FR2467401A1 - Measuring de-granulation of basophiles by flow cytometry - after staining leucocytes with Alcian blue, useful in diagnosing allergies - Google Patents

Abstract

Method for differential counting of blood basophiles in a control sample and in a sample treated with a degranulating agent, uses flow cytometry after staining with Alcian Blue (I), the degranulation and staining being carried out on the leucocytes after sepn. of cells from plasma and haemolysis of erythrocytes. Pref. a blood sample is treated with heparin, sedimented using a dextrin soln. and the cells centrifuged off. These are suspended in a buffer contg. NH4Cl (to effect haemolysis), then washed with a buffer contg. albumin as stabiliser, and resuspended in the same buffer also contg. Ca and Mg salts to facilitate degranulation. This suspension is treated with the degranulating agent under test (or with an equal vol. of buffer), incubated at 37 deg.C for at least 15 mins. to complete degranulation, then the cells washed with buffer contg. a chelating agent. They are then resuspended in buffer at a concn. of 3-4 million cells per ml, stained with (I) and after 10 mins. counted to determine the no. of basophiles per 100000 leucocytes. The method is used for diagnosis of allergies; allergens will induce a greater degree of degranulation in a sensitive person than in one who is not allergic.

Description

 It has long been known that blood basophils and tissue mast cells play a primary role in many human and animal allergies, such as allergic asthma, hay fever, allergic rhinitis, hives and drug allergies .

Blood basophils and tissue mast cells contain in the granules which characterize them a number of mediators of allergic inflammation such as histamine, serotonin or heparin.

These cells are also capable, when stimulated, of synthesizing certain additional mediators such as the "slow-reacting substance of anaphylaxis" (SRS-A) and various chemotactic factors for eosinophils (ECF-A) or for neutrophils ( NCF-A). The primordial role of blood basophils and tissue mast cells in allergy stems from the fact that two categories of antibodies accumulate on their surface, predominantly formed by allergic patients: class immuno globulins
IgE and IgG4. In an individual allergic to a given antigen such as grass pollen or drug, the interaction of the antigen with IgE and / or
IgG4 present on the surface of basophils or mast cells causes rapid expulsion of intracellular granules and the corresponding release of inflammatory mediators in the surrounding tissue.

 While tissue mast cells lend themselves by definition to in vitro investigation (since they are only accessible after excision of the tissue) it is not the same for circulating blood basophils, which form as a rule 0.5-0.8% of circulating blood leukocytes. The study of the reactivity of blood basophils of allergic patients with one or the other aatigen in vitro potentially represents an effective and safe method of diagnosis for the patient. , while the skin tests commonly practiced at the present time for the diagnosis of allergies (and which are based on the stimulation of basophils and mastocybe-in vró) are not without danger5 particularly in patients with high sensitization.

 The study of the reaction of blood basophils with an antigen can be based in principle, either on U- microscopic observation of the phenomenon of expulsion of granules ("degranulation"), or on the biochemical analysis of inflammatory mediators (in especially histamine) released into the environment. The measurement of the tannin release which has been perfected and semi-automated in recent years (EvansS DP et al. Life sciences 12 327, 1973) has found many applications in allergological research because it makes it possible to study not only the nature of the antigens to which the patient can be sensitized, but also various parameters playing a role in the allergic reaction, such as the amount of IgE and IgG4 present on the surface of basophils and the specific reactivity of these cells in the presence of antigens or other pharmacological agents capable of causing degranulation. This technique has also found its use in pharmacological research for the study of drugs making it possible to inhibit the release of inflammatory mediators. However, the complexity of the manipulations required to determine the release of histamine from isolated blood basophils, the price of the instrumentation necessary for this semi-automatic determination, the high cost of the reagents required and the fact that the number of possible analyzes per unit of time is relatively limited, has hitherto prevented the general application of this method to diagnosis routine allergies in humans.

Regarding the degranulation of basophils, the phenomenon was observed under the microscope in the presence of antigens or other agents stimulating basophils many years ago (Shelley, WB, Juhlin,
L. NATURE, 191, 1056, 1961, Hirsch, SR and Zastrow, JE, J. Allergy, 50, 338, 1972). However, microscopic examination of basophils, as a routine examination, has encountered a number of practical difficulties which have hitherto prevented the widespread adoption of this diagnostic method. These difficulties are in particular a) - the relative rarity of basophils in the circulating blood requiring
the observer has to count a minimum of 5,000 to 10,000 leukocytes, in order
detect a statistically acceptable number of control basophils.

b) - The relative fragility of blood basophils and the difficulty of
concentrate without accidentally causing their degranulation. Some
progress in microscopic examination technique has been achieved by the
development of specific coloring methods (in particular
toluidine blue) to specifically color the granules
basophils in whole blood, without prior separation of
cells. On the basis of such a microscopic method, a test of
Allergy diagnosis has recently been proposed (Benveniste, J. in
"Allergy and Clinical Immunology" (E. Mathov ed.) Excerpta Medica,
418, 1977).

c) - Each microscopic examination and counting requires attention
supported by a competent observer and the number of possible analyzes by
unit of time therefore remains very limited. To this is added
the fact that in the above methods, the total number of basophils
counted per test (and capable of partially disappearing during degranulation) is generally of the order of 30 to 50 with considerable variations from one sample to another, so that the reproducibility of the test cannot be considered as satisfactory.

 The advent of flow-cytometry techniques in recent years has led two groups of researchers to develop techniques for detecting and counting mast cells or blood basophils by an automatic method, that is to say by flow-cytometry. Flow-cytometry techniques have been described by ADAMS L.R. (J. Histochem. Cytochem.

1977, 25, 965) and by HORAN P.K. et al. (Science 1977, 198, 149). The principle is as follows: the cells suspended in a liquid are transported one by one in a glass tube where they react with a laser beam. The information from these interactions (light scattering and axial light loss) is then displayed electronically in the form of a scattering diagram, on a cathode ray tube. One of the groups of researchers who have implemented these techniques uses a fluorescent dye (berberine) which binds to heparin and requires the use of a cytofluorograph (Enerback, L. et al. J. Histochem. Cytochem. 24, 1231, 1976).

This group has so far used the berberine technique only on rat peritoneal mast cells and it is therefore not certain that it is also applicable to human blood basophils. In addition, the technique used seems to cause cell loss of 20% to 50% depending on the samples, which would not allow differential calculation to establish the presence of degranulation.

 The other group (Technicon, Saunders, A.M., J. Histochem. Cytochem.

25, 1001, 1977) who developed the flow-cytometry method for a semi-automatic basophil counting technique (on a fixed number of 1,000 to 10,000 leukocytes) used as dye the alkian blue in the presence lanthanun chloride and cethylpyridinium chloride at pH 2.2, in direct staining on whole blood. I1 does not seem to have applied or successfully used this counting technique to date for the study of degranulation phenomena.

 In fact, when trying to apply this method of counting basophils in whole blood to the study of degranulation of basophils by allergens, we quickly realized that this method does not achieve the desired result, c that is to say the calculation of the percentage of basophils having lost their granules by differential counting between a control witness and the samples of basophils subjected to the action of one or the other degranulating agent. A large number of tests have revealed that the interaction of blood basophils with degranulating agents such as antigens, anti-IgE antibodies or pharmacological agents such as ionophore A 23187, causes markedly distorting side reactions in the presence of whole blood. cytometric count of basophils.

Subsequently, using a model 6301 Biophysics (USA) cytograph, a method was developed according to the invention which makes it possible for the first time to achieve the desired goal, that is to say d '' Obtain reproducible accurate counts of basophils both in the presence and in the absence of degranulants of various natures.

 The subject of the invention is a method of differential counting of basophils between a control sample and a sample having undergone the action of a degranulation agent, using a cytograph using the flow-cytometry techniques after Alcian blue staining, process characterized in that the degranulation reaction and the staining are carried out after separation of the blood cells from the plasma and hemolysis of the erythrocytes.

 Gilbert and Ornstein (Blood 1975, 46, n02, 279-286) reported that an alkian blue staining method is suitable for counting basophils using DC sampling and electro-optical detection. Microscopic observation of the cells after staining has shown that only basophils are colored green-blue, which makes it possible to differentiate them from other non-colored cells, and in particular from other leukocytes, and from basophils having undergone degranulation.

 The coloring solution used in the process according to the invention is new compared to those previously used; it is a solution of alkian blue, pH 3.5, characterized in that it contains, for 100 ml, 120 to 200 mg, and preferably 150 mg of alkian blue; 35 to 45 ml and preferably 39 ml of physiological solution (0.9 Z aqueous solution of NaCl); 1.5 to 0.5 ml and preferably 1 ml of glacial acetic acid; 20 to 5 ml and preferably 10 ml of formaldehyde; 60 to 40 ml and preferably 50 ml of ethanol.

The method according to the invention is characterized by the following stages a) - The heparinized blood sample is sedimented by addition of dextran
in physiological solution, and the cells are separated from the plasma
by centrifugation.

b) - The cell pellet is resuspended in a buffer solution
containing ammonium chloride to hemolyze erythrocytes; c) - The cells are then washed with a buffer supplemented with albumin
as a stabilizer, then resuspended in the same
buffer than previously added with calcium and magnesium salts
to facilitate the degranulation reaction; d) - In the experimental tubes, cells are added to the suspension
the degranulating agent to be studied, and in the control tubes the
same volume of buffer solution, and incubate at 37 ° C for
at least 15 minutes, until the end of the degranulation reaction; e) - The cells are washed with a buffer solution added with an agent
chelator such as EDTA to block the degranulation reaction and
resuspends them in the same solution, at the concentration of
3 to 4 x 106 cells / ml; f) - The stain reagent with alkian blue is added to the suspension
cell, and after 10 minutes the solution is counted
stained with a cytograph and determines the number of basophils for
100,000 leukocytes.

The percentage of degranulation is calculated according to the formula
% basophils in control -% basophils in test% degranulation = x 100
% basophils in control
The buffers used during the various stages of the process according to the invention are known in themselves.

 The Tris buffers recommended for the study of histamine release by Lichsteinstein and Osler (J. Exp.

Med. 120, 507, 1964) but it is also possible to use phosphate-based buffers or the like, provided that in step b) a buffer containing ammonium chloride is used, in step c) a buffer supplemented with albumin as a stabilizer for washing cells, to which Ca ++ and Mg ++ ions are then added to carry out the degranulation reaction, finally in step 4 a buffer containing a chelating agent such as EDTA to stop this reaction.

The Tris buffers which can be used in the process according to the invention are the following 1 - in step b, a Tris-NH4Cl buffer: 0.010 to 0.030 M Tris (preferably
0.015 M); 0.5 to 1.0 Z of NH4C1 (preferably 0.75%).

2 - In step c, a Tris-A buffer: 0.01 to 0.03 M Tris (preferably
0.025 M); 0.1 to 0.15 M NaCl (preferably 0.12 M); 0.001 to 0.01 M KCl
(preferably 0.005 M); 0.1 - 0.01% human serum albumin (from
preferably 0.03%).

3 - For the degranulation reaction, a Tris-ACM buffer with the same
components as Tris-A plus 0.005 to 0.0002 M CaCl2 (preferably
0.0006 M); 0.005 to 0.0005 M MgC12 (preferably 0.001 M).

4 - In step e, a Tris EDTA buffer: with the same components as
Tris-A plus 0.05-0.005 M EDTA (preferably 0.01 M).

 The dextran used in step a) of sedimentation has a molecular weight of 150,000 to 250,000 (preferably 200,000 to 250,000) and has a concentration of 3 to 10% in physiological saline (preferably 6%).

 By way of example, a detailed operating mode of the method according to the invention is given below. 10 cm 3 of venous blood are taken in a polystyrene tube specially pretreated with EDTA and heparin.

The heparinized blood is sedimented at room temperature by the addition of half a volume of dextran at 6% in physiological solution.

After sedimentation for 60 minutes, the plasma is removed and centrifuged at 1000 rpm for 10 minutes at 40C.

The centrifuged cells are resuspended in a buffer solution of Tris-NH4Cl (0.015 M Tris, 0.747 747 Z of ammonium chloride) in order to hemolyze the erythrocytes.

The cells are washed once or twice with Tris-A buffer (0.025 M Tris, 0.12 M NaC1, 0.005 M KC1, 0.03 Z human serum albumin and resuspended in Tris-ACM buffer (Tris-A buffer plus or 0006 M calcium chloride and 0.001 M magnesium chloride at a concentration of 5 x 10 6 cells / ml.

To the test tubes are added the degranulants to be studied such as antigens at various concentrations, anti-IgE antisera or directed against other classes of allergenic immunoglobulins such as IgG4, degranulating pharmacological agents such as ionophore A 23187 etc. To the control tubes are added the same volume of buffer solution.

The control and experimental tubes are incubated for at least 15 minutes at 370C.

One to two washes with Tris-EDTA buffer solution (Tris-A buffer plus 0.01 M
EDTA) are followed by a final resuspension in Tris-EDTA buffer at a concentration of 3 - 4 x 106 cells / ml.

 Staining with an alkian blue solution (0.15 Z alkian blue pH 3.5, composed of 39 ml physiological solution, 1 ml glacial acetic acid, 10 ml formaldehyde and 50 ml ethanol): 0.9 ml of the solution of dye are added to 0.1 ml of the suspension of washed leukocytes, for 10 minutes.

 Counting the colored solution with a cytograph automatically determines the number of ungranulated basophils per 100,000 leukocytes.

 Counting basophils with a cytograph on the basis of 100,000 leukocytes (normal value 400-800 basophils) makes it possible to obtain a much better statistic than by microscopic counting and in particular to considerably reduce the variation in counting from one sample to another. .

The correlation between the values observed under the microscope and the values obtained by the cytograph is excellent, as shown in FIG. 1 where the percentages of basophils determined under the microscope are plotted on the abscissa and the percentages of basophils determined with the cytograph are plotted on the ordinate same sample (r = 0.94). It should also be added that the determination on the cytograph is approximately 5-10 times faster than on the microscope to count 10-20 times more basophils. The study in a large number of normal and allergic individuals of the effect of various degranulating agents confirms that the method used makes it possible to quickly and reproducibly establish the percentage of degranulated cells at different concentrations of degranulating agent.

FIG. 2 represents the curves of the percentage of degranulation of basophils as a function of the dilution of a degranulation agent which is an anti-serum anti-IgE (curve 1), anti-IgG4 (curve 2), anti-IgG2 (curve 3), or a normal serum (curve 0). This figure shows that 1) anti-IgE antibodies, well known for causing degranulation of
basophils and tissue mast cells in vitro (Ishizaka, K. in M.Sela
The Antigens, vol. 1, page 479 Académie Press, New York, 1973) and in vivo
(Ishizaka, K., Ishizaka, TJ Immunol. 100, 554, 1968) also cause
degranulation of basophils measurable by the cytograph (curve 1); 2) the same applies to the antibodies directed against IgG4 which represent
the second class of human immunoglobulin attached to mast cells and
partially responsible for allergic manifestations (curve 2) 3) Antibodies directed against other immunoglobulins not attached to
mast cells (eg IgG2) do not cause degranulation
measurable by cytograph (curve 3).

 FIG. 3 is a graph on which the percentage of degranulation obtained with an anti-IgG4 serum is plotted on the abscissa and on the ordinate the percentage of degranulation obtained for the same blood sample, with an anti-IgE serum, the determinations having been made. on the one hand with the blood of allergic patients (black dots) and on the other hand with the blood of non-allergic people (white dots). This figure shows that there is a definite correlation (r = 0.78) between the maximum degranulation obtained by anti-IgE antibodies and anti-IgG4 antibodies, the rate of degranulation being higher in allergic patients than in individuals witnesses without allergy.

 Other mandatory degranulating pharmacological agents such as ionophore A 23187 have a degranulating effect detectable by the cytograph but with no marked correlation with the allergic state of the patient.

 Figure 4, which is a graph on which the percentage of degranulation has been plotted as a function of the concentration of an allergen, finally shows that - in allergic patients (curve 1), the use of the corresponding antigen ( for example grass pollen) also leads to marked degranulation, which is not the case in the non-allergic patient (curve 2).

 The fact that the differential count on the cytograph actually corresponds to an extracellular expulsion of the granules and to a release of the mediators contained in the basophils is confirmed by the excellent correlation (r = 0.92) observed between the degranulation rate measured by the method on the one hand and the percentage of histamine released in the supernatant of the counted cells on the other hand, as shown in FIG. 5 where the percentage of degranulation was plotted as a function of the percentage of histamine released . Consequently, the cytograph counting method can advantageously replace, since it is much faster and less expensive, the methods for detecting released mediators such as histamine.

 In summary, the method according to the invention for differential counting of human basophils with a cytograph constitutes a new technique for diagnosing allergies in vitro, which is characterized by its simplicity, its speed of execution, its reproducibility and the large number of samples. can be analyzed per unit of time. This method calls upon a pathophysiological phenomenon directly comparable to that taking place in the allergic patient in vivo. It therefore offers better.

clinical correlation than other determining methods, for example by radioimmunological methods (RAST, PRIST), antibodies of the IgE and IgG4 type circulating freely in the serum.

Claims (10)

R E V E N D I C A T I O N S 1. Method for differential counting of blood basophils between a control sample and a sample having undergone the action of a degranulation agent, using a cytograph using the flow-cytometry method after blue staining. alcian, a process characterized in that the degranulation and staining reaction is carried out on the leukocytes, that is to say after separation of the blood cells from the plasma and hemolysis of the erythrocytes. 2. Method according to claim 1, characterized in that it comprises the following steps a) The heparinized blood sample is sedimented by addition of dextran  in physiological solution, and the cells are separated from the plasma by centrifugation b) the cell pellet is resuspended in a buffer solution  containing ammonium chloride to hemolysate erythroeytes c) the cells are then washed with a buffer containing albumin  as stabilizer, then resuspended in the same buffer  than previously added with calcium and magnesium salts to  facilitate the degranulation reaction d) in the experimental tubes, the agent is added to the cell suspension  degranulant to be studied, and in the control tubes the same volume is added  of buffer solution, and incubate at 370C for at least 15  minutes, until the end of the degranulation reaction; e) the cells are washed with a buffer solution added with an agent  chelataur such as EDTA to block the degranulation reaction.  resuspends ion in the same solution, at the concentration of 3 to  4 x 106 cells / ml f) the stain reagent with alkian blue is added to the suspension  cell, and after 10 minutes the solution is counted  stained with a cytograph and determines the number of basophils per 100,000  leukocytes. 3. Method according to one of claims 1 and 2, characterized in that the buffer used in step b) is a Tris buffer 0.010 to 0.030 M containing 0.5 to], 0 X NH4C1. 4. Method according to claim 3, characterized in that the buffer of step b) is a 0.015 M Tris buffer containing 0.75 Z of NH4Cl. 5. Method according to one of claims 1 to 4, characterized in that the cell washing buffer in step c is a Tris-A buffer consisting of Tris 0.01 to 0.03 M, NaCl 0.1 at 0.15 M, KC1 0.001 to 0.01 M containing 0.1 to 0.01 Z of human serum albumin. 6. Method according to claim 5, characterized in that the buffer consists of 0.025 M Tris, 0.12 M NaCl, 0.005 M KCl and 0.03 Z of human serum albumin. 7. Method according to one of claims 1 to 6, characterized in that, before the degranulation reaction, the cells are resuspended in a Tris-A buffer to which CaCl2 0.005 to 0.0002 M is added and MgC12 0.005 to 0.0005 M and preferably CaCl2 0.0006 M and MgC12 0.001 M. 8. Method according to one of claims 1 to 7, characterized in that one uses, in step e), a Tris-A buffer to which EDTA 0.05 to 0.005 M and preferably 0.01 M is added. . 9. coloring reagent necessary for the process according to one of claims 1 to 8, characterized in that it consists of a solution of alkian blue, pH 3.5 containing, for 100 ml, 120 to 200 mg of Alcian blue, 35 to 45 ml of physiological solution, 1.5 to 0.5 ml of glacial acetic acid, 20 to 5 ml of formaldehyde and 60 to 40 ml of ethanol. 10. Reagent according to claim 9, characterized in that it consists of 150 mg of alkian blue, 39 ml of physiological solution, 1 ml of glacial acetic acid, 10 ml of formaldehyde and 50 ml of ethanol.