DE3315194A1 - METHOD FOR SEPARATING PARTICLES FLOWING IN A FLUID SAMPLE - Google Patents

Description

33-5194

The invention relates to a method for separating particles flowing in a fluid sample.

Significant advances have been made in automating the counting of blood cells in a serum sample. The best-known device for counting blood is the so-called Coulter counter, in which blood cells are passed in a single row through an opening and are determined and counted by the way in which the electrical properties change at the opening. However, to date there have been no automated devices for analyzing and evaluating multiple cells, for example normal cells, target cells, sickle cells, etc., which can be contained in a flowing stream of a given blood sample. Thus, when information on multiple cells of this type is desired, it is normal and customary to obtain such information by preparing a slide with the cells fixed in an image plane and having an operator or machine appropriate To recognize patterns, count statistically significant numbers of cells while viewing them, one at a time, through a microscope on the slide, see e.g. US Pat. No. 4,175,860 and k 199

Attempts at the optical analysis of particles flowing in a flow stream have already been made in recent years been undertaken. Kay et al., Journal of Histochemistry and Cytochemistry, Vol. 27, p. 329 (1979) a Coulter-type opening for cells moving in a single row, which are enlarged to a vidicon. aside from that Kachel et al., "Journal of Histochemistry and Cytochemistry 1 'Vol. 27, p. 335. show a device with which Cells are moved in single row through a microscopic area where they are photographed. From these people While work has been done to automate particle analysis in a single row, it is

nothing has been reported that automated particle analysis was accomplished in a flowing river, without the particles having to be arranged in a stream in a single row, see e.g. "Flow Cytometry and Sorting ", Melaned et al., John Wiley & Sons 1979, chapt 1 and U.S. Patent 3,819,270.

In accordance with the invention, a method for separating particles flowing in a fluid sample is now provided. the Fluid sample moves in one direction through a flow cell, each perpendicular to the direction of flow has a width and a thickness. The method according to the invention provides the following steps: The fluid sample is transported through the flow cell so that the particles essentially with their minimum cross-sectional area in the transverse direction to the direction of flow and align with their maximum cross-sectional area essentially parallel to the width. Flows through the flow cell also an enveloping fluid in the same direction. Finally, a force is applied to the fluid sample to reduce the Separate particles according to a physical characteristic of the particles.

The invention is described below with further advantageous details on the basis of an exemplary embodiment shown schematically explained in more detail. In the drawings shows:

Fig »1 is a perspective view of a device for Checking a flow flow according to the invention;

FIG. 2 is a plan view of the flow chamber contained in FIG. 1; FIG.

3 shows a cross section through the device shown in FIG in the plane labeled 3-3;

FIG. K shows a circuit diagram of the electronic processor used in the case of the device according to FIG. 1;

5A is a partial side view of the flow chamber, in which the constriction force A is shown in the direction of the thickness;

5B is a partial top view of the flow chamber in which the force B is shown in the direction of width;

50 shows a partial cross section through the flow chamber, in which the resulting forces acting on the fluid sample are shown;

5D is a partial plan view of the flow chamber in which the trajectory of a large particle separated from the main stream of particles by the process of the invention is shown;

Figure 6 is a partial side elevational view of the trajectories of particles separated by the method of the present invention.

As can be seen from the drawings, in particular from FIG. 1, the device has a housing 10 which has a Flow chamber with an inlet 12 for a fluid sample, such as blood or urine and an outlet 14 · contains, wherein a channel 16 extends past an imaging or inspection area 18 between the inlet and the outlet. The channel 16 has an inlet with a conduit 20 suitable for connection to a container 22 for saline solution. As Fig. 1 and 3 show, in the inlet 12 for the fluid sample there is a needle 2, 4 in the channel 16 behind the line 20 in the direction of flow provided which is connected to a container 26 which can contain the fluid sample to be analyzed.

The cross-sectional area becomes progressively smaller with increasing distance from the inlet 12 to the imaging area 18. The thickness also decreases from the inlet 12 to the imaging area 18. The width decreases from the inlet 12 and then increases significantly to the imaging area 18. The channel l6, as can be seen from FIGS. 2 and 3, has a width and depth of approx. 5,000 μm at the inlet 12 and a width and depth of approx . 5 00 μπι at a central point 28 and a depth of 100 pm in the test area 18, where the width exceeds 5,000 μτα.

It is clear that the flow flow through the test area

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can be many times deeper than the largest cells and the width many times wider than the widest particle, e.g. more than a hundred times as wide as the widest Particle. However, in the case of a flow path configured in the manner described, that through the inlet 12 incoming current is restricted to a stable flow path of minimal shear in the test area 18. Preferably the Minimum shear cross-sectional area not significantly larger than the minimum cross-sectional area of the particles, thereby orienting the particles in the flow flow so that their minimum cross-sectional area is transverse to the direction of flow and its maximum cross-sectional area parallel to the width (i.e. visible in the plane of Figure 2). This here The term "minimal shear" used is intended to mean "minimal Describe velocity gradient ", so that a particle moving in the flow has the tendency to move with the direction of flow to align, very similar to how a tree trunk or beam drifting in a river aligns itself in the direction of the flow, when there is a flow gradient. The flow characteristics prevailing in the channel 16 can be adjusted by setting the fluid pressure in containers 22 and 26, either automatically or by adjusting the static level thereof, being controlled.

A microscope 30 is focused on the test area 18 and the test area 18 is illuminated from below by means of a strobe light 32, which is preferably the model 3018 from the US Scientific Instrument Corporation, which contains a 2UPl.5 lamp. The output of the microscope 30 is focused on a charge coupled device (CCD) camera Jk , which is preferably used as a CCD camera of model no. TCII60BD from RCA. The output of the charge coupled camera Jk is converted into a series of still images, and appropriate electronic processors are provided to evaluate these images. A processor that can be used for this is the one known as the Image Analysis System,

Model C-1285 from Hamamatsu Systems, Inc., Waltham, Massachusetts processor put on the market. Preferably, the output of the charge coupled device camera 34 is on one electronic processor 36, which is shown in more detail in FIG and to which a black and white television monitor, i.e. a monitor 38 and an imager that stores still images of the object viewed by the charge-coupled camera. The picture author 4o is preferably an imager sold as a model FGO8 by Matrox Corporation, Montreal, the output of which is on a frame buffer 42, model RGB 256 from Matrox Corporation, both with a Multibus 44

a central unit 46 are coupled, which is preferably an Intel 80/2 0 computer. The multibus 44 also comes with 48K memory 48 with direct access from Electronics Solutions Inc. and with a 16k

Dual-port memory 50 with direct access, model RM 11? Associated with the Data Cube Corporation ^. The exit of the frame buffer is also connected to a color monitor 52 which can be used to deliver digitally processed video images of individual still images for the purpose of human examination.

The second output of the dual port memory 50 with direct access is with a

Multibus 54 connected, which is also connected to a central processing unit (CPU) 56 from Applied Micro Devices, a 48K memory 58 with direct access from Electronics Solutions, Inc. and a removable memory in the form of a floppy disc controller 60, for example the

Model 8/8 of the Advanced Micro Devices and two units of Shugart floppy disk memory 62.

For further processing of images with the one shown in FIG The device can be programmed in a wide variety of ways, depending on the particular task that a

user wants to perform. As already mentioned, programming can of the Hamamatsu System 1285 can be used. However, programming is preferably carried out as follows

The tasks are first divided into those that address each pixel of a given map

and those who only need to address a small subset of the whole. As for the first If a lot of time is spent on tasks in the classroom, they are processed in assembly language on the interface processor 46 (the Intel 80/20 programmed according to Fig. 4). The output of these operations is then via the dual-port memory 50 with direct Access to the main machine, namely the central unit 56 passed. On the main engine side, almost All the necessary programming is expediently carried out in a language of a higher level, e.g. Pascal (In principle, BASIC or FORTRAN would also be suitable). On the types of tasks that are solved in assembly language, include gray scale transforms, convolutions, and gray scale histogram calculations. The tasks performed on the main machine include overall control of the others Devices, the identification and segmentation of the object of interest in the field of view, the calculation of parameters associated with such objects as well as the formatting of the output of the results obtained. Another Way of looking at the separation of tasks in this way is to do tasks with greater speed must be performed than that of a human operator, in assembly language. Tasks, that are either complicated or can be done at less than maximum speed can be seen in program the higher language. Objects are found in the field of view mainly by having a gray scale window function is set for values known to be characteristic of the desired object. These values can either be based on previous knowledge or

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can be established using well known histogram techniques. If a picture element belonging to an object in the Field of view has been located, an edge determination program is triggered to determine all of the elements associated with this image element Outline object. Once the edge has been found, many relevant parameters can be used, for example the location, area, integrated optical density and various moments can be easily calculated. Based This derived parameter can be the probability of membership using common decision theory can be determined in previously defined subgroups. Definitions for the classification of blood cell morphology are determined by trained observers. These definitions are then used as the basis for the algorithms chosen. The accuracy of the method is determined by comparing the machine results with those of trained observers, examining the same samples. The output of the results can be programmed so that they are in a wide variety of formats. Histograms, curve displays and table-like summaries are available available for special requirements.

It can therefore be seen that in the flow cell 10, the particle flow in the imaging area 18 has two dimensions. So more than one particle can be observed in a single field and different particles can be distinguished optically, which has a number of important advantages brings. E.g. two confluent cells can be optically recognized, while a Coulter counter only sees them as one can see a single double-sized cell. In addition, the still images can be processed using digital imaging techniques that have been developed for satellite photos, and the individual images can be analyzed, to get data on individual cells. For a blood sample, information about the size, cross-sectional area, shape (circular cell, target cell, sickle cell etc.) the optical density, the hemoglobin content

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Cell base, etc. can be obtained. Not only individuals can do it Cells can be analyzed and sorted optically in this way, but after analyzing and sorting it can also different types of cells are counted individually to automatically and in a single pass the number normal of red cells per volume of the sample, the number of red target cells per cubic centimeter of the sample, the number of red sickle cells per cubic centimeter of the sample, the number of white cells, the number of platelets, etc. per cubic centimeter of the Get sample.

Once a series of still images is available in digitally processed form, depending on the complex nature of computers and software available to analyze the images, a wide variety of very high quality information about the particles in the series of images.

Preferably the information derived from still images combined to obtain composite information representing the content of the multiple still images and / or predetermined Reflect reference images, the composite information thus obtained being used in a wide variety of ways can be. In the case of a simple system, the information can be printed out, e.g. to give information to the hematologist to give over multiple measurements on a blood sample. In the case of more complex systems, the combined measurements

can be used for process control, e.g. for the pressure in a homogenizing device, the temperature in a Crystallizer, or the feed rate in a microbial culture, where the plant is the particle size or number monitored.

It can be seen that the device is used for the analysis of a wide variety of optically perceptible particles moving in a stream, namely biological particles such as cells in blood or cells, bacteria,

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Cylinders / and crystals in urine or particles in gas analysis devices, etc. The output of these measurements can be used for process control, for example for the delivery of food into a stream that Contains microorganisms, as mentioned above, or controlling the growth of polymers and crystals and like ..

5A is a schematic partial side view of the flow chamber 10 which has a thickness which decreases from the inlet 12 to the imaging area 18, as previously mentioned. This creates a restriction for the fluids, both the sheath fluid and the sample fluid, as they flow from the inlet 12 to the imaging area 18. This constriction force is indicated by arrows ¹¹ A ". In Fig. 5B is a schematic partial plan view shown in the flow chamber 10. As shown in Fig. Emerges 5B, the width decreases from the inlet 12, and then substantially to 18 in the direction of the imaging region, the generates an expansion force which acts on the enveloping fluid and the fluid sample and is indicated by arrows "B." Fig. 50 shows a cross section through the flow chamber 10 viewed in the direction from the imaging area 18 to the area of the inlet 12. The constriction forces in the thickness and the expansion forces in the width are indicated by the arrows "C." The forces according to arrow "C" are the resulting forces from "A" and "B". The forces "C" limit and shape the fluid sample 26 into a flow with a Cross-sectional area which is substantially rectangular and has a thickness "D" and a width "E." It should be emphasized that the thickness "D" is extremely small and of the order of magnitude range from 0.1 to a few tens of pm.

With the method of the invention, the size of the fluid sample 26, i.e. the cross-sectional area, can be adjusted by regulating the Throughput of the fluid sample 26 or the throughput of the

enveloping fluids or the ratio between fluid sample and vaginal fluid or enveloping fluid can be varied. In particular the flow rates of either the enveloping fluid or the fluid sample or both can be adjusted so that the thickness "D" of the fluid sample is set smaller than the larger particles, for example particles 70. For the In the event that the size of the particle 70 is greater than the thickness "D" of the fluid sample 26, those of the particular one will apply Shape of the flow cell and the flow of the enveloping fluid resulting from hydrodynamic forces "C" onto the particle 70 and push it away from the main stream of particles towards the periphery of the flow chamber 10. What, on the other hand relates to particles of smaller size, e.g., particles 72, i.e. particles smaller than thickness "D" of the fluid sample 26, they are not influenced by the hydrodynamic forces "C". The path of the smaller ones Particles 72 that are affected by the hydrodynamic forces "C" are not are influenced, is denoted by 76, while the path of the larger particles is indicated by 7 ^. The result is shown in Figure 5D. Since the larger particles are along a Other path are transported, the microscope 30 can either on the larger particles 70 or on the path of the smaller particles are focused. It should be emphasized that the definition of the "size" of a particle not necessarily the largest dimension in connection with the influence of the hydrodynamic forces means. In fact, given the geometry of the flow cell 10 described above, the in the flow line 10 flowing particles with their smallest Cross-sectional area transverse to the direction of flow and with its largest cross-sectional area parallel to the width. Therefore, the larger particles 70 affected by the hydrodynamic forces "C" are those particles which have a dimension in the minimum cross-sectional plane which is transverse to the maximum cross-sectional plane and is greater than the thickness "D" of the fluid sample 26. The smaller sized particles 72 are those particles

which have a dimension in the minimum cross-sectional plane which is transverse to the maximum cross-sectional plane extends and is less than the thickness "D".

Fig. 6 shows the flow cell 10 for separating particles based on their density. In this embodiment, the flow cell 10 has a width that is a multiple its thickness and is operated so that the width is parallel to gravity, as indicated by the arrow "G" indicated. When the particle stream flows from inlet 12 to the imaging area, the main stream would be along the through the arrow "H" indicated path flow. However, the denser particles are separated from the mainstream and lose under the action of gravity "G" and sink into a lower trajectory along which they move, as indicated by the arrow "I" indicated. In view of the force of gravity on the particles, the denser particles are more affected than the less dense particles with the result that the denser particles migrate in a trajectory, the lower lies as the path along which the main stream of particles flows. In this way there can be a separation of particles due to the density.

Finally it should be underlined that other physical characteristics of the particles as a basis for the separation with Can be used with the aid of a force exerted on the flow of the fluid sample. For example, certain Particles are electrically charged before they get into the flow chamber 10. An electric field can be parallel to the Width or thickness. When an electric field is applied, the charged particles are released from the Main stream of particles is deflected out. This is done in this way a separation of particles on the basis of their electrical charge. Although this is analogous to electrophoresis, but the movement the particle in the stream is much faster. Also is

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in the method according to the invention, the use of a sheath fluid necessary to stabilize the flow of the fluid sample and to increase the flow within the laminar region hold, i.e. without any turbulence. Similarly, there can also be a separation of particles due to the magnetic Permeability can be achieved with the help of a magnetic force.

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Claims (9)

Priority: April 29, 1982 - USA - Serial No. 373 042 Claims )
Iy "Method for separating particles in a fluid sample moving in a direction through a flow cell which has a width and a thickness, respectively, perpendicular to that direction,
characterized by - That the fluid sample is transported through the flow cell in this way is that the particles are essentially transverse to said direction with their minimum cross-sectional area and align with their maximum cross-sectional area essentially parallel to the width, - That an enveloping fluid flows through the flow cell in the mentioned direction is directed, and - That a force is exerted on the fluid sample, which the particles according to a physical characteristic of the particles separates. 2. The method according to claim 1, characterized in that the force is a hydrodynamic force and then physical characteristic the Size is. 3. The method according to claim 2, characterized in that for application the force of the flow rate of the Pluidprdbe adjusted will. 4. The method according to claim 2, marked by the fact that to apply the force of the flow rate of the enveloping fluid is adjusted. 5. The method according to claim 2, marked i chnot that for applying the force, the ratio of the throughput of the fluid sample to the throughput of the enveloping fluid is set. 6. The method according to claim 1, in that the width is a multiple of the thickness. 7. The method according to claim 6, thereby g e k e η η ζ e i. c h η e t that as a force gravity is used and that the physical characteristic is density. 8. The method according to claim 7, characterized in that for application the force of the otrömungszelle is aligned so that the Width is essentially parallel to gravity. 9. The method according to claim 1, characterized in that the force is electrical Force is used, and that the physical characteristic electric Ladur.p is. 10, method according to claim 1, characterized in that magne as a force tical force is used, and that the physical characteristic magnetic permeability is.