JPS58196440A - Method of arranging grain in fluidized fluid sample - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Considerable progress has been made in automating the task of counting blood cells in serum samples. The most well-known device for counting blood cells is the so-called Coulter counter, in which blood cells are passed through an orifice in a row and detected and counted by changing their electrical properties at the orifice. However, to date, there is no device that automatically analyzes and evaluates the various types of blood cells that may be found in a given blood sample flow, such as normal blood cells, target blood cells, mechanical blood cells, etc. Therefore, if this kind of information about a large number of blood cells is desired, the standard commercial method for obtaining it is to prepare a microscope slide with the blood cells fixed in the image plane, and then observe each blood cell on the slide individually under a microscope. while the operator or pattern recognition device counts a statistically significant number of blood cells.

U.S. Patent Nos. 4,175,860 and 4,199,7
See No. 48.

In recent years, several attempts14- have been made to optically analyze particles in a flow. For example, KaV, etc.
Journal of Histochemistry and Cytochemistry J
isto-chemistry and Cyt
chemistry), vol. 27, p. 329 (197
9) shows a Coulter-type orifice that allows blood cells to flow in a single line. The blood cells are expanded on bideiron. Furthermore, Kachel et al., [Journal of Histochemistry and Cytochemistry], 27
Volume, page 335 shows an apparatus for passing blood cells in a single line through the field of a microscope and photographing them. Although these researchers had some success in automating the analysis of a single train of blood cells, they succeeded in automatically analyzing particles in a stream without having to arrange the particles into a single train of flow. There are no reports. for example,
[FIOW Cytometry and Classification J] by Melaned et al.
d Sorting), John Wiley &
5OnS, 1979, Chapter 41, and U.S. Patent No. 3,
See No. 819,270.

The present invention provides a method for aligning particles of a fluid sample moving in one direction in a flow cell.

Each cell has a width and a thickness perpendicular to the direction of flow.

Each particle of the fluid sample has a center of gravity.

The method of the invention includes passing a fluid sample through a flow cell. The sheath fluid flows through the flow cell in the direction of fluid flow to one side of the fluid sample. The sheath is such that the smallest cross section of the particle extends approximately perpendicular to the direction of flow, its largest cross section extends virtually parallel to the width of the cell, and the center of gravity of the particle lies almost entirely in one plane. Adjust the fluid flow rate.

When the center of gravity of a particle is effectively in one plane, the particle can be observed with a microscopic device focused on that plane. Furthermore, by focusing the microscope on a plane other than that one plane, particles of the size of
The particles can be optically separated so that particles of other dimensions are approximately in focus.

The invention also includes a flow cell, a microscopy device that focuses onto an imaging region of the flow cell, and an imaging pickup device that captures a substantially stationary still frame image of the particles in the imaging region. , provides a method for calibrating a pass-through analyzer. The method includes introducing a sample of particles to be calibrated into a flow cell. The particles have known parameters. A series of still frame images of the particles are formed in the imaging region by an imaging pickup device (COD camera).

Process the image to derive parameters. Calibrate the analyzer based on known and aspirated parameters.

The present invention is used in a pass-through analysis system. Also relates to calibrated fluid samples. The calibrated fluid sample includes a physical mixture of a plurality of calibration particles and a biological fluid sample containing microscopic particles. The calibration particles have known parameters.

Biological fluid samples are used for analysis with a flow-through analysis system.

The present invention also provides a method for separating particles flowing in a fluid sample. A fluid sample is moving in one direction through flow cells, each having a width and thickness perpendicular to the direction of flow. The method of the present invention is performed in a flow cell such that the smallest cross section of the particles extends perpendicular to the direction of flow and the largest cross section = 17-° plane extends approximately parallel to the width, so that the particles are approximately aligned. including the step of transporting a fluid sample. The sheath fluid also flows in the same direction through the flow cell. Finally, a force is applied to the fluid sample to separate the particles according to their physical properties.

A closer look at the drawings, particularly in FIG. 1, shows that the device shown therein has an inlet (12
) and an outlet (14), and the channel (16) has an imaging area (
18) having a body (10) including a flow chamber extending therebetween. The flow path (16) is a saline solution (
22) for the conduit (20). As shown in FIGS. 2 and 3, the fluid sample inlet (12) has a needle (24) in the flow path (16) downstream of the conduit (20), the needle (24) being connected to the fluid sample to be analyzed. It is connected to a container (2G) for storing a fluid sample.

The cross-sectional area gradually decreases from the entrance (12) toward the imaging area (18). The thickness also decreases from the entrance (12) toward the imaging area (18). The width decreases from the entrance (12) and then increases by a large amount -18= towards the imaging area (18). Therefore, as shown in Figures 2 and 3,
The channel (16) has a width and depth of approximately 5.000 microns at the blood inlet (12), a width and depth of approximately 5.000 microns at the midpoint (28), and a width and depth of approximately 100 microns at the examination area (18).
It has a depth of microns and a width of over 5,000 microns.

It can be seen that the fluid flow through the imaging (examination) region <18> can be many times deeper than the largest blood cell and the width can be many times wider than the widest particle, for example 100 times. However, if the shape of the flow path is like this, the inlet (
The flow entering from 12) is enclosed in a stable flow path with the least shear within the inspection area (18). Preferably, the cross-section with the least shear is not much larger than the smallest cross-section of the particle, such that the particle has its smallest cross-section extending perpendicular to the direction of flow and its largest cross-section extending parallel to the width (i.e., the second In this specification, the term "minimum shear" means "the velocity gradient is minimal." , as if we were looking at a river with a gradient of flow velocity (particles moving in the flow tend to align in the direction of the flow, just as dangling logs align in the direction of flow. The characteristics of the flow in the channel (16) (22
, 26) or by adjusting its static height.

Focus the microscope (30) on the inspection area,
18) from below with a strobe (32), preferably Ninis Scientific Instrument Corporation (jJ, 3.3scientifiQ I
n5trUIIlent C0rDOratiOn
) model 3018 (with 2UP1.5 lamps).

The output of microscope m1 (30) is projected onto a CCO camera <34>, preferably a CCD camera manufactured by RCA, Model No. TCl 160BD. The output of the CCD camera is converted into a series of still frame images and these images are evaluated with a suitable electronic processor. The single processor used is manufactured by Hamamatsu Systems, Waltham, Massachusetts, USA.
This processor is sold as a model C-1285 image analyzer by INC, Inc. The output of the CCD camera is an electronic processor (3G) with a black and white television monitor (38) and a frame grabber (40) that stores still frame images of the object seen by the CCD camera.
(as shown in detail in Figure 4).The frame grabber is manufactured by Matrox, Montreal.
Model FG manufactured by trox Corporation
OB frame grabbers are preferred. This output is a Matrox model RG, B256 video refresh.
A memory (42) is provided. Frame grabber (40)
and the storage device (42) are both central processing units (CPL).
l) (46) (in tel >8
0/20 computer is preferred). Multipath (44) is also an electronic
Solutions Company (E 1e (itrOnic S)
Random access memory (RAM) (48) made by 01utiOnSInc, ) and Data Cube Co., Ltd.
) model RM117 with dual port RAM on 16
(50). The output of the video refresh memory 21 is also connected to a color monitor (52) which is used to generate digitally enhanced video images of individual still frames for human inspection.

The second output of the dual port RAM (50) is connected to the multipath (54). The bus (54) is manufactured by Applied Micro Devices.
□ CPU (56) manufactured by [)6vices), 48KRAM manufactured by Electronic Solutions
(5g), and an erasable storage device, which is manufactured by Advanced Micro Devices (
Ad vanced lyl 1crol)
A floppy disk controller (60) such as the model 8/8 manufactured by
art) 7 Roybi disk storage device @ (62).

Various programming can be used to process images with the apparatus of FIG. 4 depending on the particular task desired by the user.

Programming of the Hamamatsu system 1285 can be used as described above. However, program 22-ramming should be performed as follows.

The task is to first calculate each pixel (pi×81
) into those that must be addressed and those that only address a small subset of the whole. Since most of the time is spent on tasks of the first class, they are written in assembly language as interface processors (4G>
(Intel 80/20 in Figure 4). The output of these operations is then decoal port RAM
(50) and then transferred to the host machine (56). On the host side, almost all necessary programming is preferably done in a high-level language such as Pascal (Basic or Fortran could in principle also be used). Types of tasks performed in assembly language include grayscale transformations, convolutions, and grayscale histogram calculations.

The types of tasks performed on the host side are the overall control of other devices: identification and segmentation of objects of interest within the field, calculation of parameters associated with the objects thus found, and formatting of the resulting output. including. Another way to think of this separation of tasks is to have assembly perform tasks that must be done faster than humans. Complex tasks or tasks that can be performed at less than maximum speed can be programmed in higher level languages.

The object is brought into view by setting the grayscale wind function to values known primarily as the desired object characteristics. These values can be determined by known knowledge or by well-known histogram techniques. When a pixel belonging to an object is within the field of view, an edge tracing program outlines the entire object associated with that pixel.

Once the edges are known, the location, area, overall optical density,
Many necessary parameters such as various moments can be easily calculated. The probabilities of the members of the previously defined subgroups are determined from these derived parameters by standard decision theory.

Definitions of hemomorphological classifications are determined by trained observers. These definitions are used as the basis for the selected algorithm. The quality of this method is determined by comparing the results of the machine with the results of a trained observer examining the same sample. The resulting output can be programmed into any format and can provide histograms, bar graphs, and tables depending on specific needs.

It can thus be seen that the flow cell (10) provides a two-dimensional flow of particles in the imaging region (18). Thus, more than one particle can be examined in a single field of view and different particles can be optically distinguished, yielding a number of important advantages. For example, Coulter type counter
counter), it is possible to optically distinguish between two co-flowing blood cells that might be recognized as a single blood cell twice the size. In addition, digital image intensification methods developed for sanitary photography intensify the still frame images and allow individual frames to be analyzed to provide data for individual white spheres. For blood samples, it is possible to obtain information such as size, cross section, shape (round blood cells, target blood cells, ant-shaped blood cells, etc.), optical density, hemoglobin content based on blood cells, etc. can.

In this way, individual blood cells can not only be analyzed and optically classified, but also different types of blood cells can be individually counted and automatically determined in one go, the number of normal red blood cells per unit volume of sample, the number of target red blood cells. The number of blood cells, number of sickle cells, number of white blood cells, number of platelets, etc. can be determined.

To ensure that the information extracted from the apparatus shown in FIG. 1 is constrained, the apparatus must be calibrated from time to time. In the method of the invention, calibration particles are introduced into a flow cell having known characteristics such as size, shape, color, intensity and volume of each calibration particle. Additionally, different types of calibration particles with different known properties can be introduced into the flow cell. A series of still frame images of the particles are produced in the imaging region (18) by an imaging pickup device (34). This image is processed to derive these properties. Based on the data obtained by processing the image, i.e. the derived properties, and on the basis of past knowledge of what those properties are, a 26-pass analysis system is used to correct possible errors. can be adjusted to the standard.

There are many sources of error, such as dirt accumulation and lamp aging.

More specifically, calibration particles can be used for the following purposes.

First, calibration particles can be used to calibrate the optical system to a standard for dimensional measurements.

For example, if a spherical particle with a diameter of 20 microns is determined by the electronic processor (36) to be only 19.0 microns, then for all measurements (the error is linear throughout the range of measurements) ) a correction coefficient of 5% should be applied.

Second, calibration particles can be used to orient particles in the fluid. As described above, the particles flowing through the flow cell (10) are oriented such that their largest cross section is approximately parallel to the width and their smallest cross section is perpendicular to the direction of flow. Thus, disc-shaped particles such as human red blood cells are oriented in the flow such that the disc planes are oriented approximately parallel to the width and length planes.

Calibration particles with a specific geometry can be used to flow into the flow cell (10). The predicted orientation of the calibration particles can be used to confirm the correct orientation of the particles in the fluid sample. Correct orientation of the particles occurs due to laminar flow, or lack of turbulence in the flow.

Third, the calibration particles can be used as a false tracer to indicate the position of the fluid sample flow.

For example, if the fluid sample is spinal fluid and the particles are bacteria, the number of bacterial particles per unit volume of fluid will be extremely low. That is, many images seen by the operator and the system will contain no particles at all. To ensure that the system and operator know that a fluid sample is actually passing through the field of view, calibration particles can be added to the fluid sample to track the position of the fluid sample's flow.

Finally, the number of calibration particles can be used to quantify the volume and distribution of the fluid sample.

In general, any particle can be used as a calibration particle as long as it is detected by the analysis system as different from the particles in the fluid sample.

Preferably, the calibration particles are introduced into the fluidizing hill (10) at a known flow rate. Calibration particles can be introduced separately to calibrate a flow-through analysis system, or a biological fluid sample such as blood or urine can be added to a flow cell (10
At the same time as the calibration particles are flowing through the flow cell (
10). In the latter mode of operation, the known properties of the calibration particles can be used to measure unknown values of the same type of properties of the microscopic particles in the biological fluid sample. Additionally, the density of the population of calibration particles can be used to calculate the density of the population of particles of the fluid sample.

Importantly, after the calibration particles have been mixed with the biological fluid containing the microscopic particles into a physical mixture, the entire physical mixture can be introduced into the fluid cell (10). It is.

Below are some examples of the types of calibration particles that can be introduced into the flow cell (10) and their uses.

Yes, L 29 - made from F 1uoresbrite (registered trademark) carboxylate particulate material, with a diameter of 4
The spherical, yellowish-orange calibration particles measuring 5±0.1 μ are the same size as a typical red blood cell and can therefore be used as a standard in the measurement of red blood cell size.

Calibration particles can also be quantified independently of normal blood cells to provide a volumetric calibration for measuring the concentration of red blood cells.

9L 5.5μ spherical polystyrene dyed blue.
Calibration particles consisting of latex beads were placed in a flow cell (1
0) to standardize the dimensions and color of the system, and if the bead concentration per unit volume of sample is known, it can be used to determine the volume of the fluid sample being tested. Additionally, the bead distribution can be used to indicate the location and distribution of the sample in the flow through the analyzer.

Particles of pigweed pollen approximately 20μ in diameter, distinguishable by turbulence size, optical density, and edge characteristics, were placed in a 30-vaginal tube for examination of exfoliative cells in a pass-through analyzer.
It can be mixed with aspirate (I-aspirate) to help locate the specimen flow and measure the size and number of epithelial cells.

Human red blood cells, approximately 5.0-6.0 microns in diameter and distinguishable by shape and optical density, are used in a sample of cultured yeast to confirm the correct orientation of the blood cells in the flow analyzer and to determine the orientation of the yeast cells. Measure the population.

9. Red-dyed polystyrene latex beads, spherical and 1.0 + 0.01 μ in diameter, are used to examine dorsal spinal fluid and are used as tracers in the sparse fluid to detect the presence of bacteria. judge.

Example ``L'' Avian red blood cells with an elliptical cross section with a major axis of about 7.0 to 9.0μ are used in a population of 1q human lymphocytes, which are differentiated by shape, optical density, and size. Determine properties and dimensions.

It has been found from the above that by introducing particles with known properties such as size, volume, shape and spectral properties into a pass-through analyzer, it is possible to standardize the analyzer and control its behavior. do. Accumulation of dirt on the microscope lens, whenever standardization of the system is required.
The effects of equipment aging or normal wear and tear such as electronic noise etc. can all be compensated for and corrected within this analysis system. It is also possible to measure microscopic particles in a biological sample by injecting calibration particles at the same time as an unknown biological sample and comparing them to a standard.

Once a series of still frame images is created in digital form, the complexity of the computers and software used to analyze the images provides extensive and highly complex information about the particles in the series.

It is desirable to combine information obtained from still frame images to obtain composite information that reflects the content of a plurality of still frame images and/or a predetermined reference image. The composite information thus obtained has various uses. That is, in a simple system, for example, information can be printed out to inform a hematologist of the results of a complex measurement made on a blood sample.

In more complex systems, complex measurements can be used for process control where the system monitors particle size or quantity, such as homogenizer pressure, crystallizer temperature, nutrient feed rate in microbial cultures.

Therefore, the device is suitable for the analysis of various optically perceptible particles moving in a flow, i.e. biological particles such as white corpuscles in blood, cells in urine, bacteria, casts (CaSt), crystals, and gases. The results of these measurements can be used to analyze both particles and other particles in the vessel, and the results of these measurements can be used to analyze processes such as the introduction of nutrients into streams containing microorganisms, as well as the control of polymer and crystal growth. Can be used for control.

FIG. 5 illustrates a prior art technique for testing particles in a fluid sample. As a representative example, particles such as those shown a, b and C are placed on a microscope slide (1oo) 33-. Each particle a, b and C are each A
It has a center of gravity denoted lB and C.

As is clear from FIG. 5, the centers of gravity A, B and C of particles a, b and C, which are all placed on the slide (100), are not on the same plane.

Therefore, in order to observe particles a1b and C, particle a in a plane parallel to the slide (100) and passing through the center of gravity A.
The microscope must first be focused on. To observe the particles, adjust the microscope and slide (1
00) and passes through the center of gravity B. Similarly, to observe particle C, the microscope must be adjusted.

In contrast, in the method of the present invention, particles are placed in a flow cell (1
Align when moving inside 0). particles a1b in fluid sample
and C is the flow cell (
10) Move. Sheath fluid also flows through the flow cell (10) in the direction of arrow (110). The sheath fluid encloses the sample fluid. The flow rate of the sheath fluid or the sample fluid or both fluids can be controlled 34- such that the flow of the two fluids remains in the laminar region, ie, with little or no turbulence. Additionally, the flow rate of each or both fluids may be controlled such that the smallest cross section extends substantially perpendicular to the arrow (110) and the largest cross section substantially extends through the flow cell (10).
Particles a, b and C are aligned so that they extend parallel to the width of the particle a, b and C, and the centroids A, B and C of particles a, b and C all lie substantially within one of the inspection areas (18). Plane(
120). In this way, the inspection area (
18) When particles a, b, and C are aligned, the microscope (
30) can be adjusted to focus on the same plane (120). At that time, the particles aSb and C will all be in focus when viewed through the microscope 61 (30).You can also change the flow rate of the sheath fluid to bring the particles into the focal plane with the particles in focus. It can also be moved.

Furthermore, if the centroids A, B and C of particles aXb and C lie in substantially the same plane (120), a method of optically separating the particles is also provided. As shown in Figure 6, the microscope (
30) can be adjusted to be focused on a plane (122) different from the previous same plane (12, 0). The plane (122) does not pass all the heat particles a. When viewed under the microscope (30), particle a is out of focus. However, since particles S and C are considerably larger than particle a, particles S and C remain substantially out of focus even when the microscope (30) is focused on the plane (122). Since particle a is out of focus, its gray scale level is not large enough to trigger the edgetracing routine of the electronic processor (36). Displacing the focal plane so that relatively small particles are out of focus thus results in optical separation of the particles. Furthermore, relatively small particles such as bacteria are believed to be less affected by the hydrodynamic mechanism of the flow cell (10). That is, bacteria are not greatly influenced by the shape of the flow cell <10> whose maximum cross-sectional area is made to coincide with the direction of flow. Particles such as bacteria are thought to be affected by Brownian motion. When the particles in the fluid sample affected by the hydrodynamic mechanism of the flow cell (10) lie substantially in the same plane (120), the plane in which the microscope (30) focuses is displaced and its Particles in the plane (120) can be taken out of focus so that bacteria in a different plane can be brought into focus. In this way, optical separation of particles can also be achieved.

There are numerous applications for the method of the invention, one of which is in the analysis of blood or urine as a fluid sample. In blood as a fluid sample, red blood cells are smaller than white blood cells. Thus, using the method described above, red blood cells can be optically separated from white blood cells by bringing the red blood cells out of focus and the white blood cells approximately in focus. In urine as a fluid sample, cellular particles (e.g. blood cells or epithelial cells) are separated from the same material - the latter being considerably larger than the former.
I can do it.

A variation of the method of the invention is to flow a second sheath fluid through the flow cell (10). The flow rates of both sheath fluids and sample fluid are adjusted so that the centers of gravity of the particles lie substantially in the same plane. Each sheath fluid flows in a plane substantially parallel to the width of the flow cell (10) whose width is many times wider than its thickness.

The method of the invention has many advantages. The first and biggest advantage is that maximum contrast is obtained when the particles are in focus. The highest edge intensity of each particle is in the image plane. Furthermore, if the centroids of the particles are in the same plane, there is no need to adjust the microscope (30) to observe each particle. Finally, it is also possible to bring the centers of gravity of the particles into approximately the same plane to achieve optical separation of the particles. By arbitrarily adjusting the displacement of the focal plane, it is possible to blur the edge density of small particles and leave large particles in focus. Furthermore, all the particles in the same plane (120) are blurred by the displacement of the focal plane,
It is also possible to focus the microscope on relatively small particles that are not affected by the hydrodynamic mechanisms of the flow cell (10) in different planes.

Figure 7A is a partially schematic side view 38- of the flow cell (10). The flow cell (10) has an inlet (12) as described above.
) toward the imaging area (18). For this reason, both the sample fluid and the sheath fluid enter the inlet (12
) exerts a compressive force on the fluid as it flows from the imaging region (18). This compressive force is indicated by arrow A11. Figure 7B is a partially schematic top view of the flow cell (10).

Figure 7B shows the imaging area (18) after being subtracted from the entrance (12).
). This creates an expanding force, indicated by arrow II B ++, on the sheath fluid and fluid sample. Figure 7C shows the imaging area (18)
Figure 3 is a cross-sectional view of the flow cell (10) looking towards the inlet area (12); The compressive force in the thickness direction and the expansion force in the width direction are indicated by arrows 110 ++. Force "C" is the resultant force of A" and B". The force ""C11 is the fluid sample (2
6) to form a flow of approximately rectangular cross section having a thickness 'D' and a width 'E11'. It should be noted that the thickness D'' is extremely thin, on the order of 0.1 to several tens of microns.

In the method of the invention, by adjusting the flow rate of the fluid sample, the flow rate of the sheath fluid, or the flow rate ratio of the fluid sample and sheath fluid, the dimensions of the sample fluid, ie, its cross section, can be changed. In particular, the thickness ``D'' of the fluid sample is defined as the particle (
The flow rates of the sheath fluid, fluid sample, or both can be adjusted to be smaller than relatively large particles such as those shown in 70). If the size of the particle (10) is greater than the fluid sample thickness "D'", a hydrodynamic force "C" caused by the particular geometry of the flow cell and sheath fluid flow acts on the particle (70), The particles are forced from the main stream towards the periphery of the flow cell (10).

On the other hand, like the particle (72), the thickness of the fluid sample is 11
Small particles with dimensions smaller than D++ are not affected by the hydrodynamic force II C++. Hydrodynamic force “C
The trajectory of the small particle (70) that is not affected by `` is ``
76", while the trajectory of the large particle is "74".
It is indicated by. The results are shown in Figure 7D. When the large particles are pushed to different trajectories in this way, the microscope 1 (3
0> can be used to focus on either large particles (70) or trajectories containing small particles. “dimension” of a particle, in the sense of the dimension affected by hydrodynamic forces
It is important that the definition of does not necessarily mean the maximum dimension. In fact, as mentioned above, due to the geometry of the flow cell (10), particles flowing through the flow cell (10) are oriented such that their smallest cross section is perpendicular to the direction of flow and their largest cross section is parallel to the width. Can be attached. Therefore, the hydrodynamic force II C
Large particles (70) affected by 11 are particles whose dimension in the plane of maximum cross-section, perpendicular to the plane of maximum cross-section, is larger than the thickness 'D'' of the fluid sample.Small particles (72)
) are particles whose dimension in the plane of the smallest cross section perpendicular to the plane of the largest cross section is smaller than the thickness D'' of the fluid sample.

FIG. 8 shows a flow cell (10) used to separate particles based on density. In this example, the flow cell (
The width of 10) is several times the thickness, and the width is indicated by the arrow 11 G I
It is used parallel to the gravity indicated by I. When the stream of particles flows from the inlet (12) to the imaging area, the main stream will flow along the trajectory indicated by the arrow path. However, the high-density particles separate from the mainstream and sink due to the action of gravity, as shown by arrow I.
II! Follow the downward trajectory indicated by 1. The effect of gravity acting on particles is greater for dense particles than for less dense particles, so particles with high density follow a trajectory lower than that of mainstream particles. In this way,
Separation of particles based on density can be achieved.

Finally, it is important to be able to base the separation on the basis of the force exerted on the flow of the sample fluid on other physical properties of the particles. For example, particles of species consisting of a flow cell (10
) is charged before entering. An electric field can be created parallel to the width or thickness. When an electric field is applied, charged particles are deflected from the mainstream particles. In this way, separation of particles based on charge is possible. This is similar to electrophoresis, but the movement of particles in the flow is much faster. Furthermore, using a sheath fluid in the method of the invention comprises:
It is necessary to stabilize the flow of the fluid sample and keep it in the region of laminar flow, ie, without turbulence. Similarly, separation of particles based on magnetic permeability is made possible by magnetic forces.

[Brief explanation of drawings]

FIG. 1 is a perspective view of the apparatus for inspecting the flow of the present invention, FIG. 2 is a top view of the flow chamber of FIG. 1, and FIG. 3 is a top view of the apparatus of FIG. 4 is a schematic diagram of an electronic processor used by the apparatus of FIG. 1; FIG. 5 is an enlarged side view of a prior art method of placing particles on a microscope for examination; Figure 6 is an enlarged side view of the flow of particles according to the method of the present invention in the apparatus described herein, and Figure 7A is a partial side schematic diagram of the flow cell showing the through-thickness compressive force of 8''. , FIG. 7B is a partial top schematic diagram of the flow cell showing the widthwise force B I+, and FIG. 7C is a partial cross-sectional schematic diagram of the flow cell showing the resultant force acting on the fluid sample. , FIG. 7D is a partial top schematic view of a flow cell showing the trajectories of large particles separated from the main stream of particles by the method of the invention, and FIG. 8 is a diagram of the trajectories of particles separated by the method of the invention. 10...Flow cell 12...Inlet 14...Outlet 16...Flow path 18... - Imaging (inspection) area 30...Microscope 32...Strobe 34...CCD camera 36...Electronic processor 70...Grain Child 72... Particle patent application agent Yuki Yamazaki FIG, 5゜FIG, 6゜FIG '7b. FIG, 7c, FIG 7d Procedure amendment teeth June 7, 1988 Patent Commissioner of the Agency 1. Display of the case Patent Application No. 74133 of 1982. 2. Name of the invention. Method for aligning particles in a flowing fluid sample. 3. Person making the amendment. Relationship to the case. Applicant name: International Remote Imaging Systems, Inc. Address of the agent: 1-11-28 Nagata-cho, Chiyoda-ku, Tokyo, Japan Column for the representative of the patent applicant in the application form, official drawings, and a document certifying the right of representation. 7. Contents of the amendments as shown in the attached sheet.

Claims (1)

[Claims] <1) A method for aligning particles in a liquid sample moving in one direction in a flow cell, wherein each cell has a width and a thickness in a direction perpendicular to the direction. , each of the particles has a center of gravity, passing the fluid sample through the flow cell, flowing a sheath fluid in the direction through the flow cell, and adjusting the flow rate of the sheath fluid to substantially aligning the particles such that the smallest cross-section of the particles extends perpendicular to the direction, the largest cross-section extends substantially parallel to the width, and the centers of gravity of the particles lie substantially in the same plane; , a method of having. (2) A method for observing particles in a fluid sample moving in one direction in a flow cell, the cells each having a width and a thickness in a direction perpendicular to the direction in an imaging region;
a microscope apparatus is aligned to view the particles in the imaging region, each of the particles having a center of gravity, transporting the fluid sample through the flow cell; and transporting the fluid sample through the flow cell. flowing a sheath fluid in a direction, and adjusting the flow rate of the sheath fluid so that the smallest cross section of the particle extends perpendicular to the direction and the largest cross section extends substantially parallel to the width, A method comprising: substantially aligning the particles such that their centers of gravity lie substantially in the same plane; and focusing the microscopy device on the particles. (3) A method for optically separating particles of different sizes moving in one direction in a flow cell, wherein each cell has a width and a thickness perpendicular to the direction in an imaging region. a microscope device is aligned to view the particles in the imaging region, each of the particles having a center of gravity, transporting the fluid sample through the flow cell; flowing a sheath fluid in the direction through the particles; substantially aligning the particles so that the centers of gravity of the particles in the area are substantially in the same plane; and using the microscope apparatus to bring particles of a size out of focus and particles of another size into substantially focus. The method includes the step of focusing on a plane different from the same plane. (4) A method for aligning particles in a fluid sample moving in one direction in a flow cell, each cell having a width and thickness perpendicular to the direction, each of the particles transporting the fluid sample through the flow cell; flowing a sheath fluid in the direction through the flow cell; and adjusting the flow rate of the fluid sample so that the particle has a minimum cross-section of the particle. substantially aligning the particles so that the particles extend perpendicular to the direction and have their largest cross-sections substantially parallel to the width, and the centers of gravity of the particles lie substantially in the same plane. (5) A method for observing particles in a fluid sample moving in a direction in a flow cell, wherein each cell has a width and a thickness perpendicular to the direction in an imaging region, and a microscope apparatus is aligned to observe the particles in an imaging region, the particles have a center of gravity; transporting the fluid sample through the flow cell; and transporting the fluid sample through the flow cell in the direction. flowing a sheath fluid; and adjusting the flow rate of the fluid sample such that the smallest cross-sectional area of the particles extends perpendicular to the direction and the largest cross-sectional area extends generally parallel to the width, A method comprising the steps of: substantially aligning the particles such that their centers of gravity lie in substantially the same plane; and focusing the microscopy device on the particles. (6) A method for optically separating particles of different sizes in a fluid sample moving in one direction in a flow cell, the cells each having a width perpendicular to said direction in an imaging region. a thickness, a microscope device is aligned to observe the particles in the imaging region, each of the particles having a center of gravity, and transporting the fluid sample through the flow cell; flowing a sheath stream 5 in the direction through the flow cell; and adjusting the flow rate of the fluid sample so that the smallest cross-section of the particles extends perpendicular to the direction and the largest cross-section is substantially parallel to the width. aligning the particles so that the centers of gravity of the particles in the region are substantially in the same plane; and focusing the microscope device on a plane different from the same plane. (7) The method according to any one of claims (1) to (6), wherein the one plane is substantially parallel to the width. (8) The method of claim (7), wherein the width is many times the thickness. (9) Claims (1) to (1) further comprising the step of flowing a second sheath fluid through the flow cell.
The method described in section 6). -〇- (10) Claims (2) and (2), wherein the focusing step consists in moving the microscope device.
The method described in paragraph 3), paragraph (5) or paragraph (6). (11) Claims (2), (3), (5), or (6), wherein the focusing step consists in changing the flow rate of the sheath fluid. the method of. (12) Claims (1), (2), (4), or (5), wherein the fluid sample is blood.
The method described in section. (13) The method of claim (3) or (6), wherein the fluid sample is blood. (14) Claim No. 13, wherein the one dimension is a red blood cell and the other dimension is a white blood cell.
The method described in section. (15) The method of claim (1), (2), (4), or (5), wherein the fluid sample is urine. (16) The method of claim (3) or (6), wherein the fluid sample is urine. (17) Claim No. 16, wherein the one dimension is a cellular particle and the other dimension is a cylinder.
). (18) A fluid sample of a calibration stream for use in an imaging analysis system, comprising: a plurality of calibration particles of one type having known properties; and a microscope having unknown properties to be analyzed by said system. a biological fluid sample comprising biological particles; and a physical mixture of the calibration particles and the biological fluid sample. (19) The fluid sample of claim (18), wherein the calibration particles have known properties of size, volume, shape, color, or intensity. (20) further comprising multiple types of calibration particles;
A fluid sample according to claim (19). 21. The fluid sample of claim 18, wherein the system allows the calibration particles to be distinguished from the microscopic particles. (22) The fluid sample according to claim 21, wherein the microscopic particles are blood cells. (23) The fluid sample according to claim 22, wherein the biological fluid sample is blood. (24) The fluid sample according to claim 22, wherein the biological fluid sample is urine. (25) The fluid sample according to claim 21, wherein the calibration particles have a size in the range of 0.5 to 50μ. (26) The fluid sample of claim 18, wherein the amounts of the calibration particles and the biological fluid sample in the mixture are known. (27) A method of calibrating a pass-through analyzer, the analyzer comprising: a flow cell; a microscope device for focusing onto an imaging region of the flow cell; an image pickup device for capturing a stationary still frame image; 19 = introducing a sample of one type of calibration particles having known parameters into the flow cell; forming a series of still frame images of the particles in the imaging region with an image pickup device; processing the images to derive the parameters; and determining the parameters based on the known and derived parameters. A method comprising: calibrating an analyzer; (28) Claim No. (27), wherein the charging step further includes the step of charging the sample at a known flow rate.
The method described in Section 1. (29) The claim further comprises: passing a biological fluid sample having microscopic particles through the flow cell; and measuring the parameter of the microscopic particle as a known parameter of the particle. Range number (27)
). 10-(30) The method according to claim (29), wherein the distribution step and the charging step are performed simultaneously. (31) The method according to claim (28) or (30), wherein the known parameters include the volume, size, shape, color, or intensity of each calibration particle. (32) The method according to claim 31, wherein the known parameter further includes a population density of the calibration particles. (33) The method according to claim (30), wherein the biological fluid sample is urine. (34) The method according to claim (30), wherein the biological fluid sample is blood. (35) The method of claim (27), wherein the particles have a size range of 0.5 to 50 microns. (36) The method of claim (27), wherein the step of loading comprises loading a plurality of types of calibration particles. (37) A method for separating particles in a fluid sample moving in a direction through fluid cells each having a width and a thickness perpendicular to said direction, the smallest cross section of said particles being said transporting a fluid sample through the flow cell in such a manner that the particles extend perpendicularly to the width and the largest cross section extends substantially parallel to the width so that the particles are substantially aligned; A method comprising: flowing a fluid; and applying a force to the fluid sample to separate the particles according to their physical properties. (38) The method of claim (37), wherein the force is a hydrodynamic force and the physical property is a dimension. 39. The method of claim 38, wherein applying the force includes adjusting a flow rate of the fluid sample. (40) Claim 38, wherein the step of applying the force includes the step of adjusting the flow rate of the sheath fluid.
The method described in section. 41. The method of claim 38, wherein applying the force includes adjusting a ratio of the fluid sample flow rate to the sheath fluid flow rate. (42) The method of claim (31), wherein the width is many times the thickness. (43) The method according to claim (42), wherein the force is gravity and the physical property is density. 44. The method of claim 43, wherein applying the force includes orienting the flow cell so that the width is substantially parallel to the force of gravity. (45) The method according to claim (37), wherein the force is an electrical force and the physical property is a charge. (46) The force is magnetic and the physical property is 1
3 - The method according to claim 37, being magnetically permeable.