid and ensuring its focus, is that is, the concentration of the flow of the carrier liquid in a reduced diameter jet. This requires adjusting the flow rate.  of the carrier jet at a value between one and three hundred times the square of the surface tension corresponding to the interphase of the liquid L and the enveloping fluid F, divided by the square root of the product of the density of the liquid L and the third power of the pressure increase Δp.

In order to meet the requirements of step frequency and sensor detection threshold is accurate also adjust the diameter and flow rate (linked to the frequency of passing discrete units) of the jet. Only then is the registration by the sensor of the passage of discrete units and the characterization in succession of each of them.

The envelope fluid F can be (preferably) a gas, although this requirement is not mandatory. However, choosing a gas reports great economic advantages and reduces the risk of contamination of the carrier jet by mixing or  Drag with a liquid envelope. It should be noted that the pattern FF flow totally prevents contact of the carrier jet with the exit hole (4).

As for the length of the carrier jet, This is an important parameter in order to ensure space sufficient for the measurement zone (interception by a laser beam or other measuring agent). It is possible to adjust the viscosity of the L liquid making it a product of the mixture of at least two miscible liquids of different viscosity and compatible with the discrete units in suspension, such that the length of the capillary jet is greater than 300 micrometers, preferably greater than 1 millimeter, measured from the hole (4) to the point where the hair jet disintegrates in drops.

The discrete units carried by the jet They can be diverse: cells, viruses, microcrystals, particles inert, probe-particles with affinity for substances to be analyzed or microcapsules mono- or multivesicular.

The procedure may include a stage of separation of discrete units by techniques usual sub-population disaggregation (supply of a Electric pulse to positively marked discrete units, followed by separation in an electric field).

A main aspect of the invention is the aerodynamic guidance that the focusing fluid exerts on the carrier liquid. Said focusing effect occurs without the need of any electric field; and the emission of a fine stream or filament of carrier liquid occurs for purely hydrodynamic reasons, as documented in numerous publications illustrating the flow-focusing principle.

The objective pursued is the definition of a fluidic system, applied to CMF, that ensures the following qualities:

\ circ

Low flow of carrier liquid and focusing fluid;

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Robustness of materials and driving;

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Easy to use;

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High diameter controllability and length of the carrier jet;

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Risk reduction of carrier fluid contamination;

\ circ

Avoidance of the exit hole jam (4) ( clogging ).

Claims (19)

1. Procedure for the identification and sequential characterization of discrete units in suspension in a capillary jet in laminar flow regime, by means of an optical or electromagnetic sensor (7), characterized in that it includes the following steps:

one)

drive by overpressure Δ of up to 200 atm through a hole (4), simultaneously and concentrically, of a liquid L carrying the units discrete, together with another F fluid immiscible with it, which it wraps it into a jet;

2)

jet flow setting to a value between one and three hundred times the square of the tension surface corresponding to the interphase of the liquid L and the envelope fluid F, divided by the square root of the product of the density of the liquid L and the third power of the increase of pressure Δp;

3)

adjustment of the diameter and flow rate of jet to the requirements of step frequency and threshold of sensor detection;

4)

registration by the sensor of the passage of discrete units and succession characterization of each of they.

2. Procedure for the identification and sequential characterization of discrete units in suspension in a capillary jet in laminar flow regime, by means of an optical or electromagnetic sensor, according to claim 1, characterized in that the envelope fluid F is a gas. 3. Procedure for the identification and sequential characterization of discrete units in suspension in a capillary jet in laminar flow regime, by means of an optical or electromagnetic sensor, according to claims 1 and 2, characterized in that the viscosity of the liquid L is adjusted by making it be the product of the mixture of at least two miscible liquids of different viscosity and compatible with discrete units in suspension, such that the length of the capillary stream is greater than 300 micrometers, preferably greater than 1 millimeter, measured from the hole up to the point where the hair jet disintegrates into drops. 4. Procedure for the identification and sequential characterization of discrete units in suspension in a capillary jet in laminar flow regime, by means of an optical or electromagnetic sensor, according to claims 1 to 3, characterized in that the discrete units are cells. 5. Procedure for the identification and sequential characterization of discrete units in suspension in a capillary jet in laminar flow regime, by means of an optical or electromagnetic sensor, according to claims 1 to 3, characterized in that the discrete units are viruses. 6. Procedure for the identification and sequential characterization of discrete units in suspension in a capillary jet in laminar flow regime, by means of an optical or electromagnetic sensor, according to claims 1 to 3, characterized in that the discrete units are microcrystals. 7. Procedure for the identification and sequential characterization of discrete units in suspension in a capillary jet in laminar flow regime, by means of an optical or electromagnetic sensor, according to claims 1 to 3, characterized in that the discrete units are inert particles.
tes. 8. Procedure for the identification and sequential characterization of discrete units in suspension in a capillary jet in laminar flow regime, by means of an optical or electromagnetic sensor, according to claims 1 to 3, characterized in that the discrete units are probe-particles with affinity for substances to analyze. 9. Procedure for the identification and sequential characterization of discrete units in suspension in a capillary jet in a laminar flow regime, by means of an optical or electromagnetic sensor, according to claims 1 to 3, characterized in that the discrete units are mono- or multivesicular microcapsules. 10. Procedure for the identification and sequential characterization of discrete units in suspension in a capillary jet in laminar flow regime, by means of an optical or electromagnetic sensor according to claims 1-9, characterized in that the method includes a step of separation of the discrete units . 11. Device for sequential identification and characterization of discrete units suspended in one or more capillary jets in laminar flow regime, by means of an optical or electromagnetic sensor, characterized in that it includes:

one)

a source (1) of the liquid L carrying the units discrete;

2)

a source of envelope fluid F;

3)

a waterproof barrier (5), which separates the two sources cited from the outside environment, and from which one or more jets of liquid emerge carrier L concentrically surrounded by the envelope fluid F a through one or more holes (4) correspondingly practiced in said barrier;

4)

a element that ensures an overpressure Δp of up to 200 atm of the sources with respect to the outside environment and allows all the emerging flows of the hole (4) are in the range described in claim 1;

5)

a sensor (7), located before the stable section of the jet (s) concentric, which records discrete units in transit sequential.

12. Device for sequential identification and characterization of discrete units in suspension in one or more capillary jets in a laminar flow regime, by means of an optical or electromagnetic sensor according to claim 11, characterized in that the envelope fluid F is a gas. 13. Device for the identification and sequential characterization of discrete units suspended in one or more capillary jets in laminar flow regime, by means of an optical or electromagnetic sensor, according to claims 11 and 12, characterized in that the discrete units are cells. 14. Device for sequential identification and characterization of discrete units suspended in one or more capillary jets in laminar flow regime, by means of an optical or electromagnetic sensor, according to claims 11 and 12, characterized in that the discrete units are viruses. 15. Device for the identification and sequential characterization of discrete units suspended in one or more capillary jets in laminar flow regime, by means of an optical or electromagnetic sensor, according to claims 11 and 12, characterized in that the discrete units are microcrystals. 16. Device for sequential identification and characterization of discrete units suspended in one or more capillary jets in laminar flow regime, by means of an optical or electromagnetic sensor, according to claims 11 and 12, characterized in that the discrete units are inert particles. 17. Device for the identification and sequential characterization of discrete units suspended in one or more capillary jets in laminar flow regime, by means of an optical or electromagnetic sensor, according to claims 11 and 12, characterized in that the discrete units are probe-particles with affinity for substances to analyze. 18. Device for sequential identification and characterization of discrete units in suspension in one or more capillary jets in laminar flow regime, by means of an optical or electromagnetic sensor, according to claims 11 and 12, characterized in that the discrete units are mono- or microcapsules multivesicular. 19. Device for sequential identification and characterization of discrete units suspended in one or more capillary jets in laminar flow regime, by means of an optical or electromagnetic sensor, according to claims 11 and 12, characterized in that it includes elements for effecting the separation of said discrete units of the hair jet.