FR2526164A1 - Calibration of optical analyser of particles in blood, urine etc. - using microscopic particles of selected parameter to set standard in computer - Google Patents

Abstract

Analyser has a transparent chamber through which the fluid is passed to be viewed by a microscope which transmits fixed frame images of suspended particles to an analysing computer. A fluid contg. calibration particles having a selected parameter, vol., size, shape, colour etc. is injected into the viewing chamber. Images of observed particles are transmitted to the computer for filing and for calculating a calibration value of the parameter. The biological fluid contg. particles to be analysed is passed through the viewing chamber so that the same parameter can be measured against the calibration. Pref. the calibration and biological fluids are passed simultaneously through the viewing chamber. The analyser should be capable of distinguishing between the calibration particles and the particles in the biological fluid to be analysed. The calibration particles are pref of 0.5-50 microns. Used for analysing red or white cells or platelets in blood; cells, bacteria or crystals in urine; bacteria in spinal fluid. There is no longer any need to arrange particles in single file for analysis. Results of measures taken in the analysis can be used to control a process e.g. rate of injection of a nutrient into a current of liq. contg. micro-organisms.

Description

 The present invention relates generally to continuous flow optical analyzers and relates more particularly to a method for calibrating such an analyzer and to a sample of pre-calibrated fluid intended for use in such an analyzer.

 Significant progress has been made in the area of automating the count of blood cells in a serum sample. The best known instrument for making blood counts is the Coulter counter in which blood cells pass through an orifice in a single line and are detected by the way they modify the electrical properties of 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 cells, etc. which may be in a continuous flowing current of a sample of donated blood. Thus, when it is desired to obtain information relating to the multiple globules of this type, the normal practice of the laboratories to obtain them consists in preparing a microscope slide with the globules fixed in an image plane and in employing a human operator. or a shape recognition machine to statistically count significant numbers of globules by observing the globules carried one by one under a microscope by the coverslip. On this subject, reference is made to US Pat. Nos. 4,175,860 and No. 4,199,748.

 Various attempts have been made in recent years to allow optical analysis of particles flowing in a direct current.

For example, Kay et al. Described in the "Journal of
Histochemistry and Cytochemistry ", volume 27, page 329 (1979) a device which comprises an orifice of the Coulter type for displacing the globules in a single file, the globules being reproduced magnified on a" Vidicon "tube. Similarly, Kachel and others have described in the "Journal of Histochemistry and
Cytochemistry ", volume 27, page 335, a device for moving the globules in a single file through a microscope area in which they are photographed. Although these researchers have advanced the automation of particle analysis in a single file-, no successful work that would allow automatic particle analysis in a direct current without the need to dispose of the particles in a current in a single file has been published. subject, to the work "Flow Cytometry and Sorting" by Melaned et al., John
Wiley & Sons, 1979, chapter 1; reference should also be made to US Pat. No. 3,819,270.

 According to the present invention, there is provided a method for calibrating a continuous flow analyzer which comprises a flow chamber or cell, a microscope arranged to be able to be focused on an image observation zone of the chamber or cell with circulation and sensor means for capturing fixed raster images of the particles which are in the image observation zone. The process involves injecting a sample of calibration particles into the circulation cylinder. The particles have predetermined parameters. A series of fixed raster images of the particles contained in the image observation zone are formed by the image sensor means.

The images are processed to calculate the parameters. The analyzer is then calibrated based on the predetermined parameters and the calculated parameters.

 The present invention also relates to a sample of pre-calibrated fluid designed to be used in a continuous flow analysis system. The pre-calibrated fluid sample includes a physical mixture of a series of calibration particles and a biological fluid sample containing microscopic particles. The calibration particles have predetermined parameters. The biological fluid sample must be analyzed by the continuous flow analysis system.

Other characteristics of the invention will appear on reading the description which follows and on examining the appended 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 vessel or circulation chamber of the apparatus of FIG. 1;
Fig. 3 is a sectional view of the apparatus of the
Fig. 2, taken along the plane of line 3, 3 of the
Fig. 2;
Fig. 4 is a schematic representation of the electronic processor used in the apparatus of FIG. 1.

 Reference will now be made in more detail to the drawings and, in particular, to FIG. 1 on which there is shown an apparatus which comprises a body 10 which contains a circulation chamber or vessel provided with an inlet orifice 12 for a sample of fluid, such as blood or urine, and a outlet port 14, between which extends a passage which passes through an image observation zone 18.

The passage 16 comprises an inlet orifice provided with a conduit 20 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 intended to contain the fluid sample to be analyzed.

 The cross-sectional area of the passage 16 gradually decreases from the inlet 12 in the direction of the image viewing area 18. The thickness or depth of the passage also decreases from the inlet 12 to the image viewing area 18. The width decreases from the inlet 12 and then increases sharply to the image observation area 18. Thus, as shown in Figs. 2 and 3, passage 16 has a width and a depth of about 5000 sm at the inlet 12, a width and a depth of about 500 r, in the middle 28 and a depth of 100 rm with a width greater than 5000 m 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 size being many times greater than the width of the largest particles. , for example more than a hundred times the width of the largest cell. However, since the passage is formed in the manner described above, the current flowing through the opening 12 is limited to a stable flow path with minimum shear in the examination area. cross-sectional area with minimum shear is not much larger than the minimum cross-sectional area of the particles so that the particles are aligned in the stream with their minimum cross-sectional area oriented transversely to the direction of flow and their maximum cross-sectional area oriented parallel to the width (ie visible in the plane of Fig. 2). The expression "minimal shear" is used here in the sense of "minimal velocity gradient" so that a particle which moves in the current tends to align with the direction of the current exactly like a log floating on a river aligns itself with the direction of flow, when there is a gradient of flow. The flow characteristics in passage 16 can be controlled by adjusting the pressure of the fluid prevailing in the containers 22 and 26 either automatically or by adjusting their static height.

A microscope 30 is developed on the examination zone 18 and the examination zone 18 is lit from below by a stroboscope 32 which is preferably a Model 3018 stroboscope from the company US Scientific Instrunent Corporation containing a 2UP lamp. 1.5. The output image of the microscope is focused on a camera 34 with charge transfer devices model nO TC1160BD manufactured by the company RCA
Corporation. The output signal from the charge transfer device camera 34 is converted to a series of fixed raster images and suitable electronic processors are used to evaluate these images. One processor that can be used for this purpose is the processor sold under the designation Image Analysis System Model C-1285 by Hamamatsu Systems, Inc. Waltham, Massachusetts, USA. Preferably, 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 collector 40 of raster images is preferably the image collector model FG08, 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 multiple bus 44 of the processing unit 46 which is preferably a computer Intel 80/20. The multiple bus 44 is also coupled to a 48K selective access memory (48 × 1024 bytes) sold by the company Electronic Solutions Inc.

and to a selective access memory 50 of 16K (16 × 1024 bytes) with two accesses, model RM 117 from the company Data Cube Corporation. The output signal from the video regeneration memory 42 is also transmitted to a color control television screen 52 which can be used to provide video images enhanced by digital processing of the individual still images for examination by an operator. - human writer.

The second output of the dual-access selective access memory 50 is connected to a multiple bus 54 which is connected to a processing unit 56 from the company Applied
Micro devices, with a memory with selective access 58 of 48K of the company Electronic Solutions, Inc., and with a removable memory constituted by a control unit 60 of floppy disks, such as the model 8/8 of the company Advanced Micro Devices and two diskette memories 62 of the Shugart company.

A large variety of programs can be used 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 Hamamatsu System 1285 programming. Preferably, however, the progression is carried out as follows:
The tasks are first divided into those which must fit each pixel of a given image and those which address only a small subset of the total. Since a great deal of time is devoted to the first category of tasks, these are programmed in assembly language in the interface processor 45 (the Intel 80/20 processor of FIG. 4). The results of these operations are then transferred to the host computer 56 via the random access memory 50 with two ports. As for the host computer, almost all of the programming is carried out more conveniently in an advanced language, such as Pascal (BASIC or FORTRAN could, in principle, also be used). The types of task that are performed in assembly language include gray scale transformations, convolutions and calculations of gray scale histograms. The types of tasks performed by the host include the general control of other devices, the identification and segmentation of objects of interest which are in the visual field, the calculation of parameters associated with the objects thus found and the formatting the output signals representing the results. Another way of considering the division of tasks performed in this way is to consider that the tasks which must be performed at speeds higher than that of a human operator are performed in assembly language . Tasks which are complicated or which can be executed at a speed lower than the maximum speed can be programmed in advanced language. Objects are found in a visual field mainly by setting a gray scale window function for values that are known to be characteristic of the desired object. These values can be established from prior knowledge or using well-known histogram techniques. When a pixel belonging to an object has been located in the visual field, an edge-tracing program is called to trace the outline of the entire object associated with this pixel. Once the edge has been found, many useful parameters, such as position, area, 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. Definitions of classification of morphologies of cells or cells are established by experienced observers. These definitions are then used as the basis of the chosen algorithms. The accuracy of the process is determined by comparing the machine results to those of experienced observers examining the same samples. The output signals representing the results can be programmed in any of several formats. Histograms, line plots and summary tables can be produced on demand to meet specific needs.

 It can thus be seen that, in the circulation vessel or chamber 10, the particle current which is in the image observation zone 18 is two-dimensional. Therefore, multiple particles can be examined in a single field and different particles can be distinguished optically, which has a number of important advantages. For example, two cells or globules that flow together can be recognized optically as such while a Coulter counter could recognize them as a single globule of double size. In addition, fixed raster images can be emphasized using the digital enhancement techniques that have been developed for images transmitted by satellites, and individual images can be analyzed to obtain data on individual cells or cells. In the case of a blood sample, information such as size, cross-sectional area, shape (circular blood cell, target blood cell, hematofalciform, etc.), optical density, hemoglobin content, can be obtained. base of a globule, etc ... Not only is it possible to analyze and optically sort individual globules or cells in this way but, moreover, when the globules are analyzed and sorted in this way, it is possible individually count the different types of blood cells to give, automatically and in a single pass, the number of normal red blood cells per unit of sample volume, the number of targeted red blood cells es per cubic centimeter of sample, the number of red sickle cells per cubic centimeter of sample, the number of white blood cells, the number of platelets, etc ... per cubic centimeter of sample.

 Once a series of fixed raster images have been prepared in digital form, one can obtain a very wide variety of very elaborate information on the particles contained in the series of images depending on the complexity of the hardware and software. computer that can be used for image analysis.

 Preferably, the information obtained from the fixed raster images is combined to provide composite information which reflects the content of multiple fixed raster images and / or predetermined reference images and the composite information thus obtained can be used in various ways. . Thus, in simple systems, information can be output to a printer, for example, to bring to the attention of a hematologist the results of composite measurements performed on a blood sample.

In more complex systems, composite measurements can be used by process control, for example, to control the pressure in a homogenizer, the temperature in a crystallizer, or the rate of delivery of nutrients to a microbial culture in the event that the system controls the size or number of the particles.

 Thus, it can be noted that the apparatus can be used for the analysis of a wide variety of particles which move in a current, as well as biological particles, such as the globules in the blood or the cells, bacteria, cylinders and crystals in the urine as particles in gas analyzers, etc., and the output signals representing the results of these measurements can be used for process control, for example the delivery of nutrients into a stream containing microorganisms as mentioned above, controlling the growth of polymers and crystals, etc.

 To ensure the precision and accuracy of the information obtained by means of the apparatus described with reference to FIG. 1, it is necessary to calibrate the device from time to time. According to the method of the present invention, calibration particles having predetermined characteristics, such as the size, shape, color, intensity and volume of each of the calibration particles, are injected into the flow cell. In addition, several different types of particles having different predetermined characteristics can be injected into the flow cell at the same time. A series of fixed raster images of the particles which are in the image observation zone 18 are recorded by the image sensor means34. The images are processed to calculate these characteristics. On the basis of the data which are thus obtained by processing images, that is to say calculated characteristics, and on the basis of a priori knowledge of what these characteristics are, we can standardize the continuous flow analysis system to correct any errors . The errors can come from a certain number of sources and can be due, in particular, without this list being limiting, to the accumulation of dust, to the aging of the lamp, etc.

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

 First, calibration particles can be used to normalize the optical system for measuring dimensions. For example, if the electronic processor 36 determines that a spherical particle with a diameter of 20 sm has only 19.0 ssm, a correction factor of 5% must be applied to all the measurements (assuming that the error is linear over the entire measurement interval).

 Second, the calibration particles can be used to determine the orientation of the particles in the stream. As previously indicated, a particle flowing through the flow cell 10 is oriented approximately with its maximum cross-sectional area parallel to the width of the tank and with its minimum cross-sectional area transverse to the direction of flow. Therefore, a disc-shaped particle, such as a red blood cell, moves itself in the current with the plane of the disk approximately parallel to the plane of the width and length of the cell. A particle with a particular geometric shape can be used which is passed through the flow cell 10. The expected orientation of the calibration particle can be used to confirm the correct orientation of the particles in the sample. fluid. The correct orientation of the particles is the result of laminar flow, i.e. there is no turbulence in the stream.

 Third, the calibration particles can be used as markers to indicate the position of the fluid sample stream. For example, if the fluid sample is a spinal fluid and the particles are bacteria, the number of particles formed by bacteria will be extremely small. Thus, the operator and the system will see many images without particles. To assure the system and operator that the fluid sample is actually passing through the visual field, calibration particles can be added to the sample to provide an indication of the position of the fluid sample stream.

 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 the calibration particle provided that it can be detected by the analysis system as being different from the particles contained in the fluid sample.

 Preferably, the calibration particles are injected at a known rate. The calibration particles can be injected independently into the flow cell 10 to calibrate the flow-through analysis system or the calibration particles can also be injected into the flow cell 10 along with the fluid sample biological, such as blood or urine, flows through the flow cell 10. In the latter method of use, one can use the known characteristics of the calibration particles to measure the same type of characteristics, although this is then the measurement of unknown characteristics, microscopic particles contained in the sample of biological fluid. In addition, the density of the population of calibration particles can be used to calculate the density of the population. particles contained in the fluid sample.

 It should be emphasized that the calibration particles can be mixed with the sample of biological fluid containing microscopic particles before injecting the entire physical mixture into the flow cell 10.

 Examples of the types of calibration particles that can be injected into the flow cell 10 and their uses are given below.

Example nO I
Calibration particles made up of Fluoresbrite (microcrystalline) carboxylated microspheres which have a spherical shape of 4.5 + 0.1 ssm in diameter, yellowish-orange in color, have the same size as the typical red blood cells. and, thus, they can be used to serve as standards for measuring the size of red blood cells. Calibration particles can also be counted independently of normal blood cells to provide a volumetric calibration to help determine the concentration of blood cells.

Example No. II
It is possible to use calibration particles constituted by beads of dyed polystyrene latex 5.5 ssm in diameter, of spherical shape and of blue color, injecting them into the flow cell 10 to normalize the dimensions and the colors of the system. and they can also be used to determine the volume of the fluid sample which is examined if the pearl concentration per unit volume of the sample is known. In addition, bead distribution can be used to indicate the position and distribution of the sample in the stream flowing through the analyzer.

Example No. III
Ambrosia pollen particles are used which have a diameter of approximately 20 m, and which can be identified by their size, optical density and edge characteristics with a vaginal sample taken by aspiration to carry out an exfoliative cytology examination. in the flow-through analyzer so that you can locate the position of the sample stream and help determine the size and number of epithelial cells.

Example No. IV
Red blood cells of approximately 5.0 to 6.0 am diameter are used which are identified by their shape and optical density in a yeast sample to verify the correct orientation of cells in the analyzer. continuous flow and to measure the yeast cell population.

Example nO V
Red dyed polystyrene latex beads with a diameter of 1.0 t 0.1 ssm and a spherical shape are used in the examination of spinal fluids to determine the presence of bacteria and to serve as indicator particles in a fluid which also contains few particles.

Example NO VI
Red blood cells from birds are used, which have an elliptical cross section, the major axis of which is approximately 7.0 to 9.0 ssm in a population of human lymphocytes, which can be differentiated by shape , optical density and size, to determine the flow characteristics and size of lymphocytes.

 It is thus found that by injecting particles having predetermined characteristics, with regard for example to their dimensions, their volume, their shape and their spectral characteristics, into the continuous flow analyzer, it is possible to standardize the analyzer and to order it. the operation. We can compensate and correct the effects of aging and normal wear and tear of the equipment, such as the accumulation of dust on the microscope objective, electronic noise, etc. inside the system. analysis, whenever the system needs to be standardized. In addition, the calibration particles can be injected at the same time as the unknown biological sample is injected to measure the microscopic particles of the biological sample by comparing them to a reference standard.

Claims (19)

1 - A sample of pre-calibrated fluid designed to be used in an image analysis system, this sample being characterized in that it comprises a series of a type of calibration particles, these particles having a characteristic predetermined; a sample of biological fluid containing microscopic particles with unknown characteristics, which must be analyzed by the system; l Calibration particles and the Fiant biological fluid sample mixed into a physical mixture. 2 - Fluid sample according to claim 1, characterized in that the> calibration articlles have a particular characteristic as regards their dimensions, their volume, their shape, their color or their intensity. 3 - Fluid sample according to claim 2, characterized in that it comprises several types of calibration particles. 4 - Fluid sample according to claim 1, characterized in that the calibration particles can be separated from the microscopic particles by the system. 5 - Fluid sample according to claim 4, characterized in that the microscopic particles are blood cells. 6 - Fluid sample according to claim 5, characterized in that the biological fluid sample is blood. 7 - Fluid sample according to claim 5, characterized in that the biological fluid sample is urine. 8 - Fluid sample according to claim 4, characterized in that the calibration particles have a size between 0.5 and 50 gm. 9 - Fluid sample according to claim 1, characterized in that the proportions of the calibration particles and of biological fluid sample contained in the physical mixture are known. 10 - A method for calibrating a continuous flow analyzer, said analyzer comprising a flow cell (10), a microscope (30) arranged so as to be developed on the observation area (18) image of the tank and image sensor means (34) for capturing approximately fixed raster images of the particles contained in the image observation zone, this method being characterized in that it consists in: injecting a sample of 'a type of calibration particles in the flow cell (10), these particles having a predetermined parameter; forming a series of fixed raster images of the particles contained in the image observation zone by means of the image sensing means; processing the images to calculate the parameter; and calibrating the analyzer based on the predetermined parameter and the calculated parameter. 11 - A method according to claim 10, characterized in that the injection step further comprises injecting the sample at a known rate. 12 - Method according to claim 10, characterized in that it further comprises the steps which consist in flowing a sample of biological fluid containing microscopic particles through the flow cell (10), and in measuring the same microscopic particle parameter as the predetermined calibration particle parameter. 13 - Process according to claim 12, characterized in that the flow step and the injection step are carried out simultaneously. 14 - Method according to one of claims 11 and 13, characterized in that the predetermined parameter is the volume, size, shape, color or intensity of each calibration particle. 15 - Method according to claim 14, characterized in that the predetermined parameter is also the population density of the calibration particles. 16 - Process according to claim 13, characterized in that the sample of biological fluid is urine. 17 - Process according to claim 13, characterized in that the sample of biological fluid is blood. 18 - Process according to claim 10, characterized in that the particles have a size between 0.5 and 50 ssm. 19 - Process according to claim 10, characterized in that the injection step consists in injecting several types of calibration particles.