FR2526165A1 - METHOD FOR ALIGNING, OBSERVING AND SEPARATING PARTICLES CONTAINED IN A FLOWING FLUID SAMPLE - Google Patents

Abstract

THE INVENTION RELATES TO A PROCESS FOR ALIGNING PARTICLES CONTAINED IN A SAMPLE OF FLUID FLOWING INTO A CIRCULATING TANK, TO OBSERVE THEM AND OPTICALLY SEPARATE PARTICLES A, B, C OF VARIOUS SIZES WHICH CONSIST TO CIRCULATE THROUGH IT. TANK A FLUID FORMING A SHEATH AROUND THE SAMPLE, TO ADJUST THE FLUID FLUID FORMING A SHEATH OR OF THE SAMPLE SO AS TO ALIGN THE PARTICLES WITH THEIR MINIMUM CROSS-SECTION SURFACE ORIENTED CROSS-TRANSVERSALLY TO THE DIRECTION OF THE FLUID CURRENT MAXIMUM CROSS-SECTION SURFACE ORIENTED PARALLEL TO THE WIDTH OF THE TANK, THE BARYCENTERS A, B, C OF THE PARTICLES ALL BEING LOCATED IN THE SAME PLANE 120. A MICROSCOPE CAN BE FOCUSED ON THIS PLANE TO OBSERVE THE PARTICLES OR, ON THE contrary, THE MICROSCOPE FOCUSED ON ANOTHER PLANE 122 TO OPTICALLY SEPARATE PARTICLES OF ONE SIZE FROM PARTICLES OF ANOTHER SIZE.

Description

The present invention relates to flow optical analyzers

  continuous and relates more particularly to a method for aligning particles in a sample

  circulating fluid in such an analyzer.

  Significant progress has been made in automating blood cell counts in a serum sample The best known instrument for performing blood counts is the Coulter counter in which blood cells pass through an orifice in one

  unique file and are detected by the way they modify

  depend on the electrical properties at the orifice and are counted

  that, however, no equipment has been available to analyze and evaluate multiple blood cells, such as

  normal blood cells, target blood cells, falcipidic red blood

  forms, etc. that can be in a continuous flow stream of a given blood sample Thus,

  when one wishes to obtain information relating to the

  blood cells of this type, the normal practice of

  to obtain them consists in preparing a microscope slide with the globules fixed in an image plane and

  employ a human operator or a recognition machine

  of forms for statistically counting numbers

  of globules by observing one by one under the microscope the globules carried by the coverslip Referred to this subject

  US Pat. Nos. 4,175,860 and 4,199,748.

  Various attempts have been made during the last

  years to enable an analysis to be carried out

  optical particles that flow in a continuous current.

  For example, Kay et al. Have described in the Journal of Histochemistry and Cytochemistry, Vol 27, p 329 (1979) a device which has a Coulter orifice for moving globules in a single file, the globules being reproduced enlarged on a tube "Vidicon" Similarly, Kachel et al have described in the "Journal of Histochemistry and

  Cytochemistry ", volume 27, page 335, a device for

  place the globules in a single file through a microscope area in which they are photographed Although

  these researchers have advanced the automation of the

  analysis of particles in a single file, no successful work that would allow the automatic analysis of

  particles in a direct current without the need for

  For example, the publication of "Flow Cytometry and Sorting" by Melaned et al., John Wiley & Sons, 1979, Chapter 1; we will also refer to the patent

US 3,819,270.

  In accordance with the present invention, there is provided a

  process for aligning the particles contained in a sample

  Fluid that moves in one direction through a tank or flow chamber The tank has a width and

  which both are perpendicular to the direc-

  Each of the particles contained in

  the sample has a barycenter The process of the present invention

  This intervention includes the step of passing the

  fluid sample through the flow cell A fluid forming a sheath or envelope flows through the flow cell in the direction of flow and is located on one side of the fluid sample.

  sheath fluid is adjusted so that

  are approximately aligned with their minimum cross-sectional area oriented transversely to the direction of flow and with their cross-sectional area

  cross-section oriented approximately parallel-

  to the width and with the barycentres of the particles

  all killed approximately in one and the same plane.

  When the particle centers of gravity are located

  proximately in one and the same plane, the particles can be observed by means of a microscope developed on this plane. In addition, the particles can be optically separated.

  by focusing the microscope on a plane other than the

  said plan so that particles of a particular size

  are no longer in the focal plane while

  cells of another size are approximately in the focal plane. Other features of the invention will become apparent

  upon reading the following description and review

  annexed drawings in which: Fig. 1 is a perspective view of an apparatus for examining a direct current according to the present invention;

  Fig 2 is a view of the tank or chamber circulating

  the apparatus of Fig. 1; Fig 3 is a sectional view of the apparatus of Fig 2, taken along the plane of line 3, 3 of Fig 2;

  FIG. 4 is a schematic representation of the

  electronic stopper used in the apparatus of Fig 1; Fig. 5 is a greatly exaggerated side view of the method used in the prior art for mounting

  particles on a microscope slide for the purpose of

  undermine; and Fig. 6 is a highly exaggerated side view of the

  particles in the apparatus described here in accordance with

  in accordance with the process of the present invention.

  Reference will now be made in more detail to the drawings and, in particular, to FIG. 1, in which there is shown an apparatus which comprises a body 10 which contains a chamber or flow cell equipped with an inlet orifice.

  12 for a fluid sample, such as blood or urine.

  and an outlet port 14, between which

  a passage which passes through an image observation zone 18.

  The passage 16 comprises an inlet orifice provided with a conduit

  20 to be connected to a volume of saline solution 22.

  As shown in FIGS. 2 and 3, the inlet orifice 12 of the fluid sample is formed by a needle 24 which opens into the passage 16 downstream of the conduit 20, the needle

  24 being connected to a receptacle 26 intended to contain the

  fluid sample that needs to be analyzed.

  The cross-sectional area of passage 16 decreases

  naked gradually from the inlet port 12 in the direction

  of the image observation zone 18 The thickness or depth

  the passage also decreases from the inlet

  12 to the image observation zone 18 The width

  from the inlet port 12 and then increases

  Then, as shown in FIGS. 2 and 3, the passage 16 has a width and a depth of about 5000 gm at the inlet orifice 12, a width and a depth of about 500 gir in the middle 28 and a depth of 100 gm with a width

  greater than 5000 Dom at the examination area 18.

  It will be understood that the continuous current flowing through the examination zone 18 can be many times

  deeper than the largest globule or cell, its

  being many times greater than the width of the

  larger particles, for example more than a hundred times greater than

  the width of the larger cell.

  the passage is formed in the manner described above.

  the current that enters through the opening 12 is limited

  to a stable minimum shear flow path

  in the examination area Preferably, the surface of the

  minimum shear cross-section is not much

  larger than the surface of the minimum cross-section

  particles so that the particles are

  in the current with their cross-sectional area

  minimal versale oriented transversally to the direction

  flow and their maximum cross-sectional area

  male oriented parallel to the width (that is to say,

  sible in the plan of Fig 2) The expression "shears-

  minimal "is used here in the sense of" minimum velocity gradient "so that a particle moving in the current tends to align with the direction of the current exactly like a float on a river

  aligns itself with the direction of flow, when

  that there is a flow gradient The flow characteristics can be controlled in the passage 16 by regulating the pressure of the fluid which prevails in the containers 22 and 26

  either automatically or by adjusting their static height.

  A microscope 30 is developed on the examination zone 18 and the examination zone 18 is illuminated from below by a stroboscope 32 which is preferably a Model 3018 stroboscope from US Scientific Instrument Corporation containing a lamp 2. UP 1.5 The output image of the microscope is focused on a camera 34 with charge transfer devices model NO TC 116 OBD manufactured by the company RCA

  Corporation The output signal from camera 34 at

  load transfer is converted into a series of fixed raster images and appropriate electronic processors are used to evaluate these images.

  can use for this purpose is the processor sold under the

  Signage Model C-1285 image analysis system by Hama-

  matsu Systems, Inc. Waltham, Massachusetts, USA Preferably

  Preferably, the output signal of the charge transfer device camera 34 is applied to an electronic processor 36 which has been shown in more detail in FIG. 4 and which comprises a black and white control television screen 38 and a halftone image collector 40 which stores fixed halftone images of the scene observed by the charge transfer device camera The image collector 40 is preferably the model image collector FG 08, manufactured by the company Matrox Corporation, Montreal, Canada, whose output signal is applied to a video regeneration memory 42 model RGB 256 manufactured by Matrox Corporation which are all two coupled to a bus

  multiple 44 of the processing unit 46 which is, pre-

  The multiple bus 44 is also coupled to a 48K (48x1024-byte) random access memory sold by Electronic Solutions Inc. and a 50K 16K random access memory. 16 x 1024 bytes)

  two-port model RM 117 from Data Cube Corpora-

  The output signal of the video regeneration memory

  42 is also transmitted to a television screen of

  color control 52 which can be used to provide video images accentuated by digital processing of

  individual still images for examination by an operator.

human resource.

  The second output of the two-port selective access memory 50 is connected to a multiple bus 54 which is connected to a processing unit 56 of the Applied Micro devices company, to a 48K selective access memory 58 of

  Electronic Solutions, Inc., and an amoral memory

  A floppy disk control unit 60 such as the 8/8 model of the Advanced Micro Devices Company.

  and two floppy memories 62 from Shugart.

  A large variety of programs can be used to process the images with the apparatus of FIG.

  the particular task that the user wishes to accomplish.

  As mentioned below, the program can be used

  the Hamamatsu System 1285 Preferably, however,

  In this case, the programming is done in the following way: The tasks are divided between those which must address each pixel of a given image and those which address only a small subset of the total.

  given that a lot of time is spent on the first category.

  of tasks, these are programmed in the language of

  In this case, the results of these operations are transferred to the host computer 56 via the two-port random access memory 50.

  the host computer, almost all of the program-

  This is done more conveniently in an advanced language, such as Pascal <BASIC or FORTRAN could be, in principle, also used.) The types of tasks that are

  in assembly language are particularly the trans-

  gray-scale formations, convolutions, and grayscale histogram calculations The types of tasks performed by the host include general control of other devices, identification and segmentation of objects with different characteristics. interest that are in the visual field, the calculation of parameters associated with objects

  thus found, and the shaping of the output signals

  presenting the results Another way of considering the division of tasks in this way is to consider that the tasks to be

  velocities greater than that of a human operator are ef-

  in the assembly language Tasks that are compli-

  or that can be executed at a speed below the maximum speed can be programmed in

  evolved We find objects in a main visual field-

  by setting a gray scale window function

  for values that we know to be characteristic of the ob-

  desired value can be established from knowledge

  Prerequisites or using well-known histogram-making techniques When a pixel belonging to an object has been spotted in the field of view, a program of

  tracing is called to draw the outline of the

  seems of the object associated with this pixel Once the edge

  has been found, many useful parameters, such as the

  sition, surface, integrated optical density, can be easily calculated The probability of belonging to previously defined subgroups can be determined from these parameters calculated by means of classical decision theories Classification definitions of cell morphologies or cells are established by

  experienced observers These definitions are then used

  The precision of the process is determined by a comparison of the results of the machine with those of experienced observers who examine the same samples. The output signals representing the results can be programmed in any of the various formats. histograms, linear plots and summary tables can be produced at the

  request to meet specific needs.

  It can thus be seen that in the tank or chamber to circulate

  10, the particle current in the image observation zone 18 is two-dimensional. Therefore, more than one particle can be examined in a single field and different particles can be distinguished optically, resulting in a number of particles. benefits

  For example, two cells or globules that

  Together they can be optically recognized as such while a Coulter counter could recognize them as a single globule of double size.

  accentuate fixed halftone images using

  digital accentuation programs that have been developed

  satellite images and it is possible to analyze

  use the individual images to obtain data on individual cells or cells In the case of a blood sample, information can be obtained, such as size, cross-sectional area, shape (circular globule, target cell, falciform red blood cells, etc.) optical density, hemoglobin content, based on

  of a globule, etc. Not only is it possible to analyze

  serrate and optically sort the cells or cells

  viduals in this way but, furthermore, when the globules are analyzed and treated in this way, it is possible to count individually the different types of globules to give, automatically and in a single passage, the

  number of normal red blood cells per unit volume of

  sample, the number of red blood cells

  cubic meter of sample, the number of falcipid red

  per cubic centimeter of sample, the number of

  white bules, the number of platelets, etc. per centimeter

sample cube.

  Once a series of fixed raster images have been prepared in digital form, a very

  diversity of very elaborate information on

  contained in the series of images depending on the complexity of the hardware and computer software that may be

  used for image analysis.

  Preferably, the information obtained from the

  stationary images are combined to provide information

  composite image that reflects the content of multiple fixed raster images and / or predetermined reference images and the composite information thus obtained can be used to

  In this way, in simple systems,

  training can be printed out, for example, to bring to the attention of a hematologist the results

  composite measurements performed on a blood sample.

  In more complex systems, composite measurements can be used by a process control, for example, to control the pressure in a homogenizer,

  the temperature in a crystallizer or the supply flow

  nutrients to a microbial culture if the system controls the size or number

particles.

  Thus, it can be noted that the apparatus can be used for the analysis of a large variety of particles which

  move in a current, both biological particles and

  such as blood cells in the blood or blood

  lules, bacteria, cylinders and crystals in the urine that

  particles in gas analyzers, etc., and

  output signals representing the results of these measurements can be used for process control, eg the distribution of nutrients in a

  current containing microorganisms as mentioned above.

  above, controlling the growth of polymers and crystals, etc. In Fig. 5, there is shown the method used in the prior art for examining particles of a fluid sample. Typically, particles, such as those designated as, b, and c are mounted on a coverslip

  microscope Each of the particles a, b and c has a bary-

  center, these centers of gravity being denoted by the references A, B and C, respectively, can be seen by examining FIG. that when the particles a, b and c are all mounted on a lamella 100, the centroids A, B and C are not in a common plane Therefore, to observe the

  particles a, b and cbil must first put the micros-

  cope in focus on the particle has in a foreground, pa-

  goes to the lamella 100, passing through its center of gravity A For

  observe the particle b, the microscope must be adjusted

  to be developed in a plane parallel to the lamella and passing through the barycenter B Similarly, the microscope must be adjusted again to allow observation of the

particle c.

  In contrast, in the process of the present invention, the particles are aligned as they move through the flow cell. The particles a, b and c contained in the fluid sample move in a directional direction.

  direction indicated by arrow 110 through the flow cell.

  A sheath fluid is passed through the flow cell 10 also in the direction 110. The fluid sheath envelopes the fluid sample.

  sample fluid, or the flow rates of the two fluids, from

  that the flow of both fluids is effected

  of the laminar flow range, that is, turbulence Sys at least with very little turbulence. In addition, the flow rate of one or the other fluid is controlled to cause the alignment of the particles a, b and c with their minimum transverse serving surface oriented transversely to the direction 110 and their maximum cross-sectional area oriented parallel to the

  width of the flow cell 10, the barycentres

  A, B and C particles a, b and c being all located in one and the same plane or common plane 120, in the zone

  When the particles a, b and c are thus

  In the examination zone 18, the microscope can be adjusted so that it is focused on this plane. All the particles a, b and c are then in the focal plane when viewed through the microscope. 30 According to a variant

  the flow rate of the sheath fluid can be varied, which de-

  place the particles in such a way as to move the focusing plane until the particles are in the

plan of development.

  Furthermore, when the respective centroids A, B and C are particles a, b and c, they are approximately in

  one and the same plane 120, it is also possible to realize a

  yielded to optically separate the particles

  6, the microscope 30 can be adjusted to

  to develop it on a plan 122 that is different

  Plan 122 Plan 122 does not pass at all through the

  However, because the particles b and c are significantly larger than the

? 6165

1 5 61

  particle a, plane 122 on which the microscope is put

  the point is such that particles b and c are approximately

  tivegbent in the development plan As the

  particle a is not in the development plan, its

  calf of gray scale may not be sufficient to

  turn on the processor's edge tracing routine

  Therefore, a shift in the focal plane

  killed so that the smaller particles are out of the focus plane results in a separation

  In addition, it is thought that these more

  Particles, such as bacteria, are less influ-

  In other words, they are less influenced by the shape of the flow cell 10 to align their surface with

  minimum cross-section with the direction of flow.

  These particles, such as bacteria, are thought to be influenced by Brownian motion.

  the fluid sample that are influenced by the hydrody-

  of the flow cell 10 being arranged approximately

  in a given plane 120, the plan can be modified

  of the microscope 30 in order to put the

  located in the plane 120 out of the plane of focus, while the microscope is focused on the bacteria that

  are in a different plane In this way, we ob-

  also holds an optical separation of the particles.

  The method of the present invention is capable of many applications including the analysis of blood or urine as fluid samples. When the blood is the sample fluid, the red blood cells are

  smaller than white blood cells Thus, by means of the

  described above, it is possible to optically separate the

  red blood cells by placing the red blood cells out of the focus plane while the white blood cells remain approximately in the focus plane When the fluid sample is urine one

  can separate cell particles (such as

  blood vessels and epithelial cells) cylinders

  the latter being much larger than the first.

  An alternative of the methods of the present invention is to flow a second sheath fluid through the flow cell. The flows of the two sheath fluids and the sample fluid are adjusted in a controlled manner.

  that the centroids of the particles are located practically

  in one and the same plane Each of the fluids

  The sheath flows in a plane which is approximately parallel to the width of the flow cell 10, this width being equal to many times the thickness of the vessel. The methods of the present invention have many advantages. First of all, one of the main advantages lies in the fact that, when the particles are

  found in the development plan gets the

  Maximum edge intensity The maximum edge intensity of each

  ticle is in the image plane In addition, the fact that

  the centroids of the particles are in a common plane

* mun, there is no need to adjust the microscope to observe

  Finally, because the barium

  centers of particles are located approximately in a common plane, one can also realize the separation of particles One can voluntarily produce a displacement of the focal plane to cause the diffusion of the intensity of the edges of the smaller particles while the larger particles remain in the development plan In addition,

  the displacement of the focal plane can even be such that it pro-

  the diffusion of all the particles that are in the common plane 120, the microscope being developed on smaller particles which are not influenced by the hydrodynamics of the flow cell 10 and which

find in a different plane.

Claims (11)

  A method for aligning particles contained in a fluid sample that moves in a direction through a flow cell (10), said vessel having a width and a thickness both perpendicular to said direction, each of the particles ( a, b, c) having a barycenter (A, B, C), said method being characterized by: passing the fluid sample through the flow cell; to dispense a fluid   forming a sheath in the flow cell in said direc-   tion; and adjusting the flow rate of the sheath fluid such that the particles are approximately aligned with their minimum cross-sectional area oriented transversely to said direction and with their   maximum cross section oriented approximately   parallel to said width, the centroids of the particles being located approximately in one and the same plane or common plane (120).   A method for observing particles contained in a fluid sample that moves in a direction through a flow cell (10), said vessel having a   width and a thickness both perpendicular to the-   said direction in an image observation zone (18), a microscope (30) aligned to observe the particles   located in the image observation area, each of the   particles (a, b, c) having a barycenter (A, B, C), this process   being characterized in that it consists of: passing the   fluid sample through the flow cell; discharging a sheath fluid into the flow cell in said direction; adjusting the flow rate of the sheath fluid so that the particles are approximately aligned with their minimum cross-sectional area oriented transversely to said direction and with their   maximum cross-sectional area oriented approximately   parallel to said width, the centroids of the particles being located approximately in one and the same plane or common plane (120); and to bring about the   point of the microscope on said particles.   A method for optically separating particles of different sizes contained in a fluid sample that moves in a direction through a flow cell (10), said vessel having a width and a thickness both perpendicular to said direction   in an image observation area <18), a microscope (30) -   aligned to observe the particles in the image viewing area, each of the particles (a, b, c) having a centroid (A, B, C), which process is characterized by comprising pass the fluid sample through the tank; to dispense a fluid   forming a sheath in the flow cell in said direc-   tion; adjusting the flow rate of the sheath fluid so that the particles are approximately aligned with their   minimum transverse cross-sectional area   to the said direction and with their sectional area   cross-section oriented approximately parallel-   at said width, the centroids of the particles being located approximately in one and the same plane or common plane (120); and causing the microscope to focus on a plane (122) other than the common plane (1201 so that particles <such as particle a) of a size are   outside the focus plan while the parties   cules (such as particles b and c) of another size   are located approximately in the plane of focus.   4 A method for aligning particles contained in a.   fluid sample that moves in one direction through a flow cell 110), this vessel having a   width and a thickness both perpendicular to the.   said direction, each of the particles (a, b, c) having a barycenter (A, B, C), said method being characterized in that it comprises: passing the fluid sample through the flow cell; to dispense a fluid   forming a sheath in the flow cell in said direc-   tion; and adjust the flow rate of the fluid sample to   that the particles are approximately aligned D 61 5   with their minimum cross-sectional area oriented transversely to said direction and with their   maximum cross section oriented approximately   parallel to said width, the centroids of the particles being located approximately in one and the same plane or common plane (120).   S A method for observing particles contained in a sample of fluid that moves in a direction through a flow cell (10), which tank has a   width and a thickness both perpendicular to the-   said direction in an image observation zone (18), a microscope (30) aligned to observe the particles   located in the image observation area, each of the   particles (a, b, c) having a barycenter (A, B, C), this process   being characterized in that it consists of: passing the   fluid sample through the flow cell; at   flowing a sheath fluid into the flow cell   lation in said direction; to adjust the flow of the sample   fluid so that the particles are approximately   in line with their cross-sectional area   minimal dirty oriented transversely to said direction   and with their maximum cross-sectional area ori-   approximately parallel to said width, the centroids of the particles being located approximately   in one and the same plane or common plane (120).   6 A method for optically separating particles from   different sizes in a fluid sample that is   positioned in a direction through a flow cell (10), said vessel having a width and a thickness both perpendicular to said direction in an image observation area (18), a microscope (30) aligned to observe the particles in the image viewing area, each of the particles (a, b, c) having a barycenter (A, B, C), which method is characterized by passing the sample fluid through the flow cell; to flow a sheath fluid into   the flow cell in said direction; to settle the   bit of the fluid sample so that the particles   approximately aligned with their surface area.   cross-sectional minimum transverse to the   said direction and with their maximum cross-sectional area oriented approximately parallel to said   width, the centroids of the particles being located approximately   in a single plane or common plane (120), and causing the microscope to focus on a plane (22) other than the common plane (120) so that the particles (such as particle a) d one size are located off the plane of focus while particles (such as particles b and c) of another size are located   approximately in the focus plane.   Method according to one of claims 1 to 6, characterized   in that the common plane (120) is approximately parallel to said width.   Process according to claim 7, characterized in that said width is equal to many times said thickness. 9 Process according to one of claims 1 to 6, characterized in that it comprises the step of draining   a second fluid forming a sheath through the circulating vessel tion (10).   Method according to one of claims 2, 3, 5 and 6,   characterized in that the step of focusing the microscope   (30) is to adjust said microscope.   Method according to one of Claims 2, 3, 5 and 6,   characterized in that the step of focusing the microscope   (30) is to change the flow rate of the sheath fluid.   Method according to one of claims 1 to 6, characterized   in that the fluid sample is blood.   The method of claim 12 as dependent   of one of claims 3 and 6, characterized in that   particles of the first size are red blood cells and the   particles of the other size are white blood cells.   Process according to one of claims 1 to 6, characterized   in that the fluid sample is urine.   Method according to claim 14, as dependent   of one of claims 3 and 6, characterized in that   particles of the first size are particles   and the particles of the other size are cy- urinary lindres.