FR2525915A1 - METHOD FOR SEPARATING PARTICLES IN A SAMPLE OF FLOWING FLUID - Google Patents

Abstract

THIS PROCESS FOR SEPARATING PARTICLES CONTAINED IN A SAMPLE OF FLUID 26 WHICH FLOWS IN A DIRECTION THROUGH A CIRCULATING TANK CONSISTS OF SENDING THE SAMPLE OF FLUID WITH A SHEATHING FLUID THROUGH THE TANK SUCH AS THE SURFACE MINIMUM CROSS-SECTION OF THE PARTICLES EITHER ORIENTED TRANSVERSALLY TO THE SAID DIRECTION AND THEIR MAXIMUM CROSS-SECTION AREA IS ORIENTED PARALLEL TO THE WIDTH OF THE TANK AND TO APPLY A FORCE (SUCH AS A HYDRODYNAMIC OR MAGRIC FORCE, OR MAGRIC ) TO THE SAMPLE OF FLUID TO SEPARATE THE PARTICLES 70, 72 BASED ON A CORRESPONDING PHYSICAL CHARACTERISTICS OF THE PARTICLES, SUCH AS THEIR SIZE.

Description

The present invention relates generally

  continuous flow analyzers and it relates

  more particularly to a process for separating parts

  cules in a sample of flowing fluid.

  Significant progress has been made in the area of automating the count of blood cells 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

  rely on electrical properties at the orifice and are counted So far, however, no equipment has been available to analyze and evaluate multiple cells, such as normal cells, target cells, sickle cell cells, etc which may be in a continuously flowing stream of a blood sample

  given Thus, when one wishes to obtain information re-

  Multiple globules of this type, normal practice

  Male laboratories to obtain them consists in preparing a microscope slide with the globules fixed in an image plane and in using a human operator or a pattern recognition machine to statistically count significant numbers of globules by observing one by one under the microscope. globules carried by the coverslip We will refer to this

  subject to US Patents 4,175,860 and No. 4,199,748.

  Various attempts have been made over the past

  years to allow for analysis

  optics of particles flowing in a direct current.

  For example, Kay and others have described in the "Journal of Histochemistry and Cytochemistry", volume 27, page 329 (1979) a device which includes a Coulter-type orifice for moving the globules in a single file, the globules being reproduced magnified on a tube "Vidicon" Similarly, Kachel and others 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 a-

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

  particles in a direct current without the need for dis-

  to put the particles in a current in a single file has not been published We will refer for example, on this subject, to the work "Flow Cytometry and Sorting" by Melaned and others, John Wiley & Sons, 1979, Chapter 1; we will also refer to the patent

USA 3,819,270.

  In accordance with the present invention there is provided a method for separating particles which flow in a fluid sample. The fluid sample moves in one direction through a flow cell, the cell having a width and a thickness which are all

  two perpendicular to the flow direction The pro-

  of the present invention is to pass the

  fluid sample through the flow cell of a

  so that the particles are aligned approximately

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

  their maximum cross-sectional area oriented ap-

  approximately parallel to the width of the tank Un

  sheath fluid also flows into the circulating tank

  culation in the same direction Finally, a force is applied

  as the fluid sample to separate the particles

  based on a physical characteristic of the particles.

  Other characteristics of the invention will appear

  on reading the description which follows and examining

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

  Fig 2 is a view of the tank or circulation chamber

  tion of 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 pro-

  electronic stopper used in the apparatus of Fig 1; Fig 5 A is a partial schematic side view

  of the flow chamber showing the constriction force-

  ment "A" applied in the direction of the thickness of the

  tank, Fig 5 B is a partial schematic view of des-

  above the flow chamber showing the force "B" applied

  quée in the direction of the width, Fig 5 C is a partial schematic sectional view of the circulation chamber

  tion showing the resulting forces which act on the e-

  fluid sample, and Fig 5 D is a partial schematic top view of the flow chamber showing the

  trajectory of a large particle separated from the main current

  cipal of particles according to the method of the present invention; Fig 6 is a partial schematic side view of the paths of separated particles according to the method

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 flow chamber or vessel provided with an inlet orifice

  12 for a sample of fluid, such as blood or u

  rine, and an outlet 14, between which extends

  a passage which crosses an image observation zone 18.

  Passage 16 has an inlet port provided with a conduit

  intended 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 container 26 designed to contain 1 '6-

  sample of the fluid to be analyzed.

  The cross-sectional area of passage 16 dimi-

  gradually naked from the entrance port 12 towards

  of the image observation zone 18 The thickness or depth

  passage also decreases from the inlet

  12 to image observation area 18 The width di-

  minue from inlet 12 then increases for-

  As far as the image observation zone 18 Thus, as shown in FIGS. 2 and 3, the passage 16 has a width and a depth of approximately 5000 Nom at the entry orifice 12, a width and a depth of about 500 Nom in the middle 28 and a depth of 100 gm with a width

  greater than 5000 gm at the level of examination area 18.

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

  deeper than the largest globule or cell, its lar-

  geur being many times greater than the width of

  larger particles, for example more than a hundred times larger

  laughing at 1-a width of the widest cell However,

  fact that the passage is formed in the above manner

  crite, the current entering it through the opening 12 is limited

  tee at stable flow path with minimum shear

  in the examination area Preferably, the surface of the sec-

  transverse tion at minimum shear is not much

  larger than the area of the mini cross section

  male particles so the particles are ali-

  hindered in the current with their cross-sectional area

  minimum side facing transverse to the direction

  flow and their maximum cross-sectional area

  male oriented parallel to the width {i.e. vi-

  sible in the plane of Fig 2) The expression 'shears-

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

  aligns itself with the flow direction, when

  that there is a flow gradient The flow characteristics in passage 16 can be controlled by adjusting the pressure of the fluid prevailing in the receptacles 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 the company US Scientific Instrument Corporation containing a lamp 2 UP 1.5 The output image of the microscope is focused on a camera 34 with transfer devices.

Z 525915

  load model N O TC 116 OBD manufactured by RCA company

  Corporation The output signal from camera 34 available

  charge transfer is converted into a series of fixed raster images and appropriate electronic processors are used to evaluate these images A processor that is

  can use for this purpose is the processor sold under the

  signage Image analysis system model C-1285 by Hama-

  matsu Systems, Inc. Waltham, Massachusetts, USA Preferred

  rence, the output signal from the camera 34 with charge transfer devices 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 raster image collector 40 which stores fixed raster images of the scene observed by the camera with charge transfer devices The image collector 40 The raster is preferably the image collector model 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 both coupled to a bus

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

  ference, an Intel 80/20 computer The multiple bus 44 is also coupled to a 48 K selective access memory (48 x 1024 bytes) sold by Electronic Solutions Inc. and to a 16 K selective access memory 50 ( 16 x 1024 bytes)

  with two accesses, model RM 117 from the company Data Cube Corpora-

  tion The output signal of the video regeneration memory

  42 is also transmitted to a conventional television screen.

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

  individual still images for examination by an operator

human rator.

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

  Electronic Solutions, Inc, and with an amo-

  Vible consisting of a diskette control unit 60, such as the 8/8 model from the company Advanced Micro Devices

  and two diskette memories 62 of the Shugart company.

  We can use a large variety of programs to process the images with the apparatus of Fig 4 according to

  the particular task that the user wishes to accomplish.

  As mentioned below, you can use the program-

  Hamamatsu 1285 System Preferably, however,

  In this case, programming is carried out as follows: Tasks are first divided into those which must address each pixel of a given image and those which address only a small subset of the total Being

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

  tasks, these are programmed in assembly language

  in the interface processor 46 (the Intel / 20 processor in FIG. 4) The results of these operations are then transferred to the host computer 56 via the random access memory 50 with two accesses.

  relates to the host computer, almost all of the program-

  tion is performed more conveniently in an advanced language, such as Pascal (BASIC or FORTRAN could, in principle, also be used) The types of task which are

  carried out in assembly language are in particular the trans-

  gray scale formations, convolutions and gray scale histogram calculations Types of tasks performed by the host include general control of other devices, identification and segmentation of objects with gray interest in the visual field, the calculation of parameters associated with objects

  thus found and the formatting of the output signals re-

  presenting the results Another way of looking at the division of tasks carried out in this way is to consider that the tasks which must be carried out at

  speeds greater than that of a human operator are ef-

  performed in assembly language Tasks that are complicated

  that can be executed at a speed lower than the maximum speed can be programmed in language

  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 jet These values can be established from knowledge

  Preliminary sessions or by means of well-known histogram techniques When a pixel belonging to an object has been located in the visual field, a program of

  edge tracing is called to trace the outline of the

  appears from the object associated with this pixel once the edge

  was found, many useful parameters, such as po-

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

  experienced observers These definitions are then used

  read as the basis of the chosen algorithms The accuracy of the process is determined by a comparison of the machine results with those of experienced observers who examine the same samples. The output signals representing the results can be programmed in any of the many formats. histograms, line plots and summary tables can be produced at the

  request to meet special needs.

  It is thus seen that, in the tank or circulating chamber-

  tion 10, the particle stream which is in the image observation area 18 is two-dimensional Consequently, several particles can be examined in a single field and different particles can be distinguished optically, which results in a certain number advantages

  important For example, two cells or globules that spread

  flow together can be recognized optically as such while a Coulter counter could recognize them as a single globule of double size In addition, one can

  accentuate the fixed raster images using the techniques

  digital enhancement nics that have been developed

  for images transmitted by satellites and one can analyze

  ser the individual images to obtain data on the individual globules or cells In the case of a blood sample, one can obtain information, such as the size, the cross-sectional area, the shape (circular globule, target globule, sickle cell disease, etc.) optical density, hemoglobin content, based

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

  optically sort and sort the globules or cells

  in this way but, moreover, when the cells are analyzed and sorted in this way, it is possible to N individually count the different types of cells to give, automatically and in a single pass, the

  number of normal red blood cells per unit volume of

  sample, the number of target red blood cells per cent-

  cubic meter of sample, the number of red blood cells falci-

  shapes per cubic centimeter of sample, the number of glo-

  white bules, number of platelets, etc. per centimeter

sample cube.

  Once a series of raster images have been prepared in digital form, one can obtain a very

  great diversity of very elaborate information on the parties

  cules contained in the series of images according to the complexity of the hardware and the software of computer which can be

  used for image analysis.

  Preferably, the information obtained from

  still raster images are combined to provide information

  composite image which reflects the content of multiple fixed raster images and / or predetermined reference images and the resulting composite information can be used

  various ways so in simple systems the information

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

  composite measurements made on a blood sample.

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

  the temperature in a crystallizer or the flow of

  ture of nutrients to a microbial culture in case the system controls the size or number

particles.

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

  move in a current, both bio-particles

  such as blood cells or cells

  lules, bacteria, cylinders and crystals in the urine that

  particles in gas analyzers, etc. and so-

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

  stream containing microorganisms as mentioned above

  above, controlling the growth of polymers and crystals, etc. Fig 5 A is a partial schematic side view

  of the circulation chamber or vessel 10 The circulation chamber

  lation 10 has a thickness which decreases from the orifice

  entry 12 to image observation area 18, as a pre-

  previously indicated This arrangement creates a constriction in the fluids, both the sheath fluid and the sample fluid, as they flow from the inlet 12 to the observation area 18 image This

  throttling force has been represented by the arrows "A".

  On Fig 5 B to which reference is made, there is shown a partial schematic view, from above, of the circulation chamber 10 Fig 5 B shows that the width decreases to

  from inlet 12 and then increases for-

  up to image viewing area 18 This creates an expansion force which acts on the sheath fluid and

  on the sample and is represented by the arrows "B".

  In Fig. 5C, which is a sectional view of the flow chamber 10, looking from the image viewing area 18 towards the area of the inlet port 12, the throttling forces in the thickness direction and in the expansion direction in the width direction have been represented by the arrows C The forces "C" are the forces resulting from the forces "A" and "B" The forces "C" trap and bring into play forms the fluid sample 26 in the form of a stream having a cross-sectional area which has an approximately rectangular shape with a thickness "D" and a width "E" It should be emphasized that the thickness "D" is extremely small and is around

  from 0.1 dom to a few tens of micrometers.

  According to the process of the present invention, it is possible

  modify the dimensions of the sample fluid 26, i.e.

  say its cross-sectional area, by adjusting the flow rate of the fluid sample 26 or the flow rate of the sheath fluid or the ratio of the flow rate of the sample to the flow rate of the sheath fluid More particularly, the flow rate can be adjusted of the sheath fluid, the flow rate of the fluid sample or both so as to make the thickness "D" of the fluid sample 26 smaller than the largest particles such as the particle 70 shown In the case o the particle has a size greater than the thickness "D" of the fluid sample 26, the hydrodynamic forces

  "C" generated by the particular shape of the flow cell

  lation 10 and by the flow of the sheathing fluid act on the particle 70 and push it away

  of the main stream of particles, towards the peri-

  circulation chamber 10 On the other hand, in the case of smaller particles, such as

  particle 72, that is to say the particles whose dimensions

  sions are less than the thickness "D" of the sample

  fluid 26, the hydrodynamic forces "C" have no ef-

  fet on these particles 72 The trajectory of the particles of

  smaller size 72, on which the hydrodyna-

  mics "C" have no effect has been represented by the arrow 76 while the trajectory of the particles 70 of larger size has been represented by the arrow 74 in Fig 5 D The particles of larger size being thus sent on a different trajectory, we can use the microscope by focusing it on the particles

  of larger size either on the trajectory which

  holds smaller particles We must insist

  that the definition of the "size" of a party

  cule in the context that size determines the influence of hydrodynamic forces on the particle does not necessarily mean the largest dimension In fact, as a result of the geometry of the flow cell 10 as described above, the flowing particles

  in the circulation tank 10 are oriented with their

  face of minimum cross section transverse to the di-

  flow direction and their cross-sectional area

  maximum dirty parallel to the width of the tank By cons-

  quent, the larger particles 70 on which the hydrodynamic forces "C" act are those which have

  a dimension in the plane of their cross-sectional area

  minimal versal, which is transverse to the plane of their sur-

  face with maximum cross-section and greater than the thickness "D" of the fluid sample 26 The particles

  72 of the smallest particles are the particles

  mension in the plane of their cross-sectional area

  which is transverse to the plane of their trans-

  maximum versal and is smaller than the thickness "D".

  In Fig 6 to which we will refer, we have shown

  tee use of the circulation tank 10 to separate parts

  ticles based on their density In this embodiment

  tion, the circulation tank 10 has a width which is many times its thickness and is arranged to

  so that its width is parallel to the force of gra-

  speed, indicated by arrow "G" As the particle stream flows from the inlet 12 to the image viewing area, the main stream moves

  following the trajectory indicated by the arrow "H" The par-

  denser ticles, however, are separated from the mainstream

  cipal due to the action of the gravitational force G and they sink on a lower trajectory indicated by the arrow "I", according to which they move Due to the force of gravity which acts on the particles, denser particles are more influenced than less dense particles and it follows that the denser particles move along a path which is

  lower than the path traveled by the main current

  particle chip In this way, the

  separation of particles based on their density.

  Finally, it must be emphasized that other ca-

  physical characteristics of the particles can be used as a basis for separation based on the application of a force to the sample fluid stream For example, some particles can be electrically charged

  before being introduced into the circulation chamber 10.

  We can then establish an electric field parallel to the

  width or thickness of the chamber Due to the application

  cation of the electric field, the charged particles are deflected out of the main particle current In this way, the particles can be separated on

  the basis of their electrical charge Although this is ana-

  logue to electrophoresis, the movement of particles in the current is much faster In addition, the use of a

  sheath-forming fluid in the process of the present invention

  tion is necessary to stabilize the flow of the sample

  fluid and to maintain flow in the inter-

  laminar flow valley, that is to say without any tur-

  bulence Likewise, part separation can be performed

  cules based on their magnetic permeability in use

sant a magnetic force.

Claims (8)

  1 A process for separating particles contained in   a fluid sample (26) which moves in a direction   tion through a circulation tank (10), this tank having a width and a thickness which are both perpendicular to said direction, this process being characterized in that it consists in passing the fluid sample through the flow cell in such a way that the   particles are approximately aligned with their super-   face of minimum cross section oriented transverse-   lying to said direction and their cross-sectional area   dirty maximum oriented approximately parallel to the   said width; to cause a sheath-forming fluid to flow through   towards the circulation tank (10) in said direction; and applying a force to the fluid sample to separate the particles according to a physical characteristic particles.   2 Method according to claim 1, characterized in that the force is a hydrodynamic force and the characteristic   physics is the particle size.   3 Method according to claim 2, characterized in that the step of applying a force consists in regulating the flow of the fluid sample.   4 Method according to claim 2, characterized in that the step of applying a force consists in regulating the flow sheath fluid.   Method according to claim 2, characterized in that   the step of applying a force consists in adjusting the ratio   port of flow of fluid sample to flow of fluid forming a sheath.   6 Method according to claim 1, characterized in that   the above width is many times the thickness aforementioned sister.   7 Method according to claim 6, characterized in that   force is the force of gravity and the physical characteristic density is density.   8 Method according to claim 7, characterized in that the step of applying a force consists in orienting the circulation tank (10) with the width approximately parallel to the force of gravity.   9 Method according to claim 1, characterized in that the force is electric and the physical characteristic is the electric charge. method according to claim 1, characterized in that the force is magnetic and the physical characteristic is magnetic permeability.