Process And Apparatus For Counting Biological Particles

Abstract

A method and apparatus for analyzing with a high degree of reliability the number of red cells, white cells and platelets in a sample of blood, as well as the hematocrit and average volume of the red blood cells. Two samples are successively placed in the trajectory of a laser light beam so as to obtain in the focal plane of the optical system the luminous flux diffracted by the various particles. In one sample the red blood cells have been spherized and in the other sample the red blood cells have been hemolyzed. The focal plane contains photosensitive areas which correspond to the directions which form, with the initial direction of the light beam, angles which fall between: s.sub.1 = (1.5 to 2.5) .lambda./.pi.(ad) And s.sub.2 = (3 to 5)s.sub.1 (1) s.sub.1 ' = 0.5.lambda./.pi.(ad) and s.sub.2 ' = 3s.sub.1 ' (2) where .lambda. is the wavelength of the light used, ad is the average diameter of the particles to be analyzed, and s.sub.1, s.sub.2, s.sub.1 ', and s.sub.2 " are expressed in radians. Known electronic means are connected to the photosensitive areas and permit the direct display of the analysis results.

Description

FIELD OF THE INVENTION

The present invention concerns a new process, as well as an apparatus for performing said process, to permit counting of biological particles suspended in a liquid medium and particularly to allow direct and instantaneous determination of the quantitative composition of the formed elements in the blood.

BACKGROUND OF THE INVENTION

As we know, the blood contains essentially three types of cells, namely, red blood cells, white blood cells and platelets. It is particularly important to know the concentration of these elements in order to facilitate many diagnoses of human illnesses. It is also interesting to know the hematocrit as well, i.e., the volume occupied by the red blood cells in the blood volume.

Several methods and devices are currently used to perform qualitative and quantitative measurements of the specific elements of blood.

There is, for example, the method known as electronic counting where the biological particles suspended in an electrolyte circulate between two electrodes (positive and negative), passing through a micro-orifice which is itself submerged in the electrolyte. When a particle blocks the orifice it results in an increase in resistance which is recorded and counted. This technique can be used for counting white and red cells; however, in order to determine the number of platelets, it is necessary to separate the red cells beforehand, for example, by centrifuging or sedimentation. These operations are long, delicate and prone to numerous errors as well as the serious risk of loss of platelets during the separation process mentioned above.

Another classical method, called optical counting, consists in measuring the diffusion produced by the blood particles which are examined one by one in an irradiated suspension volume. The errors in these methods of counting are high because of the background noise in the apparatus caused by the presence of the red cells. The electronic discriminations associated with this apparatus lead to numerous errors in the final count. In addition, the arrangement requires frequent calibration and constitutes a very expensive assembly for the user.

It has also been proposed to measure the diameters and numbers of fine particles (with diameters of 2-100 microns, for example) by using an optical method in which the light diffracted by the particles and coming from a source which may be a laser, for example, is eventually transformed into electrical energy (see for example: E. N. Leith, Photographic Science and Engineering, March-April 1962, pages 75-80; H. Thiry, Journal of Photographic Science, Vol. 11, page 69-77 (1963); J. Cornillault, Applied Optics, page 265-268, February 1972; H. Stark, Applied Optics, page 333-337, February 1971, etc.). Such methods, based on the diffraction of light, have also been suggested prior to the cited articles for the counting of certain blood elements. Thus U.S. Pat. No. 2,769,365 (granted Nov. 6, 1956) and 2,969,708 (granted Jan. 31, 1961) have described processes and apparatus of a rather complex nature, based on the study of the light diffused by certain blood particles at specific angles. However, the interest in these processes is limited because when they are applied to the counting of biological particles, they have thus far only been aboe to be used for counting the red blood cells, and possibly to measure the average cell volume, without being able to determine directly the hematocrit or to count the platelets and the white cells.

Hence, we are faced today with the problem of being able to have at our disposal a reliable technical method which would supply directly the parameters corresponding to the essential blood elements and would have the economic feature of being a simple and less costly apparatus that would be at the disposal of the largest possible number of laboratories and doctors.

SUMMARY OF THE INVENTION

The invention provides a simple solution and a less expensive approach to the problem. Essentially, it makes it possible for the first time to determine rapidly from a very small blood sample the quantitative proportions of the principal elments making up the blood, i.e., red blood cells, white blood cells and platelets, as well as the hematocrit and the average cll volume; the latter can be accomplished without the need to separate the red cells and the platelets, as is done in the known methods of electronic counting, and without having to resort to auxiliary determinations, which therefore eliminates the principal sources of error encountered in the known devices currently in use. Moreover, this direct determination of the principal hematological parameters of the blood is acquired with the aid of a device which is easy to use and has a less complex structure than the arrangements which are currently employed for analyzing the blood.

The process according to the invention is based on the well known principle, described below, of the diffraction of light by the microparticles of the blood and the analysis of the spatial frequency spectrum of the differences in the index of the sample which correspond to different particles of blood. However, although the counting procedures used thus far which employ this principle have not been able to count more than one part of the formed elements of the blood, it has been found that by a series of specific adaptations and operational modes, appropriately combined, it is possible to achieve directly the determination of all the investigated parameters and thus to satisfy the pressing need of the market for system which is easy to operate and yields reproducible results.

The new process is essentially charcterized by the fact that two blood samples to be analyzed, in one of which the red cells have been hemolyzed and in the other of which the cells have been spherized, are placed in the path of a beam of light emerging from a convergent optical system powered by a source of cohernet and monochromatic light so as to obtain in he focal plane of the optical system at least two photosensitive zones which receive the luminous flux diffracted by the particles, said flux corresponding to the directions which form angles with the initial direction of the beam which are located respectively between:

s.sub.1 = (1.5 to 2.5) .lambda./.pi.(ad)

and

s.sub.2 = (3 to 5)s.sub.1 ( 1)

s.sub.1 ' = 0.5.lambda./.pi.(ad)

and

s.sub.2 ' = 3s.sub.1 ' (2)

(.lambda. is the wavelength of the light which is used; ad represents the average diameter of the particles to be analyzed; s.sub.1, s.sub.2, s.sub.1 ' and s.sub.2 ' are experssed in radians); the light which is received by these zones is transformed into electrical signals, and additional electrical means of known type make it possible to determine directly and instantaneously the analytical results pertaining to the number of platelets and white cells as well as the number of red cells, the hematocrit and the average cell volume.

We know that 1 mm.sup.3 of human blood contains approximately 5 million red blood cells, 8,000 white blood cells and 300,000 platelets; the volume occupied by the red cells (or the hematocrit) amounts to approximately 45 percent of the total blood volume. The size of the particles is about 3 microns for the platelets, 6 microns for the red blood cells and 12 microns for the white cells.

The number of white cells and platelets is much lower than that of the red cells, so that it is necessary, in order to count these platelets and white cells, to deprive the red cells of the hemoglobin which they contian in advance (transforming them into "Rollet's stromata") so that they will lose their diffracting power. This result is achieved in known fashion by placing the blood sample to be analyzed in a hemolyzing fluid such as, for example, ammonium oxalate at a concentration of 1 g/liter or any other agent with the same function (see the work by E. Ponder, "Hemolysis and Related Phenomena," Gruen and Stratton, New York, N.Y.). In this fashion, it is possible to count the white blood cells and the platelets together with a high degree of accuracy.

In addition, in order to be able to count the red cells, it is necessary to spherize the latter by placing the blood sample in a solution of the appropriate liquid, such as, for example, the solution called "Gowers" which is based on sodium sulfate and acetic acid, or any other appropriate spherizing agent (cf. E. Ponder, cited above).

This is why it has been proposed in the above description of the process of the invention to subject two blood samples successively to analysis by means of the diffraction of light at a series of angles in accordance with equations (1) and (2) in order to obtain a distribution of the light representing the spectrum of spatial frequencies of all formed elements of the blood around the focus of the optical system. These samples can be placed successively in the same receptacle, as for example, a cell or, as will be explained below, in two different cells which are placed in the focal plane of the optical system. According to an interesting variation of the process of the invention, it is also possible to employ a single blood sample in a single cell and thus obtain directly a total result for the number of formed elements, providing a circuit for circulation of the sample in the cell so that the sample receives adequate amounts of the spherizing agent and then, introduced after a specific time, a supplementary quantity of this agent in order to produce in known fashion the hemolysis of the spherized red cells.

The series of diffraction angles in accordance with equations (1) and (2) above have the following correspondence: the first to the counting zones of the white blood cells, platelets and red cells (all of the particles, whose variations in diameter around an average value remain low) and the second to the zone of evaluation of the total volume occupied by the particles. The determination of a certain interval of diffraction angles has admittedly been mentioned before (U.S. Pat. No. 2,769,365 cited above, containing data from which one can derive s.sub.1 = 2.5 .lambda./.pi. (ad) and s.sub.2 = (3 to 4)s.sub.1) but it has been applied only to the determination of the red cells; the applicant stated with surprise that in selecting the intervals according to ranges (1) and (2) mentioned above he was able to create counting zones corresponding to all of the blood cells as well as the determination of the hematocrit.

In practice, the process according to the invention may be carried out in an apparatus characterized as follows:

a. a source of coherent and monochromatic light, preferably consisting of a continuous laser,

b. a convergent optical system mounted in front of said source,

c. at least one cell which serves as a container for the blood to be analyzed, said cell being placed along the trajectory of the light beam,

d. a screen placed in the focal plane of the optical system and containing at least two windows whose internal and external edges correspond to the diffraction angles which are essentially equal respectively to:

s.sub.1 = (1.5 to 2.5) .lambda./.pi. (ad)

and

s.sub.2 = (3 to 5)s.sub.1 ( 1)

s.sub.1 ' = 0.5 .lambda./.pi. (ad)

and

s.sub.2 ' = 3 s.sub.1 ' (2)

(.lambda. and ad having the same definition as given above.)

e. means of measuring the diffracted luminous fluxes which pass through said windows; and

f. means of transforming the fluxes into electrical signals and to display directly the counting results.

The laser serves particularly well as a light generator because its spatial coherence makes it possible to produce a beam which has very little natural diffraction. Moreover, its emission is strictly monochromatic and its power is high, making it possible to use it as an output amplifier.

The optical system, of the enlarger type, is preferably equipped at its internal focus with a spatial filter composed of a screen which is perforated at its center, and filters out the laser rays which are not parallel with teh axis, and therefore serves to purify the light beam by cutting down the background noise cause by parasitic diffractions or possible defects in the coherence of the beam.

The cell containing the sample to be analyzed may be composed of two transparent sheets with parallel faces. In the case described above, where the red blood cells are successively spherized and then hemolyzed within the same sample, the cell may be a receptacle with transparent walls supplied with spherizing liquid. The cell is preferably mounted between the exterior lens of the optical system and the focal plane of this system. In this position, in which the distance between the particles to be analyzed and the focal plane can be varied, the diffraction image varies homothetically relatively to its center, with the homothetic ratio being equal to the ratio of the distances. According to one version of the procedure and the apparatus according to the invention, the system may be equipped with two cells which are mounted between the exterior lens of the optical system and the focal plane of this system.

The screen which is placed in the focal plane is supplied with at least two windows with internal and external radii which are respectively calculated according to formulas (1) and (2) above. In this embodiment, the cell (or, according to another version, the two cells) containing the sample is displaced during measurement relative to the focal plane of the optical system but one cannot correct the measurement errors caused by the presence of stromata whose diffraction is very low but nevertheless perceptible. Theoretically, it is therefore preferable when selecting the diffraction angles between s.sub.1 = 1.5 .lambda./.pi.(ad) and s.sub.2 = 3 s.sub.1, to provide three windows, two of which correspond to the measurement zones for the red cells and the hematocrit (in the case where the red cells are spherized) or the platelets and the white cells (in the case where the red cells are hemolyzed), with the third window therefore serving to measure the perturbation caused by the stromata. It is likewise possible to provide four windows in the screen, notably in the case where the diffraction angle varies between s.sub.1 = 2.5 .lambda./.pi. (ad) and s.sub.2 = 3s.sub.1.

The means of measuring the luminous fluxes which pass through each window are mounted behind the latter and may be composed of photosensitive cells which transform the energy which they receive into electrical signals which are fed into an electronic apparatus of known type which acts as the calculator and make it possible to display directly on a panel the numerical results of the analysis.

The invention will be explained more clearly by a description using a sample embodiment of the aparatus as shown in FIGS. 1 and 2 of the attached drawing:

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a simplified diagram of the apparatus.

FIG. 2 is a front view of the screen with windows.

DESCRIPTION OF A PREFERRED EMBODIMENT

The light source is represented by a continuous laser 1 provided with mirrors 2 and 3 (the latter being non-reflecting). Ahead of generator 1 are two lenses 4 and 5 which magnify the laser beam 6. A screen 7, perforated at its center by an opening 8, is located at the focus of lens 4 and constitutes a spatial filter. A second shield 9, provided with a central aperture 10 behind which is a light trap 11, is equipped in the non-limiting fashion of the embodiment shown here -- with four windows 12, 13, 14 and 15 behind each of which are vacuum photodiodes 16, 17, 18 and 19. Each window is theoretically annular in shape but practically speaking the configuration need not be very strict because the apparatus according to the invention does not perform absolute determination; acting as a trap, it must be calibrated and furnishes results which are compared to reference measurements.

The blood sample to be analyzed is placed in cell 20 between lens 5 and the focal plane F of the optical system at a distance D from the latter. The radii of the internal and external edges of the window 12 are equal to r.sub.1 and r.sub.2 such that r.sub.1 = Ds.sub.1 and r.sub.2 = Ds.sub.2, s.sub.1 and s.sub.2 corresponding to the definition (1) given above with ad = 6 microns (average diameter of the spherized red blood cells). Window 13 has radii r.sub.3 = Ds.sub.1 and r.sub.4 = Ds.sub.2, with s.sub.1 and s.sub.2 corresponding to definition (1) with ad = 3 microns (average diameter of the platelets). Window 14 has radii r.sub.5 = Ds.sub.1 and r.sub.6 = Ds.sub.2, s.sub.1 and s.sub.2 corresponding to the definition (1) given above, with ad = 12 microns (average diameter of the white cells). Finally, window 15 has radii r.sub.7 = Ds.sub.1 ' and r.sub.8 = Ds'.sub.2, with s'.sub.1 and s'.sub.2 corresponding to the definition (2) given above with ad = 6 microns.

During operation, the rays in beam 6 undergo diffraction while passing through the blood and strike screen 9 within the annular zones delimited by radii r.sub.1 -r.sub.2, r.sub.3 -r.sub.4, r.sub.5 -r.sub.6, and r.sub.7 -r.sub.8 of each window. The luminous flux strikes photodiodes 16, 17, 18 and 19 which produce potentials S.sub.p (platelets), S.sub.b (white cells), S.sub.r (red cells) and S.sub.H (hematocrit) with intensities proportional to the members of formed elements present in the blood to be analyzed. However, the potential S.sub.p, for example, depends not only on the number of platelets in the sample but also on the number of white cells and stromata. In order to obtain the number of platelets, it is necessary to correct S.sub.p using signals S.sub.b and S.sub.r. These corrections can be made automatically and instantaneously by injecting, for example, in know fashion, signals S.sub.p, S.sub.b, S.sub.r and S.sub.H, coming from the cited photodiodes, across resistance bridges; in this case, the result can be read at the output in the form of a voltage. It is also possible to utilize in advantageous fashion analog or numerical modules which provide the same result. These methods of displaying the results do not themselves characterize an original characteristic of the process of the invention and therefore have not been shown in the attached drawing.

The counting of the elements corresponding to the said potentials is obtained directly by the following formulas which are translated by the calculating modules previously described:

-- after hemolysis of the red cells:

N.sub.p = aS.sub.p - bS.sub.b - c + dS.sub.r (3)

N.sub.b = a'S.sub.b - b'S.sub.p - c' - d'S.sub.r (4)

(N.sub.p and N.sub.b represent the number of platelets and white cells per mm.sup.3 of blood)

S.sub.p and S.sub.b represent respectively the platelets and the white cells but they lead to a slight interaction between the two signals which is translated by the terms b and b'. These terms, as well as a and a' and c and c' (background noise of the apparatus) are determined by preliminary calibration. Terms d and d' correspond to the influence of the stromata, and can vary as a function of the degree of completeness of hemolysis.

-- after spherization of the red cells:

N.sub.r = eS.sub.r - f (5)

N.sub.r representing the number of red cells per mm.sup.3 of blood and factors e and f being determined as above by preliminary calibration. As we said previously, once the red cells have been spherized, the window corresponding to the measurement zone for the hematocrit will also supply the hematocrit parameter H according to the formula:

H = gS.sub.H - h (g and h determined by calibration) (6)

Finally, the average cell volume is obtained by the quotient:

V = gS.sub.H - h/eS.sub.r - f, (7)

In fact, it has been found, suprisingly enough, when the apparatus according to the invention was tested, that the window corresponding to the hematocrit and the one corresponding to the counting of the white cells essentially coincide. Practically speaking, it is therefore possible to use an apparatus such as the one described in the drawing and equipped with only three windows to obtain all five parameters: N.sub.p, N.sub.b, N.sub.r, H and V which characterize the blood which is being analyzed. These parameters may be recorded directly on a numerical volt-meter or displayed on the panel of an electronic computer.

The display of the results is instantaneous, with no need to allow for a delay in calculation.

As an example of the use of the procedure according to the invention with an apparatus equipped with four windows, one can obtain, by using as a wavelength of the laser light the value .lambda. = 0.6328 microns and as the angles of diffraction of formulas (1) and (2) with s.sub.1 = 2.5 .lambda./.pi. (ad), the internal radii as follows for the four windows:

Internal radius ______________________________________ Platelets (ad: 3 microns) 0.167 D White cells (ad: 12 microns) 0.040 D Red cells (ad: 6 microns) 0.092 D Hematocrit 0.022 D

(d, as stated above, is the distance between the cell and the focal plane).

The external radii of the windows may be assumed to be equal to 3 (or 3 to 5) times the internal radii as indicated in equations (1) and (2).

In this embodiment, zones S.sub.b and S.sub.p on the one hand and zones S.sub.H and S.sub.r on the other hand are homothetic. It is therefore sufficient to have two measurement zones in the focal plane and to shift the cell when the blood is added. If the first position corresponds to a distance D, the zones of internal radii 0.167 D and 0.040 D will serve to measure the platelets and the white cells and it will suffice to shift the sample cell through a distance of about D/1.8 to measure the red cells and the hematocrit with the same window.

Finally, in order to accomplish the version of the process and apparatus according to the invention in which two cells are used and only two windows, it will suffice to place the first cell (containing the hemolyzed blood) at distance D and the second (containing the spherized blood) at a distance of approximately D/1.8, with the windows having as their internal radii the values of 0.167 D and 0.040 D.

Thus, by using a simple apparatus and combining the various possibilities of analysis described above, an apparatus has been developed which provides the complete results necessary for a blood analysis with a high degree of reliability.

It will be obvious to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown in the drawings and described in the specification.

Claims

What is claimed is:

1. A process for the instantaneous counting of the various particles in suspension in a sample of blood and for determining the hematocrit and average red blood cell volume, comprising the steps of:

placing two samples successively into the trajectory of a light beam emerging from an optical system converging on a focal plane powered by a source of coherent and monochromatic light, one of said samples being a blood sample in which the red blood cells have been spherized and the other of which being a blood sample in which the red blood cells have been hemolyzed;

detecting and measuring the luminous flux diffracted by the particles at said focal plane between the angles formed with the initial direction of the light beam:

s.sub.1 = (1.5 to 2.5) [.lambda./.pi. (ad)]

and

s.sub.2 = (3 to 5)s.sub.1,

and

s'.sub.1 = 0.5.lambda./.pi.(ad)

and

s'.sub.2 = 3 s'.sub.1,

where .lambda. is the wavelength of the light used, ad is the average diameter of the particles being analyzed and s.sub.1, s.sub.2, s'.sub.1, and s'.sub.2 are expressed in radians; and

transforming said detected and measured luminous fluxes into electrical signals, correcting to eliminate known sources of error and displaying the results for the number of platelets, the number of white cells, the number of red cells, the hematocrit and the average red blood cell volume.

2. A process in accordance with claim 1 wherein said detecting and measuring step is accomplished at three photosensitive zones corresponding as follows: the first to the white cells and the hematocrit, the second to the platelets and the third to the red cells.

3. A process in accordance with claim 1 wherein said detecting and measuring step is accomplished at four photosensitive zones corresponding to the following parameters: white cells, platelets, red cells and hematocrit.

4. A process in accordance with claim 1 wherein said placing step is accomplished by placing a single blood sample for analysis in said light beam and supplementing said blood initially by a quantity of spherizing liquid sufficient to spherize the red cells and subsequently with a quantity of hemolyzing liquid sufficient to hemolyze the red cells to form stromata.

5. A process in accordance with claim 4 wherein said hemolyzing liquid is the same as said spherizing liquid.

6. An apparatus for the instantaneous counting of the various particles in suspension in a sample of blood and for determining the hematocrit and average red cell volume, comprising:

a. a source of coherent monochromatic light;

b. a convergent optical system mounted in front of said source;

c. at least one cell which serves as a receptacle for the blood to be analyzed, said cell being placed on the trajectory of the light beam;

d. a screen placed in the focal plane of the optical system and containing at least two windows whose internal edges and external edges correspond to the diffraction angles which are essentially equal respectively to the following:

s.sub.1 = (1.5 to 2.5) [.lambda./.pi. (ad)]

and

s.sub.2 = (3 to 5)s.sub.1, (1)

and

s'.sub.1 = [0.5 .lambda./.pi.(ad)]

and

s'.sub.2 = 3s'.sub.1, (2)

where .lambda. is the wavelength of the light used, ad is the averate diameter of the particles being analyzed, and s.sub.1, s.sub.2, s'.sub.1, and s'.sub.2 are expressed in radians;

e. means for measuring the diffracted light fluxes which pass through said windows; and

f. means for transforming the fluxes into electrical signals characteristic of each type of particle and for displaying directly the results thereof.

7. An apparatus in accordance with claim 6 wherein said light source is a continuous laser and wherein said optical system includes a spatial filter at the internal focus thereof.

8. An apparatus in accordance with claim 6 including two of said cells, in one of which the red blood cells are spherized and in the other of which the red blood cells are hemolyzed and wherein the number of windows in said screen is two.

9. An apparatus in accordance with claim 6 further including means for shifting the cell in the course of the measurement in a space between the outer lens of said optical system and the focal plane thereof.

10. An apparatus in accordance with claim 6 wherein said screen has exactly three windows, two of which have internal and external edges corresponding to angles of type s.sub.1 and s.sub.2 and the third having internal and external edges corresponding to angles of the type s'.sub.1 and s'.sub.2.

11. An apparatus in accordance with claim 6 wherein said screen has exactly four windows, three of which have internal and external edges corresponding to angles of type s.sub.1 and s.sub.2 and the fourth having internal and external edges corresponding to angles of the type s'.sub.1 and s'.sub.2.

12. An apparatus in accordance with claim 6 wherein said means for measuring the diffracted light fluxes comprise photosensitive cells mounted behind each window and wherein each of said cells is associated with said means for transforming the fluxes into electrical signals.