CA2083424A1 - Kit for the rapid counting of granulocytes, and process using said kit - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The invention relates to a kit for counting the granulocytes in human blood or any other biological fluid.
This kit is made up of the following components:
a) a first receptacle containing a determined quantity of a granulocyte diluting and labeling reagent containing an arbitrarily labeled specific antibody;
b) a second receptacle containing magnetic particles to which the same specific antibody, but unlabeled, is bound; and c) a rod, called a measuring rod, provided with a magnetized terminal part.
The invention further relates to a process using said kit.

Application: counting of granulocytes.

No Figure.

Description

The present invention relates to a kit for the rapid counting of granulocytes and to a process using said kit~
Polynuclear leukocytes, also called granulocytes, comprise 3 subpopulations: neutrophilic, basophilic and eosinophilic granulocytes.
Granulocytes, which are normally present in peripheral blood, can be observed in infectious pathological conditions in other biological fluids such as urine, cerebrospinal fluid, pleural fluid or the fluid originating from a bronchoalveolar lavage.
The normal average number of polynuclear leukocytes in blood can be modified under certain circumstances. An abnormal increase in the number of polynuclear leukocytes in blood may indicate a pathological situation, for example hypergranulocytosis caused by an infectious agent during hypereosinophilia or in myeloid leukemia. Conversely, an abnormal decrease in the number of polynuclear leukocytes (granulocytopenia) can be induced by drugs or chemotherapy treatments. Certain drugs can cause substantial modifications in the number of granulocytes; this is described for example by P. Tarallo and J.F. Guelfi in Examens de Laboratoires et Medicaments (Examinations of Laboratories and Drugs), ed. G. Siest et al., Expansion Scientifique, 1985, 163- 181. The undesired effects of these drugs have to be evaluated systematically and regularly by counting the number of blood cells (total count and differential count).
Thus one of the examinations most frequently performed in hematology is that of establishing the differential count.
For the erythrocytes and each of the leukocyte populations, the average values of the total cell counts are determined and these are compared with the (lower and upper) limiting values considered to be normal, which have been determined elsewhere.
By way of example, before a chemotherapy treatment is undertaken, and in the course of the treatment, total granulocyte counts are carried out in order to observe the patient's condition and adapt the treatment accordingly. In modern therapeutic practice, the chemotherapy is often administered to hospital day patients, so it is very useful for the doctor to have a kit for the rapid counting of granulocytes which easily produces a virtually instant - result.
Hitherto, total blood cell counts have been performed by microscopy or with the aid of automatic devices. For example, it is possible to use an electronic counter of the Coulter Counter type from Coultronics~, in which the measurement is based on the variation in the resistivity of the medium in which the cells are present. Other apparatuses, such as the Hematolog D~, use cytochemical measurements.
Knowledge of the cell surface antigens or markers has advanced enormously with the development of lymphocyte hybridization and the discovery of monoclonal antibodies by Koehler and Milstein (Nature, 1975, 256, 495-497).
By international convention, the surface markers of human leukocytes have been classified in differentiation groups or classes (CD) defined by the IUIS-WHO subcommittee, 1984, described in the Bulletin de l'Organisation Mondiale de la Santé, 1984, 62(5), 813-815, and regularly updated.
European patent application 311 492 describes an immunometric method of assaying the surface antigens characteristic of a cell population. It uses on the one hand a solid support to which are bound monoclonal antibodies directed against surface antigens of the cell population to be assayed, and on the other hand a solution containing other labeled monoclonal antibodies specific for the cell population to be assayed. This method effects, in a single step, the specific immobilization of the cell population to be assayed by immunocapture on a solid support and the recognition, by a labeled antibody, of a surface antigen of ~aid population.
Several of the Use Examples described in European pa-tent application 311 492 involve the assay of surface antigens carried by blood cells. It is found that the assay proper is always preceded by a step for separating the blood cells from the whole blood, and Example 5 studies the influence of the chosen method of separation (centrifugation or lysis of the erythrocytes) on the results of the assay.
In the same Example, the influence of the incubation time for the immunocapture and labeling of the cells on the observed results is also determined and it is shown that an analytically usable specific signal is obtained after a contact time of 10 minutes. According to the Examples described, however, it is found that the effective incubation time is never less than 20 minutes.
Example 15 of European patent application 311 492 describes the assay of the CD5 antigen of human T lymphocytes by using magnetic beads coated with anti- CD2 antibodies as a solid support for the immunocapture. In this Example, the mononuclear cells are also separated from the whole blood before the incubation step for the immunocapture and the assay. Furthermore, in said Examples, the incubation step alone takes 1 hour.
Example 11 describes the assay of the CD15 antigens carried by human granulocytes. The determination is effected on granulocytes originating from whole blood: in a first stage, the granulocytes are separated from the erythrocytes by mixing with a physiological medium used in perfusion (Plasmion~); then, after standing for 45 minutes, the suspension of leukocytes is removed from the surface of the liquid medium. This prior separation step takes more than one hour. The granulocytes are then immobilized in the wells of a microtiter plate by a monoclonal antibody specific for the CD45 antigen. This CD45 antigen is present on the cell membrane of all leukocytes. The anti-CD15 antibody, labeled with peroxidase, is used for the assay.

3~4 Furthermore, the various Examples described in European patent application 311 492 show the importance, in terms of the number of manipulations and the execution time, of the step for washing the solid support to which the cell S population to be assayed is bound.
Thus, despite the concern for speed expressed in said patent application, the total execution time of the assay is always long, i.e. less than or equal to l hour. In fact, the following steps are necessary for performing the assay:
- preparation of the sample to be assayed, if appropriate with separation of the blood cells from the whole blood;
- incubation for at least 10 minutes, preferably longer;
- washing of the solid support; and - in the case of enzymic labeling, developing of the enzymic activity (20 to 25 minutes with peroxidase, longer with other enzymes).
Altogether these operations are carried out by a person skilled in the art in a time of between 45 and 60 minutes.
Finally, expression of the results requires a standardization as a function of the chosen method of measurement, this standardization being carried out with a cell preparation calibrated beforehand in respect of antigens by the quantitative cytofluorimetry method.
According to the present invention, a granulocyte counting system based on an immunometric measurement has been found which satisfies the criteria of rapid diagnostic tests, in particular the simplicity of use, the speed of execution and the reliability of the results:
- the measurement of the granulocytes is carried out using the same antibody for the capture and labeling of the cells to be assayed, so the number of cells is measured directly;
- the measurement is carried out on a sample of whole blood or biological fluid, which avoids a long prior step for separation of the polynuclear leukocytes from the whole blood or biological fluid;
- the use of appropriate magnetic particles and the method s ~3~

chosen for the capture and washing of said particles carrying the cells to be measured permit simplification and shortening of the washing time; and - the possibility of very simply adding an internal standard can lead to direct expression of a measurement result.
According to the present invention, the antibody used for capturing and labeling the granulocytes, hereafter called a specific antibody, is a monoclonal antibody directed against a surface antigen specific to the granulocytes.
Thus said antigen is present on the surface of the granulocytes but not present on other cells; furthermore, this antigen is sufficiently abundant on the surface of the granulocytes to enable the latter to be both captured and labeled by the specific antibody.
The present invention relates to a kit for the rapid counting of the granulocytes in human blood or any other human biological fluid, normal or pathological, said kit comprising the following components:
a) a first receptacle containing a determined quantity of a granulocyte diluting and labeling reagent containing an arbitrarily labeled specific antibody;
b) a second receptacle containing magnetic particles to which the same specifia antibody, but unlabeled, is bound; and c) a rod, called a measuring rod, provided with a magnetized terminal part.
Advantageously, the kit according to the invention can also contain one or more of the following elements:
d) a device for removing a determined quantity of blood or biological fluid;
e) a device containing washing liquid;
f) means for developing the marker of the specific antibody -for example, in the case of a specific antibody labeled with an enzymic probe, one or more receptacles containing the developer for the enzyme, namely one or more solutions containing the substrate for the enzyme and, if appropriate, one or more reagents necessary for measuring the activity o 6 ~ r ~ 4 Ihe enzyme; and g) at least one rod, called a reference rod, which is coated on its terminal part with a determined quantity, different for each rod, either of anti- species polyclonal antibodies, said species being the animal species from which the specific antibody originates, or of specific antigen.
In another embodiment of the invention, the kit contains neither measuring rod nor reference rod, but its use involves a magnetized bar which, on application to the outer surface of the receptacle containing the capturing and labeling reagent, makes it possible to immobilize the magnetic particles on the inner surface of said receptacle.
If the kit does not contain a reference rod, it is possible to add thereto different reference samples each IS containing a known number of granulocytes, measured by counting with the aid of an automatic apparatus.
"Device for removing a de~ermined quantity of blood or biological fluid" is understood as meaning a syringe of determined volume, equipped with a needle, for taking a sample of venous blood, or a pricker to which a calibrated capillary tube has been attached, or else any appropriate device for removing a given volume of blood or biological fluid from an existing sample.
The magnetic particles used in the kit according to the present invention have variable shapes: spherical or non-spherical. Their largest size is preferably between 0.1 and ~m. By way of example, there may be mentioned polystyrene-coated magnetic particles such as the Estapor~
particles marketed by Rhone-Poulenc, whose sizes vary from 0.1 to 10 ~m with an average value of 0.8 ,um. There may also be mentioned multilayer composite magnetic particles such as the Dynabeads~ particles marketed by Dynal, which have a size of about 2 ,um.
A further possibility is to use particles prepared according to European patent 104 101 by the polymerization of acrylic derivatives, under gamma irradiation, in the presence of a magnetic material prepared by a known method, for example using the process described by M. Ronay in American Chemical Society Symposium Series, 1982, 200 Reprographic Technology, 553-576. These particle~ have a slze of between 1 and 10 ,um.
Particularly preferably, the magnetic particles used in the kit according to the invention have a size of between 0.5 and 4 ~m.
The binding of the monoclonal antibody to the magnetic 1() particles is effected by physical adsorption or covalent bonding. It is possible to use a process proposed by the manufacturers, if the particles are commercially available, or else the process described in European patent 104 101.
The receptacle containing the magnetic particles is prepared by a customary procedure; the magnetic particles to which the monoclonal antibodies are bound are placed in a phosphate buffer at a pH of about 7, which has been rendered isotonic by the addition of sodium chloride and contains proteins such as bovine serum album~n or fetal calf serum. A

2() small quantity of this solution is placed in the receptacle, after which the latter is lyophilized, stoppered and then stored at low temperature (below 5-C). Small quantity is understood as meaning a volume about 100 times smaller than the volume of the receptacle.
~5 The labeled specific antibody is obtained in conventional manner by any one of the labeling methods well known to those skilled in the art, such as radioisotopic labeling, enzymic labeling or labeling with a fluorescent agent.
~(l Granulocytes have sizes of about lO to 15 ~um and are of irregular shape; they carry on their surface numerous cell markers or antigens. Some of these, such as the CD~5 antigen, are present on all leukocytes; others, such as CDl5, are specific to granulocytes.
'5 For one particular embodiment of the immunometric assay according to the present invention, the CD15 cell antigen was ~3 ~ 4 ~nosen for capturing and labeling the granulocytes. This antigen is very abundant on the surface of the granulocytes.
Thus the capture and labeling of the granulocytes, with a view to determining the number of these cells, are effected by an anti-CD15 monoclonal antibody whose affinity for the antigen is greater than 108 M-l.
In one particular embodiment of the present invention, the monoclonal antibody used is an immunoglobulin of isotype IgM. Such an immunoglobulin molecule possesses 10 antibody sites, whereas an immunoglobulin molecule of isotype IgG
possesses only 2 antibody sites.
By virtue of the large number of binding sites they possess (5 times more than an immunoglobulin of isotype IgG), the anti-CD15 IgMs offer a greater capacity for recognizing the CD15 antigens present on the surface of the granulocytes, and hence for capturing said granulocytes. Furthermore, the anti-CD15 IgMs can carry more tracer molecules constituting the enzymic or radioisotopic probe, which gives them a very high capacity for labeling the CD15 antigens on tne granulocytes. The SMY 15a antibody marketed by BIOSYS is an immunoglobulin IgM which is very particularly suitable for performing the assay according to the invention.
It is also possible to use the 80H5 clone anti-CD15 antibody marketed by Immunotech.
Granulocyte diluting and labeling reagent is understood as meaning the reagent containing the anti- CD15 antibody -fluorescent or labeled with an enzymic probe or a radioisotopic probe - diluted in a saline buffer with a pH of about 7, for example a phosphate buffered saline, said buffer containing proteins such as bovine serum albumin or fetal calf serum.
The reagent is used in such a way that the concentration of the anti-CD15 antibody contained in this reagent is between 0.5 and 5 ,ug/ml.
Washing liquid is understood as meaning a conventional physiological liquid for washing, such as a saline buffer 9 ~ 4 with a pH of about 7, for example a phosphate buffer rendered isotonic by the addition of sodium chloride.
The expression "a fluorescent monoclonal antibody"
means that the antibody has been renclered fluorescent with an appropriate fluorochrome such as fluorescein isocyanate.
The expression "a monoclonal antibody labeled with a radioisotopic probe" means that the monoclonal antibody carries a radioactive isotope either on an element of its structure, for example the constituent tyrosine residues, or on an appropriate radical which has been attached thereto, said radioactive isotope making it possible to assay said monoclonal antibody by counting the radioactivity associated therewith.
The expression "a monoclonal antibody labeled with an enzymic probe" means that the monoclonal antibody is coupled wi-th an enzyme which, when associated with the use of appropriate reagents, permits a quantitative measurement of this monoclonal antibody.
The substrate and the reagents are chosen so that the final product of the reaction or the reaction sequence caused by the enzyme and involving these substances is:
- either a colored or fluorescent substance which diffuses into the liquid medium surrounding the cells and which is the subject either of the final spectrophotometric or, respectively, fluorimetric measurement, or of a visual evaluation, if appropriate by comparison with a range of standardized shades, - or an insoluble colored substance which deposits on the cells and the support to which they are bound, and which can be the subject either of a measurement by reflectance photometry, or of a visual evaluation, if appropriate by comparison with a range of standardized shades.
When using an antibody which has been rendered fluorescent, the fluorescence associated with the cells is read off directly on an appropriate apparatus.
When using a radioisotopic probe such as, for example, ~ 2a~

iodine 125, the radioactivity associated with the cells is counted in a gamma counter according to any appropriate modality, for example, after solubilization of the cells with an alkaline solution (for example a sodium hydroxide solution) and recovery of the solution containing the radioactivity with the aid of an absorbent buffer.
When using an enzymi~ probe on the monoclonal antibody, the appearance of a colored or fluorescent product is achieved by adding, to the solid support to which the cell population carrying the antigen to be assayed has been bound, a solution containing the substrate for the enzyme and one or more auxiliary reagents making it possible to obtain, as the final reaction product, either a colored product soluble in the medium, or an insoluble colored product, or a soluble fluorescent product, as explained above. The light signal originating from the samples treated in this way is then measured with the aid of equipment adapted to each case: a transmission photometer, reflectance photometer or fluorimeter, respectively. Alternatively, the coloration obtained can also be evaluated visually, if appropriate with the help of a range of standardized colored solutions.
If alkaline phosphatase is used as the enzymic probe, this enzyme is coupled with the monoclonal antibody by the method proposed by Boehringer Mannheim- Biochemica. The preferred substrates for this enzyme are paranitrophenyl phosphate for a spectrophotometric final reading, 4-methylumbelliferyl phosphate for a fluorimetric reading or 5-bromo-4-chloroindol-3-yl phosphate to give an insoluble colored reaction product. Likewise, ~-galactosidase can be used as the enzymic probe, in which case the preferred substrates will be orthonitrophenyl ~-D-galactopyranoside or 4- methylumbelliferyl ~-D-galactopyranoside.
Preferably, the monoclonal antibodies can be coupled with peroxidase. In this case, the coupling process is derived from that described by M. B . WILSON and P. K . NAKANE in Immunofluorescence and Related Staining Techniques, ed. W.

~napp, K. Kolubar and G. Wicks, Elsevier/North Holland, Amsterdam, 1978, p. 215- 224. The modifications introduced by comparison with the initial protocol for the preparation of the enzymic conjugate relate to the following points:
- molar ratio peroxidase/antibody equal to 3, as opposed to 2 in the protocol, and - less rigorous oxidation of the carbohydrate units of the peroxidase by virtue of a 33% reduction in the proposed sodium periodate concentration.
The reagents used for developing the peroxidase conjugated with the monoclonal antibodies contain hydrogen peroxide, which is the substrate for the enzyme, and an appropriate chromogen, for example orthophenylenediamine or 2,2'-azinobis(3-ethylthiazoline-6-sulfonic) acid (ABTS) for obtaining a colored final reaction product which is soluble in the medium, or else 3,3'-diaminobenzidine, 3-amino-9-ethylcarbazole or 4-chloro-~-naphthol for obtaining an insoluble final reaction product, or else parahydroxyphenylpropionic acid for obtaining a fluorescent final reaction product which is soluble in the medium.
Another embodiment of the invention is the use of monoclonal antibodies coupled with acetylcholinesterase.
The acetylcholinesterase is preferably coupled with the antibody using a process derived from that described in French patent no. 2 550 799, or a process which schematically comprises the preparation of fragments of the antibody by a known technique, the modification of the enzyme by reaction with an appropriate heterobifunctional agent and, finally, the coupling of the products thus obtained. Other known processes for the construction of immunoenzymic conjugates can also be used in this case.
The developing of the enzymic activity specifically associated with the cell antigen recognized by the acetylcholinesterase conjugate is preferably carried out according to the well-known technique which employs acetylthiocholine as the substrate for the enzyme and ~ 4 ~llman's reagent, or 5,5'-dithio-2-nitrobenzoic acid, as the chromogen, according to any vari ant adapted to the case under examination, for example the variant described by Pradelles et al., Anal. Chem., 1985, 57, 1170-1173.
S The cited chromogens are used as such or in the form of water-soluble salts.
According to the present invention, the rods (measuring rod and reference rod(s)) are solid supports whose size is adapted to that of the receptacle containing the magnetic particles. In fact, the rods are intended to be partially immersed in said receptacle and then easily withdrawn. Thus the measuring rod provided with a terminal magnet makes it possible to capture all the magnetic particles contained in the second receptacle of the assay kit.
As the measuring rod, it is possible for example to choose a flat spatula about ten centimeters long, about l cm wide and one to two millimeters thick, adapted to the size of the hemolysis tubes.
The magnetic part can then cover a surface area of about l cm2 on each side of the spatula. For example, it is possible to cut out appropriately sized patches of Flexor 15~ magnetic tape, marketed by Arelec, and stick them to each side of the rod. It is also possible to mold a magnet inside the terminal part of the rod.
The reference rod or rods preferably have the same length as the measuring rod, but are narrower; their cross-section is circular (about 2 mm in diameter) for example;
furthermore, the measuring and reference rods can be firmly fixed at one end, enabling them to be manipulated together for the capturing, labeling and washing operations. They can easily be separated before the measurement proper, made on each rod, namely the developing of the enzymic activity bound to the antibody in the case of immunoenzymic labeling, or the counting of the radioactivity in the case of radioisotopic labeling.
A determined quantity of purified or unpurified ~D15 13 ~ 4 antigen has been bound to the terminal part of the reference rod or rods, for example by physical adsorption or covalent bonding. It is also possible to bind a determined quantity of anti-species antibody, said species being the animal species from which the anti- C~15 monoclonal antibodies used originate, for example anti-mouse immunoglobulin antibodies in the case where the anti-CD15 antibody is of murine origin.
In fact, according to the present invention, the reference rod is intended to capture a known and preset quantity of the anti-CD15 antibody labeled with an enzymic or radioisotopic probe, present in the diluting and labeling reagent.
The quantity of CDl5 antigen or anti-species antibody bound to the reference rod is determined in advance so that the signal which can be measured at the end of the process according to the invention corresponds to a threshold value for the number of granulocytes in the sample submitted for analysis.
When several reference rods are used, each one is coated with a different quantity of CD15 antigen or anti-species antibody so that the signal which can be measured for each reference rod at the end of the process corresponds to a different value for the number of granulocytes.
When the granulocytes in a blood sample are counted, it is possible to choose a threshold value which corresponds for example to the minimum value for the average number of granulocytes counted in healthy subjects, namely about 2000 cells per mm3 of blood. It is also possible to choose a lower value which corresponds to more severe granulocytopenia, namely between 500 and lO00 cells per mm3, for example.
The access of the cells to the magnetized part of the measuring rod must not be hindered; for this reason, the reference rod or rods are narrower than the measuring rod;
also, the rods are prevented from touching one another by choosing an appropriate geometry for each of them. For example, the presence on one of the rods of a bulge projecting towards the other rod makes it possible to keep these rods a given distance apart, for example of one to two millimeters.
The device for temporarily fixing the rods together is located either on the measuring rod, at the opposite end from its magnetized part, or on the reference rod, at the opposite end from its part carrying the anti-species antibody or the CDl5 antigen.
One particular embodiment of the two rods comprises a measuring rod provided in its lower part with the magnetized part; and a reference rod comprising in its lower part a zone coated with anti-mouse immunoglobulin antibodies.
The measuring rod and reference rod comprise male and female conjugate means enabling them to be fixed together in a separable manner.
During the assay operation, the rods are fixed together and are inserted in a receptacle, such as a tube, containing the reaction medium or the washing liquid. Furthermore, one of the rods has a bulge projecting towards the other rod.
The rods can be provided with gripping means, such as hemispheres.
In one embodiment of the invention, the washing device used can consist of different receptacles containing the washing liquid, in which are immersed the measuring rod and, if appropriate, the reference rod (or rods) to which it is (or to which they are) firmly fixed. The size of the chosen washing receptacles can be similar to that of the second receptacle of the assay kit, containing the magnetic particles, and the washing is effected by immersing the measuring rod and, if appropriate, the reference rod or rods successively in each of the receptacles containing the washing liquid.
In another embodiment, the washing device used can ~S~3~4 consist of a receptacle containing the total quantity of washing liquid necessary for one assay, said device being provided with a flow system; furthermore, it can be capable of fitting on to the receptacle containing the magnetic particles (second receptacle), into which the washing liquid flows. In ~hat case, the receptacle containing the magnetic particles also comprises a flow device. The flow of the washing liquid makes it possible to rinse the measuring rod and, if appropriate, the reference rod (or rods), which are placed in the second receptacle of the assay kit.
The present invention further relates to a process for the rapid counting of granulocytes using the kit according to the invention, which comprises:
a) the mixing of a determined quantity of a sample of human blood or biological fluid with a fixed quantity of granulocyte diluting and labeling reagent containing the specific antibody, said antibody being labeled in conventional manner;
b) the transfer of the resulting sample and the shaking thereof in a receptacle containing the magnetic particles to which the same specific antibody, but unlabeled, is bound;
c) the introduction into the receptacle of the measuring rod provided with a magnetized terminal part and, if appropriate, at least one reference rod, said reference rod being coated on its terminal part with a determined quantity either of anti-species polyclonal antibodies, said species being the animal species from which the specific antibody originates, or of specific antigen;
d) after a reaction time, the withdrawal of the measuring rod and, if appropriate, the reference rod or rods and the washing of the measuring rod and, if appropriate, the reference rod or rods; and e) the actual counting of the granulocytes.
In another embodiment of the process according to the invention, steps a) and b) can be interchanged.
The reaction time mentioned in step d) is between 1 and lfi ,~r ~ X4 3 minutes.
In step d), the washing can be carried out either with a stream of washing liquid or by successive immersions in the receptacles containing the washing liquid, for 5 to 10 S seconds in each receptacle.
In one particular embodiment of the invention, the specific antibody used is an anti-CD15 monoclonal antibody.
This is bound to the magnetic particles by physical adsorption or covalent bonding. If the specific antibody is labeled with an enzymic probe, the process according to the invention also comprises treating the measuring rod with the substrate for the enzyme and, if necessary, one or more appropriate auxiliary reagents, and, if appropriate, the same treatment is applied to the reference rod or rods.
The actual measurement of the granulocytes bound to the measuring rod depends on the mode of labeling the specific antigen. For example, it is carried out by counting the bound radioactivity or, alternatively, by transmission or reflectance photometry, or by measurement of the fluorescence emission, or by visual evaluation, if appropriate by comparison with a range of standardized signals; if appropriate, the measurement of the photometric or radioactivity signal originating from the reference rod or rods is carried out in the same manner, after which the photometric or radioactivity signals originating from the reference rod or rods are compared with those from the measuring rod.
In one particular embodiment of the invention, the reference rod or rods used are firmly fixed to the measuring rod. In this case, separation of the different rods takes place in a step dl) following step d).
According to the present invention, several rods can be used, one called the measuring rod and the other or others called the reference rods, the latter making it possible to know the results for one or several values for the number of granulocytes. Thus, when it is desired to count precisely ~he granulocytes present in a sample, the absorbance due to the labeled cells carried by the measuring rod is measured at the end of the process, the measured absorbance values originating from the reference rod or rods serving as an internal standard. If it is desired to compare the number of granulocytes in the sample to be assayed with the chosen threshold value or values, a visual comparison of the coloration obtained for each of the rods may suffice.
Conventionally, the activity of peroxidase, for example, is developed for 20 minutes, whereas the activity of alkaline phosphatase is developed for about 2 hours (European patent application 311 492). The intensity of the coloration obtained (absorbance) is then measured with the aid of a spectrophotometer.
According to the present invention, it has been found that a developing time of 2 minutes for peroxidase is sufficient for visual observation of the differences in absorbance between the measuring rod and the reference rod (or rods). It is also sufficient for measuring the absorbance of the cells carried by the measuring rod.
Likewise, for any enzyme used for labeling, the developing time conventionally proposed for immunometric assays can be substantially shortened.
In one preferred embodiment of the invention, the granulocytes in a blood sample are counted by the process according to the invention using the kit according to the invention, which contains both the measuring rod and the reference rod and in which the anti-CD15 monoclonal antibody is an immunoglobulin of isotype M, labeled with peroxidase.
3() In this case, the various steps of the process are carried out for the approximate times indicated below:
step a): 15 seconds step b): 15 seconds step d): 2 minutes 30 seconds If the anti-CD15 antibody is labeled with an enzymic probe, the step in which the measuring rod is treated with 33~
~ne substrate for the enzyme takes less than 2 minutes; the same applies to the treatment of the reference rod or rods, if appropriate.
Thus the total execution time is less than 6 minutes.
In the Examples below, the following terms or their abbreviations will be used indiscriminately:
BSA: bovine serum albumin PBS: phosphate buffered saline at pH 7.4 IgG: immunoglobulin G
IgM: immunoglobulin M
cpm: counts per minute dpm: disintegrations per minute EDTA: ethylenediaminetetraacetic acid POD: peroxidase EXAMPLE l Measurement of the granulocytes in human blood samples A) Preparation of the assay kit a) Preparation of the magnetic particles The procedure is as in European patent 104 101, magnetic oxides being added.
A solution containing the following is prepared:
- 3.8 ml of methacrylic acid _ 121.9 ml of an aqueous solution of N,N'-methylenebisacrylamide containing 20 g per liter - 88.1 ml of acrolein - 93.2 ml of hydroxyethyl methacrylate - 193 ml of water This solution, called the stock solution, thus contains 37.5~ by volume of monomers.
A fluorinated surfactant marketed by Atochem Forafac~ 1157 - diluted to 18.8 g per liter, is used.
EGM 805~, marketed by Ferrofluidics, is used as the magnetic oxide.

'~3~ 4 The following are mixed in an 18.8 ml sealing tube:
- 2.5 ml of stock solution - 0.1 ml of surfactant solution - 1 ml of EGM 805~
- the necessary quantity of water After a stream of nitrogen has been bubbled through, the reaction media are frozen in liquid nitrogen and the ampoules are sealed under vacuum.
Polymerization is caused by irradiation with y rays from a cobalt bomb (60Co) for 3 hours at 60 krad/ hour.
The diameter of the magnetic particles obtained is between 1 and 10 ~m.
b) Preparation of the tubes containing the magnetic particles The magnetic particles used are either particles prepared in step a) or Estapor~ latex particles of reference Ml.070/40, marketed by ~hône-Poulenc, which have an average diameter of 0.8 ,um. These particles are coupled with an antibody of CD15 specificity. The chosen antibody is the antibody SMY 15a, of isotype M, marketed by Biosys. The latex particles are coupled with the antibody by the procedure described below.
0.1 ml of a 10% suspension of latex particles is placed in a glass tube. 1 mg of a solution of antibody in 1 ml of PBS is added and the mixture is then shaken for 30 minutes at 56-C. After one night at 4-C, it is centrifuged (15.103 rpm, 4-C) for 30 to 60 minutes and then washed with 1 ml of PBS
and centrifuged again. Finally, the residue is saturated with 1 ml of PBS containing 0.2~ of BSA.
The particles thus obtained are placed in 1 ml of phosphate buffered saline containing 0.2~ of bovine albumin.
25 ,ul of this mixture are introduced into a hemolysis tube with a volume of 5 ml. The tubes prepared in this way are lyophilized, stoppered and then stored a-t +4-C in a sealed plastic bag.
c) Preparation of the devices containing the solution ~f monoclonal antibody/peroxidase conjugate The peroxidase ( POD ) marketed by Boehringer Mannheim Biochemica (reference 814393) is used.
The process for coupling the antibody with the peroxidase is that described by M.B. WILSON and P.K. NAKANE
in Immunofluorescence and Related Staining Techniques (ed. W.
Knapp, K. Kolubar and G. Wicks, Elsevier/North Holland, Amsterdam, 1978, p. 215-224), except that 1.5 mg of POD in 0.36 ml of distilled water is used for the oxidation of the peroxidase and that 50 ,ul of a 0.2 M sodium periodate solution are added. The resulting product is coupled with 2 mg of anti-CD15 IgM contained in 500 ,ul of carbonate buffer.
After treatment with sodium borohydride and dialysis against PBS, the IgM/POD conjugate is sterilized by filtration on a 0.22 ,um membrane.
The SMY 15a antibody/peroxidase conjugate is packaged in unit doses of 400 ,ul of ready-to-use reagent at a concentration of 2 ,ug of antibody per ml of a mixture of e~ual volumes of P8S and fetal calf serum. It can be packaged either in 5 ml hemolysis tubes or in Unopette~
devices of reference 5856, marketed by Becton-Dickinson.
d) Preparation of the developing reagent 810 mg of orthophenylenediamine dihydrochloride are dissolved in 25 ml of PBS and 5 ml hemolysis tubes each containing 50 ,ul of this solution are prepared. The tubes are lyophilized, stoppered and kept at ~4-C in a sealed plastic bag. Just before use, 600 ,ul of a developing solution, namely citrate buffer and hydrogen peroxide tsubstrate for the enzyme), marketed by Pasteur Sanofi Diagnostics, are added to each tube.
e) Preparation of the measuring rods White plastic spatulas 10 cm in length, marketed by Safaa, are used. At the end of each spatula, 1 patch of self-adhesive magnetic tape 8 mm in diameter, marketed by Arelec (reference: Flexor 15, 15/ 10e), is fixed to each side. The spatulas are washed for 1 minute with distilled water and a Manisoft~ detergent marketed by Paragem (France), rinsed with distilled water and then dried and kept in an aluminum bag.
f) Preparation of the reference rods Plastic spatulas lO cm in length and two millimeters in diameter, marketed by Somater, are used. Anti-mouse immunoglobulin antibodies are adsorbed on to one end of the rod by the following procedure: the end of the rod (5 mm in height) is immersed for 12 hours in a solution, at 4-C, containing 4 ,ug/ml of antibodies dissolved in PBS; the rod is then immersed for 10 minutes in a 0.2% solution of bovine albumin in PBS; the rod is finally dried.
g) Preparation of the washing tubes 4 ml of PBS are introduced into several 5 ml hemolysis tubes and the tubes are then closed and are thus ready to use.
B) Assay of the blood samples a) Labeling and capture of the granulocytes 44 ~ul of blood taken on EDTA anticoagulant are deposited in a tube prepared in step A c), containing the antibody/POD conjugate, and the contents are mixed for 5 seconds. The blood can be either venous blood taken by means of a syringe equipped with a needle, or capillary blood taken from the end of the finger with the aid of a Microtainer~
pricker of reference 6357, which is a device marketed by Becton-Dickinson.
When using the Unopette~ device containing the solution of the antibody/POD conjugate, the capillary tube proposed by the manufacturer (Becton-Dickinson) is used to introduce 44 ,ul of blood.
A stoppered tube containing the magnetic particles, prepared in step A b), is opened and the previously prepared mixture of blood and antibody/POD conjugate, i.e. a total volume of 444 ,ul, is rapidly deposited therein. The contents are shaken for 15 seconds, the measuring rod prepared in step A e) and the reference rod prepared in step A f) are then Lntroduced into the tube and the rods are left in the t~ 4 for 2 minutes 30 seconds.
The operation is carried out with 2 x 8 blood samples.
b) Washing and developing of the enzymic activity The rods are withdrawn from the labeling tube and then washed by being agitated up and down in 4 successive washing tubes at a rate of 5 seconds per tube, i.e. for 20 seconds in total.
The rods can also be washed with a stream of washing liquid for 20 seconds. Each rod is then immersed in the developing tube containing the orthophenylenediamine, prepared in step A d), to which 600 ,ul of developing solution are added.
c) Results After 2 minutes, it is found that the yellow coloration of the developing medium originating from the measuring rod for an analyzed sample is more intense than that originating from the reference rod.
Furthermore, the absorbance of the developing medium is measured on 200 ,ul of solution with the aid of a calibrated spectrophotometer at a wavelength of 450 nm.
The measurement was made on 2 x 8 blood samples taken from healthy donors, each donor having given 2 samples:
venous blood and capillary blood.
Also, the number of granulocytes contained in 1 mm3 of blood was measured for each sample of venous blood with the aid of automatic apparatuses. This measurement is effected by automatic counting of the cells with an Ortho ELT 8 apparatus marketed by Diagnostic System. A Hematrak~
apparatus (Geometric Data) is also used in order to determine the percentages of the leukocyte subpopulations.
The results are reported in Table 1 below.

~ ~3r 1~2~
Table 1 _ . , Number of Absorbance at 450 nm granulocytes Sample per mm3 Venous blood Capillary of venous blood blood __ _ , . _ ._ Control 0.025 0.025 0 cells ._ 1 0.253 0.315 3705 _2 0.362 0.301 4408 3 0.239 0.194 3300 4 0.261 0.256 3776 0.179 _ 0.195 1908 6 0.198 0.147 2000 7 0.274 0.203 3300 _ 8 0.363 0.357 5994 reference 0.130 _ 1700 , . __ _ Table 1 shows that the measured results are similar for venous blood and capillary blood. Furthermore, the value r of the correlation coefficients between the absorbance measured for each sample and the number of granulocytes counted for the same sample was calculated. This coefficient 10 r is 0.955 for the samples of venous blood and 0.943 for the samples of capillary blood.

Efficacy of the method of capturing the granulocytes in whole blood First of all, the number of granulocytes present in 1 mm3 was measured by automatic counting on 5 ~4 ~r,~3^~4 vlood samples taken from healthy subjects; the same measurement was then made after application of the method of capture by magnetic particles, such as described in Example 1. For each sample, the percentage of granulocytes captured 5 is obtained as the difference between the number of granulocytes counted before and after cell capture by the magnetic particles.
In these experiments, the magnetic particles and the granulocytes were separated from the reaction medium either with a magnetized rod by the procedure described in Example 1, or by means of a fixed magnet consisting of a magnetic bar placed on the outside of the tubes.
The results obtained are reported in Table 2 below.

ls Table 2 _ . . . _ Number of % of granulocytes captured by granulocytes Sample magnetic particles per mm3 of blood Magnetized Fixed magnet - . .
1 97.2 % 96.9 ~ 1890 ._ . .
94.1 % 98.9 % 3016 _ .. _ 3 98.5 % 99.5 % 3760 .. _ _ ...
4 97.3 % _ 97.5 % 3060 99-~ ~ 97.7 % 6063 _ Observation of the results expressed in Table 2 shows that the percentage of granulocytes captured by the technique according to the invention is high (97 to 100%), irrespective of the number of granulocytes present S.~4 1n the blood sample and irrespective of the type of magnet chosen for the capture.

Assay of the granulocytes in an infected urine medium 40 ~1 of infected urine and 400 ~1 of a solution of anti-CD15 antibody conjugate labeled with peroxidase, such as prepared in Example 1, step A c), are introduced into a tube containing the magnetic particles, prepared in Example 1, step A b). After shaking for 30 seconds, a measuring rod prepared in Example 1, step A e), is introduced for 2 minutes 30 seconds. Said rod is then washed by successive immersion in 4 tubes containing 4 ml of PBS, the rod being shaken for 5 seconds in each tube. Finally, the rod is immersed in 600 ~1 of peroxidase developing reagent prepared in Example 1, step A d). After 2 minutes, the absorbance is measured with the aid of a spectrophotometer at a wavelength of 450 nm. The results are compared with those observed under the same conditions for a sample of uninfected urine. Also, the number of granulocytes contained in the sample of infected urine is measured with a cell counter.
The results are reported in Table 3 below.

Table 3 . _ Sample Absorbance Number of aranulocvtes/ml . _ _ _ _ infected 0.488 + 0.073 120.103 urine _ _ uninfected 0.012 about. 0 _urine Counting of the granulocytes in various blood samples The assay kit according to the invention, comprising a measuring rod and a reference rod such as prepared in Example 1, is used and the results obtained for 8 blood samples are measured by spectrophotometry at 450 nm. By way of comparison, the number of granulocytes per mm3 of blood is measured with the aid of a counter. The results are given in Table 4.
Ta~le 4 _ . , , _ _ number of Sample Absorbance at 450 nm granulocytes _ per mm _ Measuring rod Reference rod of blood ~ _0.195 0.130 1 584 2 0.280 0.136 2 745 _ ...
3 0.272 0.127 2 880 _ 4 0.266 0.141 3 190 ....... ............ _ _ 0.310 0.128 3 192 6 0.290 0.110 3 591 ._ _ 7 0.307 0.118 3 650 _ 8 _ 0.408 0.142 _ 5 022 For each sample, it is observed with the naked eye that the yellow coloration of the measuring rod is more intense than the coloration of the reference rod.

Claims (14)

1. A kit for counting the granulocytes in human blood or any other human biological fluid, comprising the following components:
a) a first receptacle containing a determined quantity of a granulocyte diluting and labeling reagent containing a specific antibody labeled in conventional manner;
b) a second receptacle containing magnetic particles to which the same specific antibody, but unlabeled, is bound; and c) a rod, called a measuring rod, provided with a magnetized terminal part.

2. A kit according to claim 1 which also comprises:
d) a device for removing a determined quantity of blood or biological fluid;
e) a device containing washing liquid;
f) means for developing the marker of the specific antibody;
and g) at least one rod, called a reference rod, which is coated on its terminal part with a determined quantity, different for each rod, either of anti- species polyclonal antibodies, said species being the animal species from which the specific antibody originates, or of specific antigen.

3. A kit according to claim 2 wherein said reference rod or rods comprise separable means for fixing to the measuring rod.

4. A kit according to any one of claims 1 to 3 wherein the magnetic particles have a size of between 0.1 and 20 ,um.

5. A kit according to claim 4 in which the magnetic particles have a size of between 0.5 and 4 ,um.

6. A kit according to any one of claims 1 to 5 wherein the specific antibody is labeled with a fluorescent agent, an enzymic probe or a radioisotopic probe.

7. A kit according to any one of claims 2 to 6 wherein the specific antibody is labeled with an enzymic probe and the means f) consist of one or more receptacles containing the substrate for the enzyme and, if appropriate, one or more reagents necessary for measuring the activity of the enzyme.

8. A kit according to any one of claims 1 to 7 wherein the specific antibody is an anti-CD15 monoclonal antibody.

9. A kit according to claim 8 wherein the anti- CD15 monoclonal antibody is an immunoglobulin of isotype IgM.

10. A process for the rapid counting of granulocytes, which comprises:
a) the mixing of a determined quantity of a sample of human blood or human biological fluid with a fixed quantity of granulocyte diluting and labeling reagent containing the specific antibody, said antibody being labeled in conventional manner;
b) the transfer of the resulting sample and the shaking thereof in a receptacle containing the magnetic particles to which the same specific antibody, but unlabeled, is bound;
c) the introduction into the receptacle of the measuring rod provided with a magnetized terminal part and, if appropriate, at least one reference rod, said reference rod being coated on its terminal part with a determined quantity either of anti-species polyclonal antibodies, said species being the animal species from which the specific antibody originates, or of specific antigen;
d) after a reaction time, the withdrawal of the measuring rod and, if appropriate, the reference rod or rods and the washing of the measuring rod and, if appropriate, the reference rod or rods; and e) the actual counting of the granulocytes.

11. A process according to claim 10 wherein steps a) and b) are interchanged.

12. A process according to claim 10 or claim 11 wherein the specific monoclonal antibody is an anti- CD15 monoclonal antibody.

13. A process according to claim 12 wherein the anti-CD15 monoclonal antibody is an immunoglobulin of isotype IgM.

14. A process according to any one of claims 10 to 13 wherein the reaction time in step d) is between one and five minutes.