GB1560729A

GB1560729A - Counting of leucocytes

Info

Publication number: GB1560729A
Application number: GB2676876A
Authority: GB
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM
Priority date: 1975-06-27
Filing date: 1976-06-28
Publication date: 1980-02-06
Also published as: NL7607012A; DK289076A; JPS5224589A; NL171840B; NL171840C; DE2628468A1; CH613523A5; DE2628468C2; SE437079B; JPS5819063B2; SE7607282L; CA1087966A

Description

(54) IMPROVEMENTS IN OR RELATING TO THE COUNTING OF LEUCOCYTES (71) We, INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE-INSERM, a French Government Organisation of 101 rue de Tolbiac, 75645 Paris, Cedex 13, France, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The invention relates to means, more particularly a process and a composition for use in effecting a count of leucocytes, and more particularly relates to a process for selectively staining only non-degranulated basophils, such as blood basophils in man or an animal, and to an aqueous hypotonic staining composition.It relates also to a method of classifying blood samples useful in the diagnosis in vitro of certain anaphylactic disorders, and more particularly allergies.

It is known that the methods for diagnosing allergies most often used until now generally involve numerous injections of the different allergens to be tested. In addition to the unpleasantness of this method of administration, it is sometimes extremely dangerous for the patients and often gives rise to inexplicable non-specific reactions.

For some years methods have existed for making the anaphylactic antibodies in serum visible. However, they are extremely costly because they use a very complex technology in the purification, the labelling with radioactive iodine and the fixing on insoluble supports of purified allergens and antibodies obtained by immunising an animal. Furthermore, they require specialised equipment (centrifuges, and equipment for counting radioactive constituents) which makes them extremely costly and unsuitable for applications outside large urban centres of the developed countries. In addition, it is now known that the existence of serum antibodies is not sufficient to start an allergy.

In fact, it has been known for some time that the basophils in the blood play an important part in the development of symptoms of an allergy. In particular, it had been observed that allergy phenomena can largely be attributed to the degranulation of basophils in the circulating blood under the action of the allergen, a degranulation which is accompanied, inter alia, by release of large amounts of the toxic products, especially histamine, which these basophils contain.

It had also been established that the allergy reaction could be reproduced in vitro, the basophils sensitive to degranulation under the effect of the antigen studied undergoing various morphological or cellular modifications, which were not observed in the basophils of control samples or of samples previously put into contact with allergens which were unquestionably inactive with regard to those samples.

This is the reason why certain authors have already proposed methods for diagnosing allergies on the basis of a study in vitro of cellular modifications in the basophils of a blood sample from a patient in the presence of the antigens or allergens under investigation for anaphylactic reaction in that patient.

These diagnostic methods have, until now, come up against very serious difficulties. One of the prime causes of these difficulties lies in the extreme rarity of the basophils relative to the other constituents of the blood, so that the techniques which have been described generally comprise two stages: a first stage of relative concentration of the basophils in the blood samplel; and a second stage of so-called colouration of the basophils.

A standard technique is that which was described by SHELLEY W.B. and L. JUHLIN (BLOOD. 19 : 209 (1962).

Accomplishment of the first stage involved several operations including putting the blood sample to be studied into contact with a medium intended to ensure fixing of the leucocytes, including the basophils, and the destruction of the erythrocytes, this medium comprising 60 parts of ethyl alcohol, 20 parts of glacial acetic acid and 20 parts of chloroform; keeping the mixture obtained at 40C until the next day to achieve complete fixing; eliminating the supernatant liquid and putting the decanted cells back into suspension in the same medium and filtering the suspension obtained on filter paper, to obtain a residual solid formed by the leucocytes on the filter paper itself.

The second stage then consisted of immersing the filter with these cells for 30 seconds in a solution of toluidine blue obtained, for example, by dissolving 100 mg of toluidine blue in 30 ml of distilled water. The sample, the basophils of which were coloured red, was then "read" under the microscope.

This procedure of which only the essential parts have been set out above, requires extremely critical handling, and, as the authors themselves have acknowledged, presupposes enforced enthusiasm on the part of the experimenters, since most frequently differentiation between degranulated basophils and non-degranulated basophils requires evaluation of the morphological transformations which can be established in those basophils which have undergone this degranulation.

Certain authors have proposed simplified versions of SHELLEY's method. For example, KLOPSTOCK and Associates (Israel Medical Journal, September-October 1962, volume 21) have proposed new techniques for studying morphological transformations of the basophils in a blood sample under the effect of a study antigen, with a view to establishing a method for diagnosing allergies in vitro.

One of these techniques consists in incubating for 20 minutes a sample of blood, previously "heparinised" in the presence of the antigen to be studied, then diluting aliquots of this sample and of a control incubated in the presence of a normal saline solution with five times their volume of a colouring mixture formed from 0.25 % of toluidine blue and 2.5 % of acetic acid. After incubation for 5 minutes of the media obtained, a comparative study under the microscope is then effected of the proportions of the non-granulated basophils to the degranulated basophils in each of the samples, evaluation of the degranulation being based always upon morphological changes induced in the cells by degranulation.If the colouring technique is simpler, evaluation of the morphological differences between non-degranulated basophils and degranulated basophils still remains just as difficult. It is probable that the strong acidity of the medium would only add to these difficulties.

The second method proposed by KLOPSTOCK and Associates also included a stage of concentrating the basophils, usually by differential centrifuging of the blood sample following incubation in the presence of the antigen to be studied; the study of the basophils in the concentrate obtained, which contained the leucocytes and amongst them the basophils is then effected under the microscope, after colouration with a colouring reagent based upon toluidine blue, in a water/ alcohol solution containing 50 No of ethyl alcohol. Evaluation of the possible degranulation of the basophils can, however, in this method only be made even more difficult since the observer sees not only the basophils but also all the other leucocytes, the whole being enveloped in a sheet of erythrocytes.

Although these various methods or techniques present a certain degree of efficacy, they have not, however, been used in practice because of the minutiae involved in the preparation of the reagents, the length of the tests, their tedious nature and the necessity of having very well trained staff available.

Generally speaking, there are only a few visualisation methods permitting a simple count of the basophils. The difficulties inherent in these counts have been explained by James E.

MOORE and Associates (P.S.E.B.M. 1953, vol. 82, pages 601-603). These authors have explained that differentiation of basophils from the other leucocytes in a blood sample presupposed that previous haemolysis of the erythrocytes was effected without the watersoluble granules of the basophils being dissolved at the same time. They indicated in particular that acetic acid, amongst other organic acids, although effective as a haemolysis agent for erythrocytes, was not suitable because of the solubility of the basophil granules in reagents containing it. Similarly, these authors established that 15 - 30%solutions of alcohol, although effective for fixing the granules, were not effective as haemolysis agents. In order to remedy these apparently insurmountable difficulties, they proposed a quite different process for metachromate colouration of the basophils, consisting of putting a sample of blood to be studied in contact with a mixture formed from 40 parts by volume of a solution of toluidine blue in an isotonic saline solution, 11 parts by volume of ethyl alcohol and one part by volume of a 50% solution of saponin in ethyl alcohol.

The method recommended is, however, difficult to apply, especially when one needs to differentiate between the basophils according to whether they are degranulated or not, a problem which the invention aims at solving. Besides, the reproducibility of this prior art method is far from being perfect, even when it is used only to differentiate the basophils from the other leucocytes in the blood. Metachromate colouring is not distinct, and saponin tends to precipitate in the medium, All the difficulties which have arisen mean that heretofor haematology laboratories have not called for methods of studying in vitro in order to achieve diagnosis of anaphylactic sensitivity, and more particularly allergies.

There is a need for a process for staining and counting in vitro basophils, particularly non-degranulated basophils and possibly other leucocytes, which can be applied to blood samples. Preferably this should be a single stage process without previous concentration being necessary for the constituents of the samples. Any such process should desirably be easy to use, reproducible and within the scope of operation without special training. To carry out such a process it is desirable to provide a reagent which may be put into contact with the samples of blood to be tested, and quick and immediate counting, by means of counting apparatus such as a standard haemocytometer. It is advantageous if such reagents are stable over a period of time, so that it is not necessary to prepare them extemporaneously for each series of tests.The development of suitable reagents and processes would make it possible to provide methods for diagnosing in vitro anaphylactic sensitivity, particularly allergies, to which patients may be susceptible.

In one aspect this invention provides a process for selectively staining only nondegranulated basophils in a blood sample containing degranulated basophils and nondegranulated basophils, in which without first fixing leucocytes contained therein, the sample is contacted with an aqueous hypotonic staining composition comprising, per 100 ml of the composition, from 30 to 250 mg of a metachromatic agent ,from 20 to 50 ml of an alcohol fixative for the leucocytes including the basophils and an acid having lytic properties with respect to erythrosytes in an amount less than 1 ml, the relative proportions of the sample and the staining composition brought into contact being such that the pH of the formed mixture is from 3 to 5.

The concentration of the metachromatic agent is chosen being sufficiently low to allow adequate differentiation between the colouration speeds of non-degranulated basophils and of the slower colouration speeds of degranulated basophils.

The alcohol concentration in the staining composition is intended to be sufficiently low for the non-degranulated basophils to have time to undergo colouration, yet sufficiently high for the non-degranulated basophils to be fixed before they have had time to be coloured.

The water and acid contents are sufficient to bring about lysis of the erythrocytes to the extent necessary to permit further direct observation of the non-degranulated basophils, these being the only ones which are coloured. The pH conditions are very important. At a pH below 3 the colouration observed is unstable; at a pH above 5, it becomes difficult to distinguish the basophils from the other constituents of the blood, and this is all the more so since the erythrocytes are at best no more than partially lysed. Thus, in the pH bracket employed, a competition is established between the colouration speeds of intact basophils and of degranulated basophils, by means of the metachromate agent, and the fixing speeds of at least the basophils, and possibly also the other leucocytes.The concentrations of fixative and metachromate agents used ensure that only the intact basophils have time to be coloured, before being fixed by the fixative, whereas the degranulated basophils are fixed before they have time to become coloured to an extent that will later be visible to the naked eye.

It is interesting to note that the non-degranulated basophils appear to the observer to be coloured throughout their whole mass, which can perhaps be attributed to the basophil granules beginning to dissolve in the medium which has penetrated into these basophils, but inside the cells, fixing of the latter having the effect of preventing dissolved matter from diffusing to the outside.

The absence of visible colouration in the basophils, even when only partly degranulated, could be interpreted in the same manner, the possible weak colouration induced in the granules and still existing in these basophils ceasing to be observable because of its excessive dilution, even if it is only in the heart of these basophils.

It is to be understood that hypotheses put forward herein in an attempt to explain the invention are not in any way intended to be limiting upon the scope of the invention, and only seek to provide a rationale for the observation that the invention terms what was previously regarded as a disadvantage to an advantage in so far as the effect, previously deemed to be disastrous, of acetic acid on the water-solubility of the basophil granules is concerned.

The invention also provides an aqueous hypotonic staining composition for selectively staining only non-degranulated basophils in an unfixed of, or derived from, blood and containing basophils, the composition comprising, per 100 ml of the composition, from 30 to 250 mg of a metachromatic agent, from 20 to 50 ml of a alcohol fixative for leucocytes including the basophils and an acid having lytic properties with respect to erythrocytes in an amount less than 1 ml.

When in the process of the invention the blood sample is diluted by an integral number of volumes of the staining composition, it appears that the respective proportions of the constituents of the staining composition and its pH will extend approximately to the staining composition/blood sample mixture to be studied, and vice versa.

In the composition of the invention, the alcohol fixative is preferably miscible with water and capable of fixing leucocytes and stabilising basophils. The water acts both as diluent for the alcohol and as a lytic agent for the erythrocytes. The acid is selected from amongst those which are compatible with the leucocytes, and particularly the basophils. Preferably the reagent is free from any mineral salts of the type which contribute to making an aqueous solution isotonic - such as sodium chloride.

In its preferred form the staining composition contains, per 100 ml of liquid: - from 20 to 50 ml of alcohol; - from 80 to 50 ml of water (preferably distilled, and if desired double-distilled); - less than 1 ml of an acid; and - an appropriate amount of metachromate agent.

A preferred metachromate agent is toluidine blue. Other metachromate colourants can, of course, be used - for example those described in the work "Mast-cells and basophils", Annals of the New York Academy of Sciences, Vol. 103, 1963. The preferred acid and alcohol are respectively ethanol and acetic acid.

The staining composition of the invention, may be used to bring about destruction of the erythrocytes in the blood sample, fixing of the leucocytes and stabilisation of the basophils, and finally and most importantly selective red (when toluidine blue is used) colouration of the non-degranulated basophils. This colouration is practically immediate and when the mixture is transferred to the chamber of a haemocytometer it is possible to count quickly, by methods well known in this technology, the number of basophils contained in one unit by volume of blood sample, which have not undergone degranulation.

When the pH of the mixture is of the order of 4.5, colouration of the cytoplasm of single basophils is observed. A very slight blue colouration of the nuclei of the other leucocytes is also noted, but the cytoplasm of cells other than the basophils is never coloured, so that the only cells which appear to be coloured red, throughout their mass, are the basophils. It is pointed out, that the granules contained in the cytoplasm of the basophils, and coloured by the metachromate colourants, are specific to basophils, for which reason the cytoplasm of other polynuclear cells and leucocytes is also not coloured, so that it is possible for the basophilic polynuclear cells to be counted and for the other polynuclear cells and lymphocytes to be separately counted.

The pH resulting from the direct mixture of the staining composition and blood may be adjusted by varying the proportions of these components to give the pH value desired. The pH of the mixture can also be adjusted by modifying the pH of the staining composition before mixing - for example, using acetic or hydrochloric acid or a base such as sodium hydroxide, according to whether it is wished to raise or lower the pH value of the mixture. It is, of course, particularly simple to regulate the acid content of the reagent beforehand, to produce the pH desired in the final mixture.

The process is an extremely simple operation. It can be applied to whole blood, without any necessity to separate its constituents beforehand. It can be applied to natural blood or blood previously washed with an isotonic buffer, for example that known by the term "Tyrode buffer" a buffer comprising 0.195 g KCI, 8.0 g NaCl, 0.605 g TRIS, 1.040 g tetrasodium EDTA, 0.385 g heparin, 1.0 g glucose and 1.000 ml water, to which is added 2.5 g of human serumalkumin and sufficient HC1 to adjust the pH to 7.35 at 370C). It can be used directly on fresh blood or on blood containing an anticoagulant, such as heparin, if this has been kept for some time.

The proportions of blood and staining composition which are mixed depend, inter alia upon the initial pH of the staining composition and on the pH desired in the mixture. In the preferred case using the "optimum" reagent described hereinafter, the mixture of 1 volume of blood with 9 volumes of staining composition gives a pH of the order of 4.5 which permits selective colouration of single basophils which have not undergone degranulation.

The simplicity of the method, complemented by the excellent legibility of the slides (the red basophils stand out easily on the light background of the treated sample), quickly renders it within the scope of any untrained person. In addition, the reagent is not very expensive, bearing in mind the nature of its constituents.

Finally, an appropriate choice of the proportions of the constituents of the staining composition leads to a perfectly stable mixture, for periods which can last for several months at ambient temperature, although it is better to keep such reagents at a temperature of the order of 4"C, so as to reduce the risk of the alcohol evaporating.

In this respect, preferred staining compositions are those which contain the following proportions by volume - or by weight as far as the metachromate agent is concerned - of the preferred constituents, per 100 ml of liquid: - 20 to 50 ml of ethanol; - 80 to 50 ml of water; - between 150 and 600 microlitres of glacial acetic acid, such that the pH of the final mixture is from 3 to 5; and - 30 to 250 mg of toluidine blue.

The compatibility of the constituents of this mixture, its great stability as well as its efficacy in metachromate colouring of the basophils under the conditions indicated, have been shown in testing.

The particularly preferred proportions for the various constituents are as follows: - 25 to 40 ml of ethanol; - 75 to 60 ml of water; - between 150 and 600 microlitres of glacial acetic acid, such that the pH of the final mixture is from 3 to 5; and - 30 to 100 mg of toluidine blue, per 100 ml of the composition.

The optimum staining composition has a pH of from 3.4 to 3.6 and comprises; - 30 ml of absolute ethanol; - 70 ml of distilled water; - 70 mg of toluidine blue; and - 400 microlitres of glacial acetic acid.

The invention also relates to a method of classifying blood samples on the basis of whether the patient from which the sample is taken displays anaphylactic sensitivity to an allergen by assessing the degranulant effect of the allergen on basophils in the samples. This method may be used in the in vitro diagnosis of anaphylatic sensitivity, and particularly allergies, and maybe carried out on blood samples of human or animal patients.

According to this method each sample is divided into two fractions, the fractions are incubated in the presence respectively, of the allergen and of a control antigen (believed substantially inactive with respect to the basophils of the patient) for a given period sufficient for at least partial degranulation of basophils in the sample incubated in the presence of the allergen to take place if the blood sample is sensitive to this antigen, each fraction is treated by a process of the invention selectively to stain non-degranulated basophils in the fraction, the stained basophils in each fraction are counted, preferably by means of a standard haemocytometer, and the two counts are compared and the sample is classified on the basis of the difference between the counts, a significant difference between the counts being indicative of anaphylactic sensitivity of the patient from whom the blood sample was taken with respect to the allergen tested. Thus, the method enables blood samples to be classified, so that the classification of a sample enables allergies to be diagnosed.

It is to be understood that the invention is applicable to diagnosing the sensitivity of a patient to any allergen - antigen or chemical agent - liable to cause degranulation of the basophils.

The great advantage of this method of diagnosis lies in its simplicity. It is based solely upon counting techniques, without it being necessary to become involved in morphological transformations of the basophils. It also avoids the necessity of concentration operations which can only be employed in specially equipped laboratories and by trained technicians.

The counts are preferably carried out on blood samples immediately after the incubation operation, in the presence of the allergen to be tested. They can, however, be postponed until later. However, it is then convenient to block the reactions and to avoid later aggregation of the cells, for example by adding an anti-aggregating agent or anticoagulant such as an ethylenediaminetetracetic acid (EDTA) salt.

The sensitivity of the method of the invention, in the majority of cases enables the sensitivity of a sick person with regard to an allergen to be diagnosed as soon as a significant reduction is observed in the counts carried out on blood samples incubated in the presence of this allergen as compared to counts carried out under similar conditions on a control sample.

But due to the simplicity and the sensitivity of the method of the invention, the existence in certain patients of an apparent discrepancy between what could be interpreted as an absence of degranulation of the basophils, especially when the count is carried out on a sample of whole blood, and an obvious anaphylactic sensitivity in these patients with regard to the allergen studied, could be established. It has been possible to elucidate the nature of the phenomena giving rise to these discrepancies and to obtain additional information about the behaviour of certain factors contained in the blood with regard to the allergens studied. In particular, another embodiment of the method of the invention has been evolved to cope with the fact that the plasma could in certain cases, contain factors, hereinafter called "blocking factors", which contradict the allergen/basophil reaction.

In this arrangement, each blood sample is divided into a sample of whole (or total) blood and a sample of washed blood (as defined hereinafter), and in which each of these two samples is divided into two fractions which are incubated, stained and counted as defined hereinbefore and the counts are compared, a significant difference between the counts taken from the two fractions incubated in the presence of the allergen being indicative of the plasma in the blood sample counteracting the degranulant effect of the allergen with respect to the basophils.

As the two types of samples are distinguished from one another by the presence or absence of plasma, the observation of a difference in the counts can only come from the plasma, and more precisely from the constituents or factors which it contains and which protect the basophils, especially the immunoglobin of class IgE fixed on their surface, against attack by the above-mentioned antigens. When these factors are present in the plasma of the samples of whole blood, breaking down of the allergen in these samples is inhibited and the number of degranulations observed is no longer indicative of the actual degranulant effect of the allergen.

By contrast, the number of degranulations observed in the samples of washed blood will be all the more representative of the actual harmfulness of the allergens tested with respect to the basophils, their action then being no longer counteracted by the factors which, in this type of blood sample, were present in the plasma.

When the counts obtained on the samples of washed blood and of whole blood from the same sample do not display any significant difference, it may be inferred that, in the plasma of the patient from whom the blood sample comes, blocking factors with respect to the allergens are absent of course insofar as a comparison of these counts with those of the control fractions coming from the same blood sample and incubated in the absence of these same antigens (or in their presence, but in the absence of calcium, as will be discussed further on) reveals a difference.

A prime, important advantage of the above-mentioned embodiment of the invention lies in the fact that in practice it enables comparisons previously deemed necessary between the effects of the allergen to be tested and the control antigens, especially non-allergens with regard to patients from whom the blood sample studied comes, to be abolished, in order to conclude in favour of the existence of an anaphylactic reaction in these patients with regard to the allergens tested.

As used herein, the term "sample of washed blood" is used to mean a sample which consists of a suspension of the cellular constituents of blood in an isotonic buffer compatible with these cell constituents. In other words, an isotonic buffer is, in a sample of washed blood, substituted for plasma in the sample of whole blood. Advantageously, this isotonic buffer contains, in addition, constituents such as carbohydrates, especially glucose, and proteins (preferably as human serum albumin) so as to maintain in the washed sample the cell constituents of the blood in a medium approaching that in which the cell constituents of the whole blood are immersed.

Advantageously, this buffer has a pH of the order of 7.4 to 7.6 and is based upon (tris-hydroxymethyl)-aminomethane, also known as "TRIS". It may also contain a small amount of anticoagulant, such as heparin and/or EDTA. EDTA has, inter alia the effects of stabilising reactions, of rendering the blood uncoagulable, and of preventing cell aggregations and the deposition of cells on the walls of tubes used to contain the samples. Heparin also contributes to these functions, and may also protect EDTA from chelation when calcium is added. Of course, these constituents can be replaced by other anticoagulents insofar as they are suitable for performing the same functions.

Advantageously, to reduce even further the influence of parameters such as may result from different physical treatments of the cell constituents of the sample of whole blood and the sample of washed blood during the physical separation operations to which the cell constituents of the latter sample and the plasma in which they are previously immersed have been subjected the sample of whole or total blood is preferably a sample of reconstituted whole blood obtained by separating the cellular constituents and the plasma in the blood sample being studied, under conditions identical to those used for the preparation of the sample of washed blood, the separated cellular constituents being then reunited with the plasma. The volume of buffer used to constitute the sample of washed blood is equal to the volume of plasma previously separated from the cell constituents with which it is formed.

The separation of the cell constituents and the plasma into the fractions under consideration can, of course, be carried out in any manner knownper se, and especially by centrifuging.

The cell constituents of the different samples may be, if necessary, washed with the isotonic buffer the cellular constituents free from plasma thus obtained being associated respectively with the plasma from which they had been separated to form samples of whole blood, and with isotonic buffer in volumes equal to the volumes of separated plasma to form samples of washed blood.

According to an additional embodiment of the invention, the fact that the degranulation of the basophils under the effect of certain antigens can only take place in the presence of calcium is turned to advantage. In this embodiment of the method of the invention the incubations are carried out in the presence of calcium, and for checking purposes, further incubations of the samples of whole blood and of washed blood are carried out in the presence of excess calcium, but in the absence of allergen.

A comparison of the results obtained enables an anaphylactic sensitivity with respect to the allergen studied to be distinguished from any other possible form of sensitivity of the basophils under consideration. In particular, a degranulation observed in the samples incubated in the presence of calcium alone can only mean sensitivity of the basophils to factors other than the allergen tested.

According to an additional preferred feature of the method of the invention, there is also carried out, again to provide a control, incubations of the samples of whole blood and of washed blood in which the calcium content of the samples is masked, especially by chelation before being incubated in the presence of the allergen.

An absence of degranulation in the samples tested in the presence of the allergen alone, together with the observation of degranulation of the basophils in the presence of the same allergen, but in the presence of unmasked calcium, enables the anaphylactic sensitivity of the patient from whom the blood samples are obtained, to be confirmed with regard to this allergen. On the other hand, degranulation of the basophils in the presence of the allergen alone and in the absence of calcium, shows toxicity of the allergen with regard to the basophils which do not cause the IgE anaphylactic system to come into play. This toxicity can be due to many factors, and may be a result of the presence in the medium of contaminants or of degraded products or other factors.

The above observations thus show the importance of the gains made by the method of the invention, both as regards carrying out the tests more sensitively and as regards the safety of the diagnosis. The invention finally relates to a presentation in the form of a test kit, in which are assembled the reagents necessary for the diagnosis of allergies.

The test kit comprises, each in a separate container, an aqueous staining composition of the invention, and samples of each allergen in respect of which the test kit is to be used to test for anaphylactic sensitivity, and especially for allergies.

The essential constituents of the liquid reagent may be presented separately, each in different containers and, if desired, in volumes corresponding to the proportions required in the composition, so that the person handling the kit will only have to mix them in order to form this reagent which can then be kept for a prolonged period because of its stability, especially if the above-mentioned proportions correspond to the preferred compositions set out above.

The allergens which are brought into contact with the blood samples to be tested can be in the form of a solution. However, in a preferred form the basic allergens are presented in small tubes or on slides or microslides having wells or depressions, these tubes or depressions containing predetermined amounts of the different allergens in a dry state. These amounts are selected having regard to the dilutions subsequently desired in the samples of blood introduced into these wells or depressions. Deposition of the allergens in these tubes or depressions can be carried out in any manner known per se, for example starting from suspensions or solutions of these allergens previously places in tubes or depressions, by lyophilisation or evaporation of the solvent. A preferred form of presentation is on a microslide.

By having recourse to this type of presentation, diagnoses of allergies can be effected very quickly. It is, in fact, sufficient to introduce into each of these tubes or depressions containing allergens a predetermined amount of blood - for example, using a micropipette - to carry out incubation of the blood in contact with these allergens at a temperature and for a period appropriate to bring about degranulation of those blood basophils which give such a reaction in the presence of the corresponding allergen. Then there is added, to each of these tubes or depressions, a predetermined volume of the staining composition of the invention, and the basophils in each of these tubes or depressions is counted under the conditions which have been described above.

In order to enable the comparisons described above between whole blood and washed blood samples to be carried out, it is advantageous to employ a test kit comprising on the one hand the staining composition of the invention, or the constituents necessary to prepare it, and on the other hand the allergens to be tested, preferably contained in wells or depressions on a slide or microslide, the test kit being such that each of these allergens is associated with an amount of the calcium salt sufficient to promote degranulation of a part at least of the basophils in a blood sample contacted with the allergen by being introduced into the corresponding wells or depressions during incubation under conditions suitable for ensuring this degranulation.Advantageously, there is provided on each slide or microslide containing allergen with associated calcium salt a series of wells or depressions containing, respectively, different (increasing) doses of the same allergen, so as to permit preparation of respectively, increasing (or decreasing) dilutions. It will thus be possible to determine the existence not only of a possible sensitivity of a patient with regard to a given allergen, but also the degree of the sensitivity of his/her basophils with regard to this allergen.

According to one form of test kit of the invention, each of the abovementioned slides or microslides also includes wells or depressions containing the antigen alone in the absence of calcium and/or wells or depressions containing the calcium salt alone in the absence of the antigen.

In another form of the test kit, each of the above-mentioned microslides is subdivided into two microslides, one containing a series of wells or depressions containing only calcium and a series of wells or depressions containing both calcium and the corresponding allergen as indicated above, the other microslide incorporating a corresponding series of wells or depressions containing corresponding doses, respectively increasing (or decreasing), of the same allergen in the absence of calcium.

The series of wells or depressions in the first microslide which contain, as the main ingredient, only calcium, could of course alternatively be formed in the second microslide.

The above description is not exhaustive, and further variations falling within the scope of the invention are Certain aspects of the invention will now be described, though only by way of illustration, in the following Examples, with reference to the accompanying drawing, which shows a microslide for use in connection with the invention.

It will be noted that the blood used in the tests described below was collected on pure heparin (10 units of heparin per millilitre of blood in siliconised plastics tubes). Whilst awaiting completion of the tests, the blood is kept either in ice or under agitation in order to avoid non-specific aggregation of the leucocytes.

EXAMPLE 1: Preparation of a staining composition according to the invention.

70 ml of 95% ethanol are mixed with 30 ml of distilled water, then 75 mg of toluidine blue, such as that marketed by K and K, Plainview Lab., NY., USA are added. This mixture is subjected to agitation until total dissolution is attained. While continuing this agitation, 400 microlitres of freezing acetic acid are added, which takes the pH of the solution to 3.4 - 3.6.

This solution is filtered forty-eight hours later. The filtrate can be kept for several months at ambient temperature. However, in order to reduce evaporation of the ethanol, it is preferable to keep it at 4"C. It is desirable also to filter the solution about every two months.

EXAMPLE 2: Metachromatic colouring, and counting, of blood basophils.

0.50 to 1 ml of blood (human or rabbit) is poured into a tube containing 10 units per ml of heparin or 25 microlitres of sodium EDTA, 0.2 M of pH 7.4. This blood sample is then diluted in nine times its volume of the staining composition of Example 1, which leads to a pH in the order of 4.5.

The mixture is gently agitated and one proceeds, after 4 - 5 minutes, to count of the basophils, in a manner known per se, in the chamber of a hemocytometer.

In the same way, it is possible to work on smaller volumes, for example on 10,us of blood (taken from the human finger). To 90 CL of reagent contained in the bottom of a "rhesus"type microtube are added 10 ILl of blood, and the red flecks resulting from metachromatic colouring of the basophils are counted, as in the preceding case.

The results obtained with blood taken from a healthy human and the rabbit were as follows: In Man: 23 to 70 basophils per mm3 of blood (12 samples).

which corresponds to an average + a standard deviation of 44' 8; In the Healthy Rabbit: 298 1 23 basophils per mm3 of blood (38 samples).

The technique described above could be modified to enable other leucocytes to be counted, by operating at a higher pH. In particular it is established, when the pH is progressively increased, that, in succession, the nuclei of the small lymphocytes, then the larger lymphocytes, the monocytes and finally the polynuclear leucocytes progressively stain blue.

The higher pH values are obtained either by operating with a reagent containing less acetic acid, or by adding sodium hydroxide to the reagent before mixing the reagent with the blood sample to be studied.

EXAMPLE 3: Study of the action of allergens.

A fixed amount of blood (250 to 500 microlitres) is introduced into two series of tubes.

Increasing quantities of the allergen to be tested are added to the tubes of the first series and increasing quantities of a control antigen, to which the patient is not sensitive, and added to the second series. After gentle agitation, the contents of the tubes are incubated at 37"C for 10 minutes. A quantity of sodium EDTA is then added to obtain a final dilution of 2 x 10-4 M.

the EDTA arrests the reaction and avoids subsequent aggregation of the cells. The colourations may be carried out in the following six hours, for example by taking a sample from each of the tubes of 10 microlitres of blood and by mixing each of these samples with 90 microlitres of staining composition.

Counting the basophils is then carried out as indicated above. By way of example, there is given in the table below average counts of basophils obtained from the blood of two patients A and B, following incubation in the presence of, on the one hand, pollen, and on the other, penicillin. The counts in the presence of pollen are given in the first horizontal line, and those in the presence of penicillin in the second horizontal line of the table. These results establish the fact that patient A displays an allergy to pollen, whilst patient B displays an allergy to penicillin.

Table A B Pollen .......................... 7 50 Penicillin ........................ 50 12 EXAMPLE 4: Use of microslides in the study of the action of allergens.

Increasing dilutions of the allergen to be tested in distilled water enriched in proteins - for example, 0.01 to 0.1% of human serum albumin - are placed in the indentations of Teresakitype microslides. The liquid solvent is evaporated. These microslides are ready for use. 10 microlitres of blood are added to each of the indentations of the slide, the whole is agitated and the microslide is placed in a humid incubator at 370C for 10 minutes. The blood is then introduced into capillary tubes containing dry EDTA and passed rapidly back into the indentations. 90 microlitres of the staining composition are then added to each of the indentations and the count of the basophils is carried out as described above. The readings can be effected in the cells known by the term "Fuchs Rosenthal cells".

In place of the allergen, any degranulant agent can be tested in this manner. Degranulation of the basophils in the presence of an anti-IgE antiserum can thus be evaluated, which provides a further parameter of the allergic reactivity of the patient.

The whole of the reaction can be carried out on whole or "total" blood or on blood washed twice with Tyrode buffer or other isotonic buffer. By employing allergen levels necessary to bring about degranulation in total and washed blood, it is possible to reveal the existence of "blocking" factors in the plasma - that is to say, factors which consume the antigen and which are of very great importance in allergic pathology.

EXAMPLE 5: In the sole figure of the drawing, a microslide for use in a particular embodiment of the invention has been schematically represented.

It comprises a plurality of indentations arranged in eight rows, denoted respectively by the letters A to H, and twelve columns, denoted respectively by the numbers 1 to 12.

By way of example, the wells of the horizontal row, denoted by the letters A and H, contain a dose of a calcium salt free from antigen, intended to permit the production of the control incubation solutions described hereinbefore.

The wells of the remaining six rows B to G contain decreasing doses of allergen, making it possible to attain subsequently dilutions of allergen of similarly decreasing concentrations.

For example, the wells of row B contain doses which permit concentrations of 10-5 g of allergen to be subsequently attained, those of row C contain doses which permit concentrations 10-6 g of allergen to be subsequently attained, the wells of the remaining rows D, E, F and G contain, respectively, doses which permit concentrations of 10-7, 10-8, 10-9 and 10 g of allergen, respectively, to be obtained. It is pointed out that, of course, these doses are given only by way of example, inasmuch as they may vary from one allergen to another.

The indentations of columns 1 to 12 all contain the same allergen (with the exception of the indentations which form part of rows A and H). Such a slide or microslide permits, therefore, comparison of the counts of non-degranulated basophils resulting from six different samples, after incubation of samples of washed blood and of total blood from each of these samples respectively, in the presence of decreasing concentrations of allergen. For example, the wells of column 1,3,5,7,9 and 11 will receive determinate volumes of samples of total blood from six separate samples and indentations 2, 4, 6 8, 10 and 12 the same volumes of washed blood obtained from the same samples.

A second slide or microslide contains preparations of the same allergen in corresponding indentations, but in every case in the absence of calcium.

After incubation of the whole slide at 37"C, for example for 10 minutes, determinate volumes ot the contents will be taken from each of the wells of the microslide and the counts carried out on each of them under the conditions described hereinbefore.

By operating in the manner which has been described, namely with different doses of antigen (in the well pairs 1,2; 3,4; 5,6; 7,8; 9,10; and 11,12 respectively), it will be possible, not only to ascertain the number of degranulations in those samples taken from wells of one column of the microslide in the presence of the allergen, to the extent to which the latter would be active, but also to determine the intensities of degranulations which can be induced in the samples studied. These intensities are then expressed in terms of the correlation of the dose of antigen to the proportion of basophils which have undergone degranulation.

It is, of course, possible to use microslides having far greater numbers of wells than the microslide shown in the drawing.

In the method of realization which has just been described, the test kit comprised one or more slides or microslides carrying allergen each of these slides permitting counting of the basophils in blood samples from different samples.

Variations within the scope of the invention will be readily conceivable by the person skilled in the art. For example, it is quite possible to prepare test kits containing "mixed" slides or microslides, each of these slides or microslides comprising increasing doses of various allergens. For example, a slide such as the one shown in the drawing could comprise six separate allergens occupying the pairs of columns 1-2; 3-4; 5-6; 7-8; 9-10 and 11-12, respectively. Such a slide or microslide would therefore permit the comparison of the counts of non-degranulated basophils in samples (of total blood and washed blood, respectively) from the same sample, in the presence of six different allergens.For example, the wells of columns 1,3 , 5,7 , 9 and 11 would receive determinate volumes of total blood, and indentations 2,4, 6,8, 10 and 12 the same volumes of washed blood from the same sample.

The wells of the slides or microslides containing calcium salts may each also contain an equal weight of a magnesium salt.

EXAMPLE 6: Preparation of microslides according to Example 5.

Solutions having the following composition were used: Solution 1: - Ca Cl2, 6 H2O 200mg -Mg Cl2, 6 H2O 200 mg - human serum albumin ..................................... 100 mg - distilled H2O ........................................ 100 ml Solution 2: The same as Solution 1, minus, however, the calcium and magnesium salts.

In the preparation of slides or microslides intended to receive each of the allergens to be tested in the presence of calcium, there is introduced, into each well of rows A and H, 10 microlitres of solution 1, without allergen.

Then, into the indentations of rows B, C, D, E, F and G, appropriate doses of the allergen to be tested are introduced, diluted in 10 microlitres of solution 1, so as to be capable of giving suitable dilutions of the allergen in the blood samples to be tested for example, dilutions of 10-5, 10-6, 10-7, 10-R, 10-9 and 10-l".

Corresponding slides or microslides intended to receive the allergen are prepared in the same way, but without the calcium, by carrying out the same operations as for the first slides, but with solution 2 in place of solution 1.

The solutions are left to evaporate at 373C overnight and microslides for use in the invention are consequently obtained. They may be covered with a film of adhesive plastics material to be removed at the time of use.

Advantageously the test kit of the invention comprises in addition one or more tubes prepared in advance to be suitable to receive the blood samples to be studied, these tubes being coated with anticoagulant, preferably heparin and/or EDTA, in an amount sufficient to prevent coagulation of the samples which will be introduced therein during use.

In a preferred embodiment, the tubes may be prepared by using a solution with an ethylenediaminetetraacetic acid and/or heparin base and by leaving the solution to evaporate in the tubes at 37 C. An appropriate solution contains, in 100 ml of distilled water: - 3.7 g of disodium EDTA, - 4.3 g of tetrasodium EDTA.

- 0.4 g of heparin.

A complete test kit of the invention may thus be constituted of: - microslides comprising the afore-mentioned deposits, - tubes, optionally coated with anticoagulant, - washing buffer solution, which is advantageously provided in the lyophilized or concentrated state - for example, concentrated by a factor of 10 - in ampoule or analogue form, reconstitutable with distilled water, - distilled water, and - the staining composition of the invention.

The washing buffer may, for example, have the following composition: - (hydroxymethyl) aminomethane, buffer TRIS ............. 3.03 g - NaCL ....................... 7.00 g - KCl ........................ 0.370 g - glucose (D-glucose + anhydrous glucose) ..................... 1.00 g - pure heparin ..................... 0.040 g - disodium EDTA ........................... 0.46 g - tetrasodium EDTA ....................... 0.54 g - human serum albumin ........................ 2.50 g - distilled H2O to bring up to ........................... 1.000 ml EXAMPLE 7: Investigation of an anaphylactic reaction, particularly of an allergic reac tion, in a patient, with the aid of a microslide according to Example 5.

a) Taking of Blood Samples 5 ml of blood are taken from the patient and introduced into one of the tubes such as are defined above. The tube is placed in an apparatus which permits gentle agitation of its contents until the tests are carried out.

b) Preparation of the Fractions of Total Blood and of Washed Blood Two blood samples of 1 ml are taken [and placed] in two of the afore-mentioned tubes.

These are centrifuged at 800 g for 10 minutes at laboratory temperature, and the upper level of the plasma after centrifugation is marked on the tubes.

The contents of the tubes are made up and washed with a total volume of washing buffer of 4 ml and the washing buffer is separated by means of centrifugation. It is advantageous to repeat the washing, and, after the final separation, the centrifuged deposit of one of the tubes is suspended, under gentle agitation, in the plasma from which it had been separated up to the original level, in order to reconstitute the fraction of total blood, whilst the centrifuged deposit of the other tube is suspended in the washing buffer up to the original level, in order to form the fraction of the washed blood.

c) Process of Incubation on the Microslide Samples of 10 microlitres from the afore-mentioned total blood samples and from washed blood samples are introduced into the wells of each of the microslides containing an allergen under test with regard to the said samples - that is to say, the samples of washed blood are introduced into the wells of vertical rows 2, 4, 6, 8, 10, 12, and those of total blood into wells 1, 3, 5, 7, 9, 11.

The volume of blood introduced into each well is gently sucked in and out by means of a micropipette to ensure good mixing and the uncovered slide is then placed in a humid incubator at 37 C for 10 minutes.

d) Coloring Process At the end of the study, 90 microlitres of the staining composition of the invention are placed in each well, mixing of the colouring solution and the blood being ensured by agitation by means of sucking in and out with the aid of a micropipette.

e) Reading The mixtures from each well are removed and placed in the chambers of a hemocytometer, for example that known under the name "Fuchs Rosenthal cell". It simply remains then to take the reading by microscope.

It has already been indicated hereinbefore how conclusions can be drawn especially from the comparison of the number of degranulations observed in the presence of one or more allergens, on the one hand with the results established in the presence of allergens which have produced no degranulation and, on the other hand, with respect to the various controls.

By using increasing proportions of antigens in the rows B to G, it is equally possible from the results observed in the presence of an allergen which has brought about degranulations to establish curves representative of the variation in the numbers of degranulations observed as a function of dilution. Information may also be obtained on the toxicity level of the tested allergens or the degree of sensitivity of the basophils of the patients from whom the samples come with regard to these allergens.

Having once established the dilution which, in the presence of calcium, brings about a minimum level of degranulation, it may be possible to limit the aforementioned comparisons to observation of the behaviour of the basophils in those indentations of the other microslide which contain the same dilutions of allergen, but in the absence of calcium.

WHAT WE CLAIM IS: 1. A process for selectively staining only non-degranulated basophils in a blood sample containing degranulated basophils and/ or non-degranulated basophils, in which without first fixing leucocytes contained therein, the sample is contacted with an aqueous hypotonic staining composition comprising, per 100 ml of the composition, from 30 to 250 mg of a metachromatic agent, from 20 to 50 ml of an alcohol fixative for the leucocytes including the basophils and an acid having lytic properties with respect to erythrocytes in an amount less than 1 ml, the relative proportions of the sample and the staining composition brought into contact being such that the pH of the formed mixture is from 3 to 5.

2. A process as claimed in claim 1, in which the aqueous staining composition is free from mineral salts which if present would contribute towards making the composition isotonic.

3. A process as claimed in claim 1 or claim 2, in which the metachromatic agent is toluidine blue, the alcohol fixative is ethanol and the acid is acetic acid.

4. A process as claimed in claim 3, in which the aqueous staining composition comprises, per 100 ml of the composition: between 150 and 600 micro-litres of glacial acetic acid; and from 80 to 50 ml of distilled water.

5. A process as claimed in claim 4, in which the aqueous staining composition comprises, per 100 ml of the composition: from 30 to 100 mg of toluidine blue; from 25 to 40 ml of ethanol; and from 75 to 60 ml of distilled water.

6. A process as claimed in claim 5, in which the aqueous staining composition comprises, per 100 ml of the composition: 30 ml of absolute ethanol; 70 mg of toluidine blue; 400 micro-litres of glacial acetic acid; and 70 ml of distilled water, the pH of this composition being from 3.4 to 3.6 7. A process as claimed in any of the preceding claims, in which the blood sample is a sample of total blood.

8. A process as claimed in any of claims 1 to 6, in which the blood sample is a sample of washed blood as hereinbefore defined.

9. A process according to any of claims 6 to 8, in which the pH of the sample/staining composition mixture is adjusted to a value of substantially 4.5 either by simple mixing of the staining composition with the blood sample in appropriate proportions, or by addition of a basic or acidic reagent.

10. A process as claimed in claim 9, in which the blood sample and the staining composition are mixed in a ratio of 1 volume of blood sample to 9 volumes of staining composition.

11. A process as claimed in claim 1 and substantially as described herein with reference to Example 2.

12. An aqueous hypotonic staining composition for selectively staining only nondegranulated basophils in an unfixed sample of, or derived from, blood and containing basophils, the composition comprising, per 100 ml of the composition, from 30 to 250 mg of a metachromatic agent, from 20 to 50 ml of an alcohol fixative for leucocytes including the basophils and an acid having lytic properties with respect to erythrocytes in an amount less than 1 ml.

13. A composition as claimed in claim 12, which is free from mineral salts which if present would contribute towards making the composition isotonic.

14. A composition as claimed in claim 12 or claim 13, in which the metachromatic agent is toluidine blue, the alcohol fixative is ethanol and the acid is acetic acid.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. It has already been indicated hereinbefore how conclusions can be drawn especially from the comparison of the number of degranulations observed in the presence of one or more allergens, on the one hand with the results established in the presence of allergens which have produced no degranulation and, on the other hand, with respect to the various controls. By using increasing proportions of antigens in the rows B to G, it is equally possible from the results observed in the presence of an allergen which has brought about degranulations to establish curves representative of the variation in the numbers of degranulations observed as a function of dilution. Information may also be obtained on the toxicity level of the tested allergens or the degree of sensitivity of the basophils of the patients from whom the samples come with regard to these allergens. Having once established the dilution which, in the presence of calcium, brings about a minimum level of degranulation, it may be possible to limit the aforementioned comparisons to observation of the behaviour of the basophils in those indentations of the other microslide which contain the same dilutions of allergen, but in the absence of calcium. WHAT WE CLAIM IS:

1. A process for selectively staining only non-degranulated basophils in a blood sample containing degranulated basophils and/ or non-degranulated basophils, in which without first fixing leucocytes contained therein, the sample is contacted with an aqueous hypotonic staining composition comprising, per 100 ml of the composition, from 30 to 250 mg of a metachromatic agent, from 20 to 50 ml of an alcohol fixative for the leucocytes including the basophils and an acid having lytic properties with respect to erythrocytes in an amount less than 1 ml, the relative proportions of the sample and the staining composition brought into contact being such that the pH of the formed mixture is from 3 to 5.

6. A process as claimed in claim 5, in which the aqueous staining composition comprises, per 100 ml of the composition: 30 ml of absolute ethanol; 70 mg of toluidine blue; 400 micro-litres of glacial acetic acid; and 70 ml of distilled water, the pH of this composition being from 3.4 to 3.6

7. A process as claimed in any of the preceding claims, in which the blood sample is a sample of total blood.

15. A composition as claimed in claim 14, which comprises, per 100 ml:

between 150 and 600 micro-litres of glacial acetic acid; and from 80 to 50 ml of distilled water.

16. A composition as claimed in claim 15, which comprises, per 100 ml: from 30 to 100 mg of toluidine blue; from 25 to 40 ml of ethanol; and from 75 to 60 ml of distilled water.

17. A composition as claimed in claim 16, which comprises, per 100 ml: 30 ml of absolute ethanol; 70 mg of toluidine blue; 400 micro-litres of glacial acetic acid; and 70 ml of distilled water, the pH being from 3.4 to 3.6.

18. A composition as claimed in claim 12 and substantially as described herein with reference to Example 1.

19. A method of classifying blood samples taken from patients on the basis of whether or not the patient displays anaphylactic sensitivity to an allergen by assessing the degranulant effect of the allergen on basophils in the samples, in which method each sample is divided into two fractions, the fractions are incubated in the presence respectively, of the allergen and of a control antigen for a given period sufficient for at least partial degranulation of basophils in the sample incubated in the presence of the allergen to take place if the blood sample is sensitive to this allergen, each fraction is treated by a process as claimed in any of claims 1 to 11 selectively to stain non-degranulated basophils in the fraction, the stained basophils in each fraction are counted and the two counts are compared, and the sample is classified on the basis of the difference between counts, a significant difference between the counts being indicative of anaphylactic sensitivity of the patient from whom the blood sample was taken with respect to the allergen tested.

20. A method as claimed in claim 19, in which each blood sample is divided into a sample of total blood and a sample of washed blood (as defined hereinbefore), and in which each of these two samples is divided into two fractions which are incubated, stained and counted in the manner defined in claim 19, and the counts are compared, a significant difference between the counts taken from the two fractions incubated in the presence of the allergen being indicative of the plasma in the blood sample counteracting the degranulant effect of the allergen on basophils.

21. A method as claimed in claim 20, in which the sample of washed blood is obtained from the blood sample taken from the patient by separating the plasma from the cellular constituents of the blood, recovering and washing the cellular constituents with an isotonic buffer and suspending these washed cellular constituents in the isotonic buffer, the formed suspension constituting the sample of washed blood, and in which the sample of total blood is obtained by returning the separated and washed cellular constituents to the plasma from which they had initially been separated.

22. A method as claimed in claim 21, in which the isotonic buffer has a pH comprising between 7.4 and 7.6 and contains glucose and proteins in the form of human serum albumin so that the cellular constituents are maintained in a glucose and protein environment comparable to that of the total blood.

23. A method as claimed in claim 22, in which the buffer comprises (tris-hydroxy-methyl) aminomethane.

24. A method as claimed in claim 22 or claim 23, in which the buffer contains an anticoagulant.

25. A method as claimed in any of claims 20 to 24, in which the incubations are carried out in the presence of calcium, and further incubations of samples of total blood and washed blood are carried out to provide a first control in which the calcium content of the samples is masked by chelation before being incubated in the presence of the allergen, and a second control which is incubated in the presence of excess calcium but in the absence of allergen.

26. A method of classifying blood samples as claimed in claim 19 and substantially as described herein with reference to any one of Examples 3, 4 and 7.

27. A test kit for use in the diagnosis of allergies, comprising, each in a separate container, an aqueous staining composition as claimed in any of claims 12 to 18 and samples of each allergen in respect of which the test kit is to be used to test for anaphylactic sensitivity.

28. A test kit as claimed in claim 27, in which the constituents of the aqueous staining composition are presented separately, each in different containers and in volumes corresponding to the proportions defined in any of claims 15 to 17.

29. A test kit as claimed in claim 27 or claim 28, in which the allergen samples are each contained in a well on a microslide.

30. A test kit as claimed in any of claims 27 to 29, in which each of the allergens is associated with a calcium salt in an amount sufficient to promote degranulation of basophils in a blood sample contacted with the allergen.

31. A test kit as claimed in claim 30, in which each allergen with its associated calcium salt is contained on a slide or microslide having a plurality of wells, with each well containing a different dose of the allergen.

32. A test kit as claimed in claim 30 or claim 31, which also comprises samples of allergen not associated with a calcium salt, each of these allergens being contained on a slide or microslide having a plurality of wells, each well containing a different dose of the allergen, corresponding to the doses contained in the wells of the slide or microslide defined in claim 31.

33. A test kit as claimed in any of claims 30 to 32, in which each slide or microslide also comprises wells containing calcium salt in the absence of any allergen.

34. A test kit as claimed in claim 30, which comprises a plurality of slides or microslides having a plurality of wells, the wells of each slide or microslide containing different doses of various antigens.

35. A test kit as claimed in any of claims 30 to 34, in which those wells containing calcium salt also contain an equal weight of a magnesium salt.

36. A test kit as claimed in any of claims 30 to 35, which also comprises, in a separate container an isotonic buffer, as defined in any of claims 22 to 24, or a lyophilisate or concentrate which reconstitutes the isotonic buffer on addition of an appropriate volume of distilled water.

37. A test kit as claimed in any of claims 30 to 36, which also comprises one or more tubes coated with an anticoagulant.

38. A test kit as claimed in claim 27 and substantially as described hereinbefore with reference to either of Examples 5 and 6 and the accompanying drawing.