CA1087966A

CA1087966A - Process and a metachromatic composition for effecting a count of leucocytes, and more particularly of basophiles

Info

Publication number: CA1087966A
Application number: CA255,681A
Authority: CA
Inventors: Jacques Benveniste
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: 1976-06-25
Publication date: 1980-10-21
Also published as: DE2628468C2; SE7607282L; GB1560729A; NL171840C; DK289076A; SE437079B; DE2628468A1; JPS5819063B2; CH613523A5; NL171840B; JPS5224589A; NL7607012A

Abstract

A B S T R A C T

The invention relates to a process for counting in vitro leucocytes, more particularly non degranulated basophils contained in a blood sample which comprises contact of the blood sample to be tested with a reagent containing a metachromate agent, a fixative fox leucocytes and basophils, and an acid present in such proportions that the final pH of the sample/reagent mixture is between about 3 and 5. Said process can be used for diagnosing in vitro anaphylactic sensitivity more particularly allergies.

Description

~Q~37~ti6 The invention relates to means~ more particularly a process and a composition for effecting a count of leUCoCyteB 9 and more particularly of basophils, such as blood basophils in man or an animal7 and it relates also to a proCe~s for dia~noBing in vitro the causes Of Certain anaphylactic disorders, more particularly allergies, employing the means indicated above.
It is known that the methods for dlagnosing allergies, mo~t often used until ~oday, involve, in ~eneral, numerous in~ections of the different allergens to be tested. In addition to the unpleasantness presented by this method of administration, it is sometimes shown to be extremely dangerous for the patients and often gives rise to non-specific reactions which are difficult to explain.
~ or some years methods have existed for making evident anaphylactic antibodies in serum. But, on the one hand7 they are extremely costly, because, they use a very complex technology for purification, for marking with radioactive iodine and for fixing on insoluble supports, purified allergens and antibodies obtained by immunising an animal and then purified. On the other hand, they require specialised equipment (centrifuges7 and equipment for counting radioactive constituents), which makes them extremely costly and unsuitable outside large urban centres of the developed countries. In addition, it is now known that the existence of serum anti-bodies is not su~ficient 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 the symptoms of the allergyO It had? in particular, been observed that the a~ergy phenomena can9 to a great extent, be attributed to the degranulation of the basophils in thq circuIatin~ blood, under the action of the allergen agent, a -- 1 .

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degranulation which is accompanied, inter alia, by relaase of large amounts of toxic products, especially histamine~ which these basophils contained.
It had also been established that the allergy reaction could be reproduced in vitro, the basophils sensitive to degranula-tion under the effect of the antigen studied under-going various morphological or cellular modifications, which were not observed in the basophils in control samples or previously put into contact with allergen agents which were unquestionably inactive with regard to these samples.
This is the reason why certain authors have already proposed methods for diagnosing allergies based upon the study in vitro of cellular modifications which the basophils of a blood sample originating from a patient are liable to undergo, in the presence of antigens or allergens, of which the possibility of inducing anaphylactic reactions in thisi patient is investigated.
~ hese diagnostic methods have, until now, come up against very serious difficulties~ One of the prime causes of these difficulties lies in the extreme, relative rarity of the basophils with respect to the other constituents of the blood, so that the techniques which have been described comprise, in general, two stages, that is to say a first stage of relative ~`
concentration of the blood sample in basophils, then the stage of colouration of the latter.
A standard technique is that which was described by SHELLEY W.B. and L. JUXLIN (B~OOD. 19 : 209 (1962). -Accomplishment of the firs-t stage involved several operations including pUtting the blood sample to be studièd into contact with a medium intended to ensure the fixing of ~the leucocytes, including the basophils, and the destruction of the ~ , .......................................... .

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~Q8~Y9~6 erythrocytes, this medium compri8ing 60 parts of ethyl alcohol, 20 parts of glacial acetic acid and 20 parts o~
chloroform; keepin~ the mixture obtained at 4C until the next day to achieve complete fixin~; eliminating the super-natant liquid and putting the decanted cells back into suspension in the same medlum and filtering the suspension obtained on filter paper~ to obtai~ a residual solid formed by the leucocytes on the filter paper itself.
~ he 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. ~he sample, the basophils of which were coloured red, was then "read" under the microscope.
~ his technique, o~ which only the essential parts have ~ :
been recalled to mind above, is, however, extremely critical to employ, and, as the authors themselves have acknowledged, presupposes enforced training on the part of the experimenters, to the extent that most.frequently differentiaticn between the degranulated basophils and the non-degranulated basophils lies in evaluation of the morphological transformations which can be established in those basophils whlch have undergone this degranulation.
Certain authors have proposed simplified versions :
of SHELLEY's method. For.example, KLOPS~OCK 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, -in order 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 anti~en to be studied, then diluting aliquot -' :1~8~79~;6 parts of this sample and of a control incubated in thepresence of a normal saline solution with five time~ their volume of a colouring mixture formed from 0.25% Of toluidine blue and 2.5% of acetic acid. A~ter incubation for 5 minute~
of the media obtained, a comp~rative study under -the microscope is then effected, of the proportions of the non-degranulated basophils to the basophils which have under~one degranulation in each o~ the samples, evaluation of the degranulation being ~ -based always upon the morphological changes induced in the cells under the effect of degranulation. If the colouring technique is simpler, evaluatio~ of the morpholbgical differ-ences between non-degranulated basophils and degranulated basophils still remains ~ust 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 again included a stage of concentrating the basophils, espedially by differential centrifu~ing of the blood sample previously incubated in the presence of the antige~ to be studied, the study of the basophils in the concentrate obtained, which contained the leucocytes and amongst them the basophils, being then effected under the microscope, after colouration with a colouring reagent based upon toluidine blue, in a water/alcohol solution containing 5~/a of ethyl alcohol. ~valuation of the possible degranulation of the basophils can, however, ln this method, only be made even more difficult, to the extent that the observer sees not only the basophils but also all the other leucocytes appear, the whole being envelopped in a sheet of erythrocytes~
Although these various methods or techniques present a certain degree of efficacy~ they have not, however, bee~ used :`:
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in practice, because of the minutiae with which the reagent~
have to be prepared, the length of the tests, their tedious nature and the necessity of having very well-trained staff availableO
Generally speaking, there are only a few visualisation methods permitting a simple count of the basophils. ~he difflculties inherent in these counts have, indeed1 be~n explained by James ~. MOOR~ and Associates (P~S~BoMo 1953 ~
vol~ 82, pages 601-603). These authors have already explained ' :
that differentiation of the basophils with respect to the other . ~ .
leucocytes in a blood sample presupposed that previous haemolysis of the erythrocytes is effected~ without the water~soluble granules of the basophils being dissolved at the same time.
He indicated in particular that acetic acid, amongst other organic acids, however 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 had to establish that 15 - 30~ solutions of alcohol, however effective for fixing the granules, were not effective as haemolysis agents.
In order to remedy these apparently insurmountable difficulties, they proposed, consequently, a quite different process for metachromate colouration of the basophils, consist-ing of putting a sample of blood to be studied in contact with a mixture formed from ~0 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 s~ponine in ethyl alcohol.
~ he method recommended remains, however, difficult to apply, especially when it is a question of applying~it to the differentiation of the basophils from one another, accordin~
to whether they are degranulated or not, a problem which the , 10~'7966 :~, , . - , ::
invention aims at solving.
- Besides, the reproducibility of this method is far from being perfect, even when it is used only to differenciate ;
the basophils from the other leucocytes in the blood. Meta-chromate colouring is not distinct. Saponine tends to precipitate in the medium.
A11 the difficulties which have arisen mean that until the invention, current haematology laboratories have not called for methods of studying in vitro in order to achieve diagnosis of anaphylactic sensitivity, more particularly allergies.
~ he aim of the invention is to provide means, especially processes for visualising and counting in vitro basophils, more particularly non-degranulated basophils, excluding those which have undergone degranulation~ and possibly leucocytes, too, which means can be applied to blood samples, in a single stage, without any previous ;
concentration belng necessary for certain of the constituent9 of these samples, and which are extremely easy to use, reproducible and within the scope of anyone handling them and who is not specially trained. Its aim is also to provide a reagent which permits, after being put into contact with the samples of blood to be tested, quick and immediate counting, by means of counting apparatus, especially standard haemocyto-; meters. Its aim is also to produce such rèagents which are very 9table over a period, so that it will no longer be necessary to prepare extemporaneously the reagents appropriate for each series of tests. ~inally the invention aims at perfecting a method for diagnosing in vitro anaphylactic sensitivity, more particularly allergies, b~ which patients can-be ~ffected.
~ he process according -to the invention of visualising .

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the basophils and, possibly too the other leuGocytes contained in a biological medium1 especially a blood sample, is characterised by the direct mixture of this sample, preferably in a single stage~ with a hypotonic aqueous .:
reagent containing a metachromate agent t a fi~ative for leucocytes and basophils, an acid suitable for contributing to the.destruction, caused at least in paxt by the water contained in the reagent, of the erythrocytes in the blood sample, this acid being present in the reagent in such proportions that the final pH of the sample/reagent mixture is between about 3 and about 5.
More particularly, the process according to the invention for visualising the basophils contained in a sample of blood or the like, using a metachromate agent~ is characteris .
ed by putting this sample, in which the basophils have not bee~
prevlously fixed, into direct contact with a hypotonic a~ueous reagent containing together the metachromate agent, an alcohol suitable for permitting the basophils to be fixed and an acid having lytic properties with respect to erythrocytes, in such proportions that the pH of the sample/reagent mixture is between 3 and 5;
the metachromate agent concentration of this reagent .
being sufficientl.y low to allow adequate differentiation of the colouration speeds of the non-degranulated basophils, on the one hand, and of the slower colouration speeds of the basophils which have undergone previous degranulation, on the other hand;
the alcohol concentration in the reagent being sufficiently weak for;the non-degranulated basophils to have time to undergo colouration, yet sufficiently high for the non-degranulated basophils to be fixed.before they hav~e had time to be coloured in turn.

~Q8~966 ~ he water and acid contents of the reagent thus formed are sufficient to warrant lysis of the erythrocytes to the extent necessary to permit further direct observation of the non-degranulated basophils, the only ones of all the basophils which are coloured-.
The pH conditions are very important. At a pX below 3 the colouration ob~erved 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 ~ythrocytes are, at heat, no more than partially lysedO
~he invention thus sets in action, in the pH bracket -indicated, a competition between the colouration speeds of ~-the intact basophils on the one hand, and of degranulated ~ -basophils on the other hand, b~ means of the metachromate agent, and the fixing speeds of the basophils, possibly t~o, of the other leucocytes. Due to the concentrations of fixative and metachromate agents used respectively, only the i intact basophils have time to be colouredt before being fixed . .
by the fixative, whereas the degranulated basophils are fixed be~ore they have time to become the subject of a colouration which will later be visible to the naked eye.
It is interesting to note that the non-degranulated baso-phils appear to the observer to be coloured throughout their hole mass, which can, perhaps, be attributed to the basophil granules begining to dissolve in the medium which has pene- ;
trated into these basophils, but inside the cells, fixing of the latter having ~he 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 ~ame manner, the possible weak colouration induced in the g~anules and still existing in these basophils ceasing to be observable , . . ,:, .. : . . .. ~ . .

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9~d6 because of its too great dilution, even if it is only in the heart of these basophils.
Naturally t it goes without saying that there it is only a question of hypotheses which have no other claims than that of making it seem that the invention apparently derives advantage from what was previously considered to be a disadvan-tage, more particularly in so far as the effect, previously deemed to be di~astrousil of the acetic acid on the water-solubility of the basophil granules is concerned.
The invention also relates to the reagent itself which is characterised in that it is formed from a hypotonic aqueous solution containing a metachromate agent, especiall~ toluidine blue, a fixative for the leucocytes and the basophils, and an acid suitable for contributing to the destruction of the erythrocytes in the blood samples, this acid being present in the reagent in such proportions that the final pH of the reagent is between about 3 and about 5.
It i9 noted that the reagent responds appreciably to the conditions which were indicated with regard to the process according to the invention. Given that, generally speaking, the blood sample must be diluted in a volume which is a multiple of the reagent~ it appears that the respective proportions of the constituents of the reagent and the pH
conditions will extend approximately to the reagent/blood sample mixture to be studied, and vice versa.
In the reagent according to the invention, the fixative is preferably constituted by an alcohol miscible with water and suitable to achieve fixing of the leucocytes and stabili-sation of the basophils. ~he water acts both as diluent for the alcohol and as lytic agent for the erythrocytes. ~he acid is selected from amongst those which are compatible _ 9 - - ~V~ 79 6 6 .
with the leucocytes, more particularly the basophils. Prefer-ably, the reagent is free from any mineral salts of the type of those which, ~uch as sodium chloride, can contribute towards making an aqueous solution isotonic.
In its preferred form, the reagent according to the invention contains~ for a volume of 100 ml of liquid : -- from about 20 to abou~ 50 ml of alcohol, - from about 80 to about 50 ml of water, preferably distilled, if desi~double-distilled~ water, - less than 1 ml of an acid and - the necessary amount of metachromate agent.
A preferred metachromate agent is constituted by a toluidine blue. Other metachromate colourants can, of course, be used, for example tho~e described in the work !'Mast-cells and basophils", Annals of the New York Academy o~ ~ciences9 Vol. 103, 1963. The preferred acid and alcohol are constituted, respectively, by ethanol and acetic acid.
It has been established that it is thus possible to achieve, simultaneously in a single stage9 using the reagent according to -the invention9 the destruction of the erythrocytes in the blood sample, the fixing of the leucocytes and the stabilising ~f the basophils, and finally and in particular de the 9elective red colouration of the nont~ranulated basophils when the me~achromate colouring used is constituted by a toluidine blue. This colouration is immediate in practice and when the mixture is transferred to the chamber of a haemocyto-meter 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, and which have not undergone degranulation.
When ~he pH of the mixture is of the order of 4~5, colouration of the cytoplasm of the single basophils is . ,, ., ', .. .''' ~ '., : . ' : ' ~ ;
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observed. A very slight blue colouration Or -the nuclei of the other leucocytes is also noted, but the cytoplasm of cells other than thè basophils i5 never coloured, so that the only cells which appear to be coloured red,throughout their mass, are the basophils. It is recalled, in fact, that the granules contained in the cytoplasm of the basophils, and coloured by the above-mentioned metachromate colourants, are specific of basophils, for which reason the cytoplasms of the other polynuclear cells and of the leucocytes are also not coloured, whence arises the possibility of counting the basophilic polynuclear cells on the one hand, and the other polynuclear cells, and the lymphocytes on the other hand.
~ he pH selected, results from the direct mixture of proportions of reagent and of blood, adjusted depending upon the pH value desired. Possibly, the pH of -the mixture can also be adjusted by modifying the pH of the reagent before mixing, for example using acetic or hydrochloric acid or a base constituted, for example, by soda, according to whether it is wished to reduce or, on the contrary, to increase the pH value of the mixture. It is, of course, particularly simple to regulate previously the acid content of the reagent, depending upon the pH finally desired in the mixture.
~ he process is an ex-tremel~ simple operation~ It can be applied to whole blood, without any necessity to separate previously certain of its constituents. It can be applied to natural blood or blood previously washed with an isotonic bu~fer, for example that known by the term "~yrode buffer".
It can be used directly on fresh blood or on blood containing an anticoagulant, such as heparine, in the case of blood which has been kept for some time.
~ he proportions of blood and reagent which arb mixed depend, inter alia upon the initial pH of the reagent and on ~ Q8~9~;
~he pH desired in the mix-ture~ In the pr~ferred case called to mind ~urther on, with the ~'optimum" reagent, the mixture of 1 volume of blood with 9 volumes of reagent leads direct to a pH of the order of 4.5 permitting selective colouration in practice of the single basophils which have not undergone degranulation~
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~ he simplicity of the method, complemented by the excellent "legibility" of the slide~ (the red basophils stand out easily on the light background of the treated sample), put it quickly within the scope of any untrained person handling it.
Finally, the reagent i5 not very expensive, bearing in mind the present nature o~ its constituents.
Finally, a wise choice of the proportions o~ the constituents of the reagent leads to a perfectly stable mixture, for periods which can last for several months at ~ ambient temperature, although it is of interest to k~ep i- such reagents at a temperature of the order of 4C, with the aim of reducing the risk of the alcohol evaporating.
In this respect, preferred reagents are -those which contain the following proportions by volume - or by weight ~ as far as the metachromate agent is concerned - of the said ; preferred constituents, for a volume of 100 ml of liquid:
- 20 to 50 ml of ethanol, - 80 to 50 ml of water, - an amount between 150 and 600 microlltres of glacial ~; acetic acid 9 such that the pH of the final mixture is between about 3 and about 5, and - 30 to 250 mg of ~oluidine blue~
~ he compatibility o~ the constituents of this mixture, the great stability of the latter as well as its efficacy :.

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in metachromate colouring of the basophils under the conditions indicated, have been proved.
The proportion~ which are even more p~erred Of the various constituents in the reagent according to the invention are as follow~:
- 25 to 40 ml of ethanol, 'r ~ 75 to 60 ml of water, - an amount between 150 and 600 microlitres of glacial acetic acid, such that the pH of the final mixture is between about 3 and about 5, and - 30 to 100 mg of toluidine blue, for a total volume of 100 ml of reagent.
~ Optimum results are obtained with a reagent at pH
3.4 - 3.6 containing, for example, the above-mentioned constituents in the above-mentioned proportions:
- about 30 ml o~ absolute ethanol~
- about 70 ml of distilled water, - about 70 mg of toluidine blue, - about ~00 microlitres of glacial acetic acid.

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~ he invention also relates to a process for diagnosing in vitro anaphylactic sensitivity, more particularly allergies, based upon counting the leucocytes, in particular the basophils, such as the blood basophils in man or an animal.
According to this process, samples of a blood sample are incubated, which were given in the presence, respectively, of an antigen of which it is desired to verify the degranulating activity, and of a control antigen held to be inactive with respect to the basophils of the sick person under consideration, and after the time necessary to achieve at least partial degranulation of the basophils in the presence of the antigen to be tested, insofar as the blood :
sample tested is sensitive to the latter antigen, the various fractions are mixed with a reagent as defined above, under the conditions also indicated above, then in each of the fractions counts of the visible basophils are effected by means of a standard haemocytometer and the counts obtained are compared.
By comparlng the results obtained on the samples tested with those obtained on a control sample, it is decided immediately whether or not the sick person is sensitive to the antigens tested, according to whether or not the counts carried out on the samples tested differ in a 9ignificant manner from those obtained with the control fraction.
It is understood that the invention applies to diagnosin~ the sensitivity of the patient to any antigen or chemical agent liable to cause degranulation of the basophils, even if, in the most frequent cases, these agents or antigens are constituted b~ allergens.
The great advantage of the method ~ diagnosis accord-.
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8~96;~i ing to the invention lies in its simplicity. It is based solely upon counting techniques, without it being :
necessary to become involved in morphological trans-formations o~ which the basophils which had undergone degranulation formed the subJect. It also avoids having recourse to concentration operations which can only be employed in specially-equipped laboratories and by trained technicians.
~he counts are preferably carried out on blood samples immediately after the incubation operation, in the presence of the allergen to be testedO ~hey can also 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-ag~regating agent such as the sodium ethylene diamine tetracetic acid (EDTA) salt.
~he sensitivity of the method according to the arrangement set out above, in the majority of cases, enables a conclusion to be made clearly in favour of the sensitivity of a sick person with regard to an antigen tested, as soon as a significant reduction is observed in the counts carried out on blood samples incubated in the presence o~ this antigen with respect to counts carried out under similar conditions on a control sample.
But due to the simplicity and the sensitivity of the method according to 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 the sensitivity, which is, moreover, v obvious, of these patients with regard to the antigen studied, could be established. I
In addition, thanks to the method of the invention, it .

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was possible to elucidate the nature of the phenomena, whichwere the source of these discrepancie~ and to ob-tain additional informatlon about the behaviour of certain factors contained in the blood with regard to the antigens studied. In particul.ar, another arrangement of the proceeds from the statement that the plasma could in certain cases, contain factors, hereinafter called "blocking factors", which conbradict the allergen/basophil reaction.
~ his arrangement, according to which the incubation of various blood samples to be studied is always carried out in contact with different antigens under the conditi.ons set out above, and in which, after the period which is necessary eventua~lly to obtain, in one or other of these .
samples, at least partial degranulation of the basophils in the presence of the correspond.ing antigens, a count is made of the basophils which have not undergone degranulation, :
preferably having recourse to the reagents of the invention, ls characterised by dividing each of the blood samples on which the above-mentioned operations are to be carried out, on the one hand into a sample of whole blood and, on the .:-other hand, into a sample called hereinafter "sample of :.
washed blood", from which -the plasma has previously been removed; b~ carrying out for each of these blood samples, the above-mentioned incubations and counts, both on the sample of whole blood and on the sample of washed blood, the possible obtension of a different meaning between the counts obtained for each blood sample expressing then both ::
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the anaphylactic sensitivity of the patient with respect " to the corresponding antigen studied and the presence i~ :
the plasma of factors contradicting the degranulating action ... .

~tl8'7966 .~, .

of the antigen with respect to the basophils.
In fact, as the two types of samples are distinguished from one another by the presence or absence of plasma9 the observation of a difference in the result can only come from the plasma, more precisely from the constituents or factor3 which it contains and which protect the basophils, more pàrticularly the immunoglobuline 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 antigen in these samples ls inhibited and the number of degranulations observed is no longer indicative of the actual degranulating action of the antigen.~
On the contrary, the number of degranulations observed in the samples of washed blood will be all the more representative of the actual harmfulness of the antigens tested with respect to the basophils, their actlon then being no longer contradicted by the factors which, in this type of blood sample, were present in the plasma.
! On the contrary, when the counts obtained on the samples sample of washed blood and of whole blood from the same/do not display any significant difference, the absence, in the plasma of the patients from whom the blood sample comes, of ~` bloc~ing factors with respect to the antigens studied can reasonabl~ be inferred, of course in so far as, moreover, the comparison of the counts is observed on these samples and on control samples 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).

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- A prime, important advantage of the above-mentioned -~ arrangement of the invention lies~ then, in the fact tha~
in practice it enables comparisons prevlously deemed necessary between the ef~ects of the antigen to be tested and the control antigens, especially non-allergens with regard to patient~ 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 antigens tested.
Generally speaking~ the sample of washed blood is, in ~act, constituted by a suspension of the cèll constituents, especially erythrocytes and leucocytes, in suspension in an isotonic buffer compatible with these cell constituents.
In other words, this isotonic buffer is, in the sample of washed blood, substituted for plasma in the sample of whole blood. Advantageously, this isotonic buffer con-tains, in addition, constituents such as carbohydrates~ especially glucose, and proteins, 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.l~ to 7.6 and is based upon(tris-hydro~ymethyl) aminomethane.
Possibly it also contains a small amoun-t of anticoagulant, such as heparine and ED~A. ~he EDTA has, inter alia the effect o~ stabilising the reactions, of rendering the blood uncoagulable, and of preventing cell aggregations and the j deposition of cells on the walls of the tubes. The heparine ` - contributes to these functions, too, and protects the EDTA
from chelation by the added calcium. Of course, these .
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constituents can be replaced by others, in so far as they are suitable for performing the same functions.
Advantageously and so as to reduce even further the influence of parameters such as those which would result from the difference in the physical treatment to which the cell constituents of the sample of.whole blood and the sample of washed blood are subjected previously, because of the physical separation operations to which the cell constituents of the latter and of the plasma in which they are previously immersed have been subjected, recourse is made to a sample of whole blood constituted, in fact, by a sample of reconstituted whole blood, obtained by separating the cell constituents and the plasma in an initial sample originatlng from the blood sample to be studied, under conditions identical to those used for the preparation of the sample of washed blood, the cell constituents thus separated being then reunited again with the plasma. ~he 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.
~ he separations of the cell constituents and the plasma into the fract1ons under consideration can, of course, be carried out in any manner known per se, especially by centrifuging, the cell constituents of the different samples being, if necessary, washed with the same buffer, the cell cons-tituents ~ree from plasma finally obtained being then associated with the amounts of plasma from which they had :.
been separated respectively, as far as the samples of whole b~ood are concerned, and with volumes of isotonic buffer equal to those of the previously-separated voluml~e of - 19 ~

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plasma, as far as the samples of washed blood are concerned.
According to an additional arrangement of the invention, the fact that the degranulation of the basophils, under the èffect of certain antigens9 Can only work in the presence of calcium, is turned to advantage. ~he improvement attributed to the process according to the invention, in which the above-mentioned incubations are carried out in the presence .. . . .
of calcium, is more particularly characterised in that, for checking purposes, incubation of the samples, respectively of whoIe blood and of washed blood, is carried out, moreover, in the presence of an excess of calcium, but in the absence of the antigens studied.
A comparison of the results obtained enables an anaphylactic sensitivity with respect to the antigen studied to be distinguished from an~ other possible form of sensitivit~
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 ~actors other than the antigen to be tested.
According to an additional preferred feature of the improved process for diagnosi~ according to the invention, there is also carried out, still for control purposes, incubation of the above-mentioned samples of whole blood and of washed blood, of which the possible calcium contents were previously masked, especially by chelation, in the presence of the antigen to be tested, but in-the absence of calcium.
An absence of degranulation in the samples tested in the presence of the antigen alone, together with the observation of degranulation of the basophils in the presence of the same antigen, but in the presence of calcium, en~bles the anaphylactlc sensitivity of the patient from whom come '~
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the blood samples, to be confirmed with regard to this antigen. On the contrary, degranulation of the basophils~
in the presence of the an-tigen alone and in the absence of calcium, shows to~icity of the antigen with regard to the basophils which do not cause the IgE anaph~lactic s~stem to come into play. ~his to~icity can be due to multiple factors, whether it is a question of the presence in the medium of contaminants or of degraded products, etc.
i ~: . ~ ,., . : , . . ,: , ~

: .:. : ' : . '~,: : ': ' - ': : :: . . :' , . ., . ' ': .' . :.-' .:

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.
~879~6 : . .
The observa-tions above thus show the importance .-. of the gains made by the improved process according to 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 of the~ .
"kit" type, in which are assembled the reagents necessary for the diagnosis of allergies or the like.
In one of its preferred forms, the pharmaceutical presentation, comprises. on the one hand a liquid reagent according to .
; . .. .
the invention, and on the other hand the basic allergens necessary for carrying out a diagnosis of anaphylactic sensitivity, especially of the allergy type, in receptacles which are9 respectively, separate. Possibly the essential constituents of the liquid reagent can also be contained in separate receptacles, if desired in volumes corresponding to the proportions required in the reagent, so that the person handling this will only have to mix them in order to constitute this reagent which can then be kept for a prolonged period because of its stability, especially if the above-mentioned propor-tions correspond to the preferred cases which were set out above. ~he antigens which are brought together with the blood samples to be tested can be in the.form of a solution. However, in a preferred form of presentation of the basic allergens, recours~is made to small tubes or to slides, microslides or the like having depressions, these tubes or depressions containing predetermined amounts of th~ese different allergens, in the dry staten These amounts are adapted to the dilutions subsequentl~ desired in the samples of blood introduced into these wells or depressions. Deposi-tion of these allergens in these tubes or depressions can be carried out in any manner known ~ se, for example st~rting . . . - , 7~

from suspenslons or solutions of these allergens previously places in tubes or depressions, by lyophilisation or evapora-tion of the solvent.
By having recourse to this latter form of presentation, diagnoses of allergies can thus be effected very quickly.
It is, in fact, sufficient to introduce into each of these tubes or depressions a predetermined amount of blood, for example using a micropipette9 to carry out incubation of the blood in contact with these allergens at a temperature and for a period suitable for carrying out, in one or other of these tubes or depressions, the degranulation of the blood basophils? in so far as the latter would give~rise to such a reaction in the presence of the corresponding allergen, -then ~ ~`
to add, to each of these tubes or depressions, a volume, also predetermined, of the reagent according to the in~ention, and then to carry out a count of the basophils in each of these depressions, under the conditions which have been set out above.
In order to enable the comparisons set out above between the whole blood and the washed blood to be carried out, recourse is advantageously made to a pharmaceutical presentation containing on the one hand the liquid reagent of the invention, or the constituents necessary to prepare it, and on the other hand the antigens contained in wells br depressions in a slide or in a microslide, this pharmaceutical presentation being characterised in that each of these antigens is associated with an amount of the calcium salt necessary for the degranulation of a part at least of the basophils, in the sample of blood introduced into the corresponding wells or depressions, during incubatiorl under conditions suitable for ensuring this degranulation oflthe _23 _ ~0~'796 Ei antigen under considera-tion. ~dvantageously, there is provided in this slide or microslide with antigen, and for each of the slides, a serie~ of wells or depressions containing, respectively, increasing doses of this same antigen, ~n or-der to permitting preparation of respectivel~, 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 antigen, but also the degree of the sensitivity of his/her basophils with regard to this antigen D
According to one form of making such a presentation or "kit" according to the invention, each of the above-mentioned microslides includes, in addition, wells or depressions containing9 respectively, the antigen alone1 in the absence of calcium, and wells or depressions containing the calcium salt alone, in the absence of the antigen.
In another form of making this "kit", each of the above-mentioned microslides lS subdivided into two microslides, one containing a series of wells or depressions containing only calcium and series of wells or depressions containing both calcium and the corresponding antigen, under the conditions indicated above, the other microslide incorporating corresponding series of wells or depressions containing corresponding doses, respectivel~ increasing (or decreasing), of the same antigen, in the absence, however, of calcium.
~ he series o~ wells or depressions in the first microslide, which contain, as the main ingredient, only calcium~
could ofcourse also be borne by the second microslide.
Obviously the invention is not limited to the cases mentioned. It is clear that all kinds of variations can be imagined, which~ of course, wi]1 not be outside the 'scope of the invention.

Other characteristics of the inven-tion will become apparent later in the course of the specification which follows ways of implementing the process and the reagent according to 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 plastic tubes). Whilst awaiting realization of the tests, the blood is kept either in ice or under agita~ion in order to avoid non-specific aggregation of the leucocytes.
EXAMPLE 1 : Preparation of the colouring reagent according to the invention.
70 ml of 95/ ethanol are mixed with 30 ml of distilled water, then 75 mg of toluidine bluei such as that marketed by K and ~, Plainview Lab., NY, USA are added. This mixture is sub~ected to agitation until total dissolution is attained~ While continuing this agitation, 400 micro-litres of freezing acetic acid are added, which takeslthe pH of the solution to 3.4 - 3.6. This solution is filtered fort~-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~ It is desirable also to filter the solution about every two months.

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~ . - .

'7~ 6~

EXAMPLE_2 : Metachromatic colouring, and counting, of blood basophils.
0~50 to l ml of blood (human or rabbit~is poured into a tube containing lO units per ml of heparin or 25 microlitres of sodium ~D~A, 0~2 M of pH 7.4. ~his blood sample is then diluted in nine times its volume of the colouring reagent of example l, which leads to a pH in the order of 4~5~
~he mixture is gently agitated and one proceeds, after 4 - 5 minutes, to the counting of the basophils, -in a manner known per se, in the chamber of a hemocyto-meter.
In the same way, it is possible to work on smaller volumes, for example on lO ~1 of blood (taken from the human finger). ~o 90 ~lof reagent contained in the bottom of a "rhesus"-type microtube are added lO ~l of blood, and the red flecks resulting from metachromatic colouring o~ 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 health~ rabbit 298 ~ 23 ba~ophils per mm3 of blood (38 samples).
~ he technique described above equally enables the other leucocytes to be counted, when one operates 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, 8~7966 the monocytes and finally the polynuclear leucocytes progressively stain blue.
~ he highest pH Lvalues] are obtained either b~
operating with a reagent containin~ less acetic acid, or by adding sodium hydroxide to the reagent before mixing the reagent with the blood sample to be studied.
EX~MP~E 3 : Study~of the action of allergens.
A fixed amount of blood (250 to 500 microlitres) is introduced into two series of tubesO Increasing quantities of the antigen to be tested are added to ~ -the tubes of the first series and increasing quantities of the control antigen, to which the patient is not sensitive. A~ter gentle agitation, the contents of the tubes are i~cubated at 37C for 10 minutes. A
... .
quantity of sodium ED~A is then added to obtain a final dilution of 2 x 10 4 M. The ED~A 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 the colouring -reagent.
Counting the basophils is then carried out as indicated above. By way of example, there is given in the table below the average counts of basophils obtained ~rom the ~lood of two patients A and B, following incubation in the presence of~ on the one hand, pollen, and on the other, penicillin~ ~he counts in the presence of pollen are given in the first horizontal line, and those in the presence o~ penicillin in -the second horizontal line of the table. ~hese results establish the fact that patient A displays an allergy to pollen, - , ' -796~
whilst patient B ~isplays an allergy to penicillin.

- ~able A __ Pollen O.~.......... ~ 7 50 Penicillin ~7~00~ 50 12 ~ _ ,,,.___. , .... ~ ~

EXAMP~E 4 : Use of microslides in the study of the action of allergens.
Increasing dilu-tions of the antigen to be tested in distilled water enriched in proteins, for example 0.01 to Ool% of human serum albumin; are placed in the cups of ~eresaki-tgpe microslides. ~he liquid solvent is evaporatedO ~hese microslides are read~ for use. 10 microlitres of blood are added to each of the --cups of the slide, the whole is agitated and the microslide is placed in a humid incubator at 37C for 10 minutes. ~he blood is then introduced into capillary tubes containing dry ED~A and passed rapidly back into the cups 90 microlitres of the colouring reagent are then added to each of the -- cups and the count of the basophils is carried out as described above. ~he 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 patien-t.
The whole of the reaction can be carried out on "total" blood or on blood washed twice with some Tyrode~

, . ..
, ' ~,' ~ ,~ - .

796~

buffer or other isGtonic bufferu By incorporating the allergen levels necessary to bring about degranulation in total and washed blood, it is possible to reveal the existence of "blockingl' factors in the plasma, that is to say, which consume the antigen, [and] which are of very great importance in allergic pathology.

, EXAMPL~ $ : In -the sole figure of the drawing, a micro-slide according to a particular method of carrying out the invention has been schematically represented.
It comprises a plurality of ~---cups 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 which were the subject of the preceding description~ ~
~ he wells of the remaining six rows B to G contain decreasing doses of antigen, making it possible to attain subsequently dilutions of antigen of similarly decreasing concentrations. For example, the wells of row B contain doses which permit dilutions of 10 5 g of antigen to be subsequently attained, those of row C
CGontain] doses which permit dilutions containing 10 6 g of antigen to be subsequently attained, the wells of the remaining rows D, E9 ~ and G contain, respectively, doses which permit dilutions of 10 7, 10 8, 10 9 and 10 10 g of antigen, respectively, to be obtained. It goes without s~ying, of course9 that these doses are given only by way of e~ample, inasmuch as they ma~ naturally vary from one antigen to another.
The cups of columns l to 12 all contain the same antigen (with the exception of the cups 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 lncubation of samples of washed blood .

8~g~

and of total blood from each of.these ~amples, respectively, ::
in the presence of decreasing dilutions of antigen, for -;
example such as those indicated above. For example, the wells of column 1, 3, 5, 7, 9 and 11 will receive determinate volumes of samples of total blood from 8iX separate samples and cups .: . 2l 4, 6, 8, lO and 12 the same volumes of washed blood obtained from the same samples.
The second slide or microslide contains preparations :
of the same antigen, in corresponding . ..cups in every case, however, in the absence of calcium.
After incubation of the whole of the slide at 37C, for example for lO minutes, determinate volumes of the ~ cups. ' contents will be taken from each of the wells o~ the microslide and the counts carried out on each of them, more especially in the conditions described in the preceding part [of this documentJ. .:-.
By operating in the manner which has been des.cribed, namely with different doses of antigen (in the well pairs 1~2; 3,4; 5,6; 7,8; 9,lO and 11,12 respectively), .
it will be possible, not only to ascertain the :
degranulations in those of the samples taken from. ~ :
certain of the wells of one of the columns of th'e microslide, in the presence of the antigen~ to -the extent to which the latter would be active, but also to determine the intensities of degranulation which it can induce on~
the samples studied. ~hese intensities are then . .
expressed by the value of the - ra~io .of the dose .::
of antigen to the proportion of basophils which have undergone degranulation.
It goes without saying, o~ course, that it is possible to use microslides havingfar greater numbers of wells than the microslide described by way of example.

` " :L08796~

In the method of realization which has just been described, the "kit" comprised a slide or microslide with antigen, each of these slides permitting namel~ the counting of the basophils in blood samples from different samples.
It goes without saying, of course, that the invention covers all the variants which are readily conceivable by the person skilled in the art~ For example, it is quite possible to prepare "kits" containing "mixed" slides or microslides, each of these slides or microslides comprising increasing doses of various antigens. ~or example~ a slide such as the one represented could comprise six separate antigens 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 ~herefore 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 antigens to ;~
be tested. For example, the wells of columns 1,3 ~ 5,7 , 9 and 11 would receive determinate volumes of total blood, an~ cups 2, L~ , 6~ 8 ~ 10 and 12 the same volumes of washed blood from the same sample.
Example 6 : Preparation of microslides according to example 5.
Solutions having the following composition were used:
Solut,ion 1 ., - Ca C12~ 6 H20 ~ O~ 200 mg - Mg C12t 6 H20 ~ O~O~ 200 mg - human serum albumin ~ O~ O~ 100 mg - distilled H20 .......... ~........... ~..... 100 ml Solution 2 : ~ I
~he same as Solution 1, minus, however, the calcium 0~'79 ~ 6 and magnesium salts~
. With re~ard to the preparation of a slide or micro-slide intended to receive each of the antigens 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 antigen Then~ into the :. cups of rows B, C, D, ~
and G, doses of the antigen to be tested are introduced, diluted in 10 microlitres of solution 1, and suitable to subsequently obtain dilutions of the antigen in the blood samples to be tested, introduced into these .
. . cups in the adequate volume, of respectively equal dilutions, for example of 10 5, 10 6, 10 7, 10 8, 10 9 and 10 10.
Corresponding slides or microslides intended to receive the antigens are prepared in the same way, however, without the calcium, by carrying out the same:operations as for the first slides9 but.with solution 2 in place of solution l.
~ he solutions are Ieft to evaporate at 37C overnight and microslides correspondi.ng to the invention are :
consequently obtained. They may be covared with a film of adhesive plastic material to be removed at the time o~ use.
Advantageously the presentation of the "kit'1 type ~ccording to the invention comprises in addition tubes ::
prepared in advance, suitable to receive the bl.ood samples to be studied, these tubes being more particularly coated with a quantity of anticoagulant, namely with heparin and ED~A, sufficient to prevent coagulation of the samples which.will be introduced therein at the time of use. ~ ~.
In a preferred example of application according to ~ 33 ~

.. . - . ... . ~ .

~o~g~

the invention, the tubes may be prepared by using a solution with an ethylene~diamine tetra acetic acid and/or heparin base and by leaving the solution to evaporate in the tubes at 37C. ~or example7 one could make use of a ~olution containing, in 100 ml of distilled water : .
- 3.7 g of disodium ED~A, - 4.3 g o~ tetrasodium ED~A, - 0.4 g of heparin.
A complete presentation of the "kit" type according ~ `
to the invention will thus be constituted of :
- microslides comprising the afore-mentioned deposits, - tubes9 possibly coated with anticoagulant, ~ washing buffer solution, which is advantageousl~ :
provided in the lyophilized or concentrated state, ~or example 10 times, in ampoule or analogue form, reconstituable with distilled water, - distilled water, and - the reagent according to the invention.
The washing buffer may, for example? have the following composition ~
- (hydroxymethyl) aminomethane, buffer TRIS ... 3.03 g - ~aCl ..... ~........ ~......................... 7.00 g - Kal ...... O............... ~.............. 0... 0.370 g - glucose (D-glucose ~ anh~drous glucose) ..... 1.00 g ~ pure heparin ~ O~ 0-040 g - disodium EDTA ............................... 0.46 g - tetrasodium ~D~A ............................ 0.54 g - human serum albumin ......................... 2.50 g - distilled H20 to bring up to ................ 1.000 ml Example 7 : Disclosure of an anaphylactic reaction, more particularly of an allergic reaction, in a patient, with ~ 34 -. , .

the aid of a microslide according to example 5.
a) Takin ~of blood samples 5 ml of blood are taken from the patient and intro-duced into one of the tubes such as are defined above.
The tube is placed in an apparatus which permits ~entle agitation of its contents until realization of the tests.
b) Preparation of the fractions of total blood and of ~-washed blood -Two blood samples of 1 ml are taken Land placed] in the afore-mentioned tube. ~he whole is centrifuged at 800 g for 10 minutes at laboratory temperature, and the upper level of the plasma after centrifugation is marked on the tubes.
~ he contents of the tubes are made up to completion and washed with a total volume of washing buffer of 4 ml and the washing buffer lS separated by means of centri-fugation. 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 of the corresponding plasma, 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 antigen~ the action of which one wishes to study with regard to the said samples, that is to sa~, the samples of washed blood [are introduced] into the wells . . ...

1(J8';19~
.

of vertical rows 2, 4, 6, 8, 10, 12, and those of total blood into wells 1~ ~ 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 ~7C for 10 minutes.
d) Colourin~ ~rocess After-the--incubator s~p 90 microlitres of the colouring reagent described hereinbefore application are placed in each well, mixing of the colouring solution and-the blood being ensured by , agitation by means of suckin~ in and out with the aid of a micropipette.
e) Reading ~ he mixture from each well is removed and placed in the chambers of a hemocytometer, for example in that known under the name "Fuchs Rosenthal cell". It simply remains then to ta~e the reading by microscope.
In the preceding part of this document the conclusions have already been indicated which can be drawn especially from the comparison of the number of degranulations observed in the presence of one or more antigens, on the one hand with the results established in the presence of antigens which have produced no de~ranulation and, on the other hand, with respect to the controls, placed in the example just described in indentations A and H, as regards only calcium, and in the cups of rows B to G of the microslide prepared with the~antigen, but in the absence of calcium.
By having recourse also to increasing proportions of antigens in the differing rows B to G, it is equally~
possible in this way, from the results observed in the ';'9~
presence of an antigen which has brollght about such degranulations, to establish curves representative of the variation in the numbers of degranulations observed as a function of the dilutions, whence [there exists]
the possibility, equally, of obtaining information on the toxicity level of the tested antigens9 or the degree of sensitivity of the basophils of the patients from whom the samples come, with regard to these antigens.
Having once established those of the dilutions ~hich, in the presence of calcium, bring about a minimum level of degranulation, it may be possible to limit comparisons of the afore-mentioned kind to possible observation of the behaviour of the basophils in those cups of the other microslide which contain the same dilutions of antigen, but in the absence of calcium.
As is self-evident and as, moreover, is apparent already from the preceding part of this document, the invention is in no way limited to those of its modes of application and realization which have been more particularly considered; on the contrary, it includes all the variants~

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: . ' . .:
:~.
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Claims

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:

1. A process for selectively staining only non-degranulated basophils in a blood sample selected from whole blood or plasma-free blood and containing degranulated basophils and/or non-degranulated basophils, in which without first fixing leucocytes contained therein, said blood 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 said blood 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, 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 according to claim 5, 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.

8. 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 compos-ition.

9. An aqueous hypotonic staining composition for selectively staining only non-degranulated basophils in an unfixed blood sample selected from whole blood or plasma-free blood and containing basophils, the composition comprising, per 100 ml of the composition, from 30 to 250 mg of a meta-chromatic 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.

10. A composition as claimed in claim 9, which is free from mineral salts which if present would contribute towards making the composition isotonic.

11. A composition as claimed in claim 9, in which the metachromatic agent is toluidine blue, the alcohol fixative is ethanol and the acid is acetic acid.

12. A composition as claimed in claim 11, which comprises, per 100 ml:
between 150 and 600 micro-litres of glacial acetic acid; and from 80 to 50 ml of distilled water.

13. A composition as claimed in claim 12, 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.

14. A composition as claimed in claim 13, 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.

15. 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, contacting each of said fractions 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 leucocytes including the basophils and an acid having lytic properties with respect to erythrocytes in an amount less than 1 ml thereby selectively staining the non-degranulated basophils contained in each fraction, counting the stained basophils in each fraction and comparing the two counts,classifying the blood sample on the basis of the difference between the counts, a significant difference between the counts being indicative of anaphyletic sensitivity of the patient from whom the blood sample was taken with respect to the allergen tested.

16. A method as claimed in claim 15, in which each blood sample is divided into a sample of total blood and a sample of plasma-free blood, 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 15, 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.

17. A method as claimed in claim 16, in which the sample of plasma-free 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 suspend-ing 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 return-ing the separated and washed cellular constituents to the plasma from which they had initially been separated.

18. A method as claimed in claim 17, 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.

19. A method as claimed in claim 18, in which the buffer comprises (tris-hydroxy-methyl) aminomethane.

20. A method as claimed in claim 17 or claim 18, in which the buffer contains an anticoagulant.

21. A method as claimed in claim 15, 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.

22. A test kit for use in the diagnosis of allergies, comprising, each of the following in a separate container, from 35 to 250 mg of a metachromatic agent, 20 to 50 ml of an alcohol fixative for leucocytes including the basophils, between 150 to 600 micro-litres of an acid having lytic properties with respect to erythrocytes in an amount less than 1 ml and 80 to 50 ml of distilled water, and sample of each allergen in respect of which the test kit is to be used to test for anaphylactic sensitivity.

23. The test kit of claim 22, wherein each of the following is in a separate container:
from 30 to 10 mg of toluidine blue;
from 25 to 40 ml of ethanol;
from 150 to 600 micro-litres of glacial acetic acid; and from 75 to 60 ml of water.