METHOD OF DETERMINING THE ALLERGENIC RESPONSE OF A SAMPLE OF MAMMALIAN BLOOD TO AN ANTIGEN

Abstract: A method is provided for determining the allergenic response of a sample of mammalian blood to an antigen, typically a food antigen. The method includes probing the blood sample after incubation with the antigen to determine the amount of degranulation of one or more of the granulocyte populations in the sample relative to an incubated antigen-free control; typically, the neutrophil population is probed.

Description: METHOD OF DETERMINING THE ALLERGENIC RESPONSE OF A SAMPLE OF MAMMALIAN BLOOD TO AN ANTIGEN DESCRIPTION The present invention relates to a method of determining the allergenic response of a sample of mammalian blood to an antigen, particularly a food antigen. The invention also embraces an in vitro method of diagnosis for detecting an allergenic response to an antigen in a patient. Some patients are allergic to certain foods. The particular foods vary from patient to patient, and although the mechanism of the allergenic response to food allergens is not fully understood, it is thought that allergenic reactions to foods may in some patients be at least partly responsible for symptoms as diverse as migraine, asthma, eczema and arthritis. A particular disease in which food intolerance may be implicated is obesity. In some patients, obesity is a result of fluid retention. It is thought that in some patients an allergenic response to certain food antigens may give rise to an increase in the permeability of their blood capillaries; the capillaries become leaky, and water in the blood escapes into tissue juxtaposed the capillaries. As a result, the -patient may feel thirsty and drink more liquid than normal to compensate. However, the additional consumed fluid ultimately also escapes from the capillaries, and the patient becomes "fluid retaining" and puts on weight. Once an allergenic response to a particular food has been diagnosed in a patient, an effective treatment is the elimination of the food from the patient's diet. A typical daily diet, however, includes many different types of food; there can be considerable difficulty therefore in determining which foods cause an allergic response in a particular patient and should, therefore, be avoided. Furthermore, there is a general prejudice among medical professionals and the public alike against the recognition of a causal link between the symptoms mentioned above and allergic intolerance. In consequence testing for food intolerance has hitherto been confined outside mainstream general medical practice, and has included techniques for which the scientific basis has not been satisfactorily proven. Testing for food intolerance has conventionally been carried out by a "skin-prick" test in which a patient is pricked with a sterilized point carrying a small quantity of a food antigen. The patient's response to the food antigen is monitored visually to determine whether or not an intolerance is exhibited. Although a patient can be tested for intolerance to five or more antigens simultaneously by subjecting the patient to a plurality of pricks at different skin locations, the "skin-prick" test is time consuming, and testing for tens of foods can take many weeks for a single patient. The prick test also suffers from the disadvantage that visual monitoring is required; small and/or transient reactions may therefore be unnoticed. It has been observed that the total number of leucocytes in a sample of blood from a patient may decline if the sample is challenged with an antigen to which the patients exhibits an allergenic reactions. US-A-4,614,722 discloses a method and apparatus for the determination of the degree of reaction between a suspected allergen and a blood sample; the method involves comparing the total number of white blood cells present in the sample before and after reaction with the suspected allergen. It is now thought that the various symptoms which may result from an allergenic response in a patient to one or more foods may be caused by the release of granules from one or more of the granulocyte populations in the blood of the patient, typically the neutrophils, into the patient's blood stream. In particular, it is thought that certain food allergens may interact with the granulocytes in the patient's blood causing some of the granulocytes to rupture and release granules into the blood stream. It will be appreciated by a person skilled in the art that the chemicals normally released by a granule in the cytoplasm of a granulocyte may, if released directly into the blood stream of a patient, cause inflamation or damage to tissue which may, in turn, lead to the symptoms associated with those medical conditions thought to be affected by an intolerance to foods. According to one aspect of the present invention therefore there is provided a method of determining the allergenic response of a sample of mammalian blood to an antigen; which method comprises the steps of:- a) adding the antigen to the blood sample to challenge the white blood cells in the blood sample; b) allowing the blood sample/antigen mixture to incubate; c) thereafter probing the mixture to determine the amount of degranulation of one or more of the granulocyte populations in the blood sample relative to an incubated antigen-free control blood sample; and d) comparing the amount of degranulation with a statistical mean degranulation derived from testing a plurality of other blood samples, thereby to determine whether the amount of degranulation is significant as compared with said mean degranulation. The invention also comprehends an in vitro method of diagnosing an allergy in a patient, the method comprising taking a blood sample from the patient and determining the allergenic response of the sample to an antigen utilising a method in accordance with the present invention. In another aspect of the invention, there is provided apparatus for determining the allergenic reponse of a sample of mammalian blood to an antigen; which apparatus comprises:- means for adding the antigen to the blood sample; means for probing the blood sample/antigen mixture to determine the amount of degranulation of one or more of the granulocyte populations in the blood sample relative to antigen-free control blood sample; and means for comparing the amount of degranulation with a statistical mean degranulation derived from testing a plurality of other blood samples thereby to determine whether the amont of degranulation is significant as compared with the said mean degranulation. Typically, the blood sample/antigen mixture may be probed to determine the amont of degranulation in the neutrophil population. The amount of degranulation may be determined in some embodiments by directly measuring the number of granules leaked by the granulocytes in the sample into the blood plasma. Alternatively, the amount of degranulation may be determined indirectly by measuring the loss of granules in the granulocytes in the sample In some embodiments, the amount of degranulation may be determined using a haematology analyser. The haematology analyser may probe the white blood cell populations of the blood sample using a combination of radio frequency alternating current and direct current. Typically a haematology analyser of the type which is commerically available from Messrs. Toa of Japan under the trade name SYSMEX NE 8000 may be used. The principle and method of operation of this haematology analyser is comprehensively described in its accompanying manual available from Toa under the reference NE-8000/11.88 (revised 08.89,12.89); see particularly Section 4 "Principle of Operation" . The blood sample may be divided in to a plurality of aliquots: a first aliquot may be treated with a lysing agent to lyse all the cells therein except the white blood cells; a second aliquot may be treated with a lysing agent to lyse all the cells except the basiphils; and a third aliquot may be treated with a lysing agent to lyse all the cells except the eosinophils. Each aliquot may be suspended in an electrolytic diluent and disposed in a transducer chamber having a partition dividing the transducer chamber into two regions. The partition may have a small aperture extending therethrough between the two regions; each region may be provided with an electrode. The size of the aperture may be of the same order as the size of the white blood cells in the blood sample; the electrolytic diluent may be selected to have a significantly different electrical conductance from said white blood cells. The white blood cells in each aliquot may be aspirated through the aperture from one region of the transducer chamber to the other region. A substantially constant electrical current may be applied across the electrodes; as a white blood cell passes through the aperture the voltage between the electrodes may change transiently by a detectable amount; said voltage may be monitored continuously during aspiration of the cells to count the number of cells passing through the aperture. In the case of the second and third aliquots, the applied constant current may be a constant direct current; the total volume of each cell passing through the aperture may be proportional to the transient change in the voltage. The numbers of basiphils and eosinophils in the second and third aliquots respectively may thus be counted. On aspirating the white blood cells in the first aliquot, a constant direct current and a constant radio frequency alternating current may be applied across the electrodes; as before the transient change in the d.c. signal may be proportional to the total volume of a white blood cell as it passes through the aperture; the total nuclear and cytoplasmic granule volume of the cell may be proportional to the transient change in the r.f. signal as it passes through the aperture. Thus, a scattergram may be produced from the aspiration of the first aliquot in which the position of each white blood cell passing through the aperture may be plotted against (i) a total cell volume axis (d.c. pulse) and (ii) a nuclear and granule volume axis (r.f. pulse). It will be appreciated by a person skilled in the art that intact lymphocytes, granulocytes and monocytes may show up in different regions of the scattergram as a result of their different overall volume to nuclear/granule volume ratios. Based on experience, therefore, "lymphocyte", "granulocyte" and "monocyte" regions of the scattergram may be delineated by applying calculated "discriminator" lines; the total number of cells represented in each region may then be counted. The apparent neutrophil population in the blood sample may be calculated by subtracting the counted numbers of basiphils and eosinophils in the second and third aliquots from the calculated apparent granulocyte population in the first aliquot. In accordance with the present invention it will be appreciated that a degranulated granulocyte may appear under d.c. and r.f. analysis as described above as a "monocyte" or lymphocyte". Thus, in one aspect of the invention, degranulation of one or more of the granulocyte populations in the blood sample may be detectable as an apparent increase in the number of monocytes or lympocytes and an apparent decrease in the number of granulocytes, typically neutrophils. In the scattergram, this may show up as an apparent shift of the leucocyte population from the "granulocyte" region to the "monocyte" region. A control sample which does not contain any antigen may be probed, and the amount of degranulation in the blood sample challenged with antigen may be determined by calculating the apparent increase or decrease respectively in the monocyte/lymphocyte and granulocyte populations of the blood sample relative to the control. Typically, a plurality of blood samples may be prepared; each blood sample may be challenged with a different antigen. In a particular aspect of the invention the antigen may be a food antigen. In some embodiments a plurality of food antigens may be selected which include sufficient foods to constitute a complete and balanced diet. The foods antigens may be presented to the blood sample in suspension. The suspension medium may be glycerol or a benzoyl solvent. In one embodiment, glycerol suspensions of food antigens obtained from Messrs. Dome/Hollister-Stier may be used; the antigen suspensions may be of the type typically used for conventional 'skin-prick' testing. The food antigen/glycerol suspension may be further diluted. The dilutant may be a saline solution, typically saline BP. The food antigen suspension may include a small proportion of human serum albumen (HSA); about 0.01% to 0.05% HSA may be used, typically 0.03%. Where food antigens obtained from Messrs. Hollister-Stier are used, the glycerol suspension may be diluted with saline BP in the ratio about 1:9, but it will be understood that the present invention comprehends dilution ratios of the Hollister-Stier food antigens to saline in the range about 1:5 to 1:15. The diluted food antigen suspension in glycerol may be added to an undiluted blood sample. The correct ratio of blood to food antigen may be of importance; if too little blood is used, the analysis may be non-specific. In some embodiments, the food antigens obtained from Messrs. Hollister-Stier may be diluted with nine parts saline BP and include 0.03% HSA and be mixed with undiluted blood in the ratio 50:50. When using this diluted glycerol suspension of food antigen, a ratio of antigen to blood in the range 40:60 to 60:40 may be satisfactory. After introduction of the food antigen into the blood sample, the mixture may be incubated. The period of incubation may be important to obtain a satisfactory result. If the mixture is incubated for too long, the reaction of the granulocytes to the food antigen may be non-specific. The mixture may be incubated for a period in the range 30 minutes to 90 minutes, typically 60 minutes. The respective increase and decrease in the apparent monocyte/lymphocyte and granulocyte populations of the blood sample for a given food aritigen may be compared respectively with the mean increase and decrease obtained from a plurality of blood samples. A significant increase or decrease respectively as compared with the obtained mean, as background, may indicate an intolerance to the antigen concerned. The obtained mean increase or decrease in the monocyte/lymphocyte and granulocyte populations respectively may vary from food antigen to food antigen, but typically a decrease of about 5% - 10% in, say, the neutrophil population may indicate an intolerance. Once an intolerance to a particular food antigen has been determined for a given blood sample, treatment of the patient may be effected by advising the patient to eliminate the food from his/her diet. Alternatively, in accordance with one aspect of the present invention, the patient may limit his/her diet to foods which do not apparently provoke an allergenic reaction; foods not tested may be reintroduced into the diet one by one over a period of time and the patient's reaction over time noted. The present invention provides a method of testing a blood sample for an allergenic reaction against one or more food antigens. The method may be substantially automated using an haematology analyser and a microprocessor to analyse the results. A large number of antigens may be tested in a relatively short period of time as compared with "skin-prick" testing known in the prior art. The invention aslo provides a method for measuring relatively small changes in the white blood cell populations in response to challenge by food antigens, and provides means for comparing the changes with a mean response based on a plurality of tests. The invention may therefore provide a more accurate and sensitive method for determining the allergenic response of a given blood sample to a particular antigen. Following is a description by way of example only and with reference to the accompanying drawings of methods of carrying the present invention into effect. In the drawings:- Figure 1 is a scattergram of an antigen-free control blood sample. Figure 2 is an histogram of the blood sample of Figure 1. Figure 3 is a scattergram of a blood sample from the same donor as the sample of Figure 1 after challenge with a food antigen. Figure 4 is a histogram corresponding to the scattergram in Figure 3. Figure 5 is a scattergram of a different sample of blood from the same donor as Figures 1 and 3, which different sample has been challenged with a different food antigen. Figure 6 is a histogram which corresponds to the scattergram in Figure 5. Food antigens of the kind suitable for use in a conventional "skin-prick" food intolerance test were obtained from Messrs. Dome/Hollister-Stier. Each food antigen was supplied in a glycerol suspension and was subsequently diluted with nine parts saline BP solution and 0.03% human serum albumen (HSA). The food antigens were as follows:- MEATS PULSES beef bean, haricot chicken bean, kidney duck bean, runner lamb bean, soya pork chocolate rabbit coffee turkey lentil pea FISH NUTS cod almond crab brazil nut haddock cashew mackerel coconut plaice hazel nut prawn peanut salmon olive sardine sesame seed sole sunflower seed trout walnut/English tuna GRAINS FRUIT barley apple maize/corn avocado malt banana oats blackcurrent rice date rye grape wheat grapefruit lemon mango MILK AND DAIRY melon/canteloupe orange cheese, cheddar peach milk, cow's, whole pear milk, evaporated pineapple raspberry rhubarb EGGS strawberry egg white egg yolk VEGETABLES FUNGI beetroot Candida albicans broccoli mushroom cabbage yeast/bakers carrot yeast/brewers cauliflower celery chilli cucumber SWEETNERS egg plant/aubergine garlic honey green pepper (bell) saccharine leek sugar, beet lettuce sugar, cane onion parsnip potato ADDITIVES radish spinach black/white pepper mix sprout monosodium glutamate tomato salicylates turnip tea A sample of blood was taken from a patient and divided into 100 portions. Each portion was mixed with one of the food antigen mixtures in a ratio one part blood to one part food antigen mixture. One portion of blood was mixed with an antigen-free mixture containing one part glycerol, nine parts saline BP and 0.03% HSA as a control. The blood/food antigen mixtures and the control sa ple were then incubated at room temperature for about one hour. The control and blood/food antigen mixtures were then analysed using an haematology analyser obtained from Messrs. Toa of Japan under the trade name SYSMEX NE 8000. The haemotology analyser divides a blood sample into a plurality of aliquots: A first aliquot is treated with a lysing agent to lyse substantially all the cells in the aliquot except the white blood cells; a second aliquot is treated with a lysing agent to lyse sustantially all the cells except the basiphils; a third aliquot is treated with a lysing agent to lyse substantially all the cells except the eosinophils. Each aliquot is then suspended in an electrotytic diluent having a significantly different electrical conductance from white blood cells and is disposed in a first region of a transducer chamber. The cells are then aspirated through a small aperture formed in a partition in the chamber to a second region, and a substantially constant electrical current is applied between first and second electrodes disposed respectively in the first and second regions of the transducer chamber; the aperture has a diameter of the same order of magnitude as the size of the white blood cells in the aliquot, thus as a blood cell passes through the aperture the resistance of the electrical path between the electrodes in the chamber increases significantly; in consquence the voltage drop across the electrodes increases transiently by a detectable amount. On aspirating the second and third aliquots, a substantially constant direct current (d.c.) is applied between the electrodes; the transient change in d.c. voltage as a blood cell is aspirated through the aperture being proportional to the total cell volume. On aspirating the first aliquot, a substantially constant direct current and a substantially constant radio frequency alternating current (r.f.) are applied. The transient change in the r.f. voltage as a leucocyte passes through the aperture is proportional to the total nuclear and cytoplasmic granule volume and density of the leucocyte. The position of each leucocyte aspirated in the first aliquot is plotted on a scattergram of the type shown in Figures 1, 3 and 5 of the accompanying drawings against a first d.c. pulse (total cell volume) axis and a second r.f. pulse (total nuclear and granule volume) axis. Intact granulocytes, monocytes and lymphocytes show up in different regions of the scattergram as a result of their different overall volumes and nucleus/granule constitution; the scattergram can therefore be divided into "granulocyte", monocyte" and "lymphocyte" regions by applying calculated "discriminator" lines to the scattergram, and the total number of cells plotted in each region can then be counted. With reference to Figure 1, the granulocytes generally show up in the region marked 10, and monocytes are indicated in box 12. The apparent neutrophil population of the blood sample can be calculated by subtracting the numbers of basiphils and easinophils counted on aspirating the second and third aliquots from the total apparent granulocyte population counted in the first aliquot. The haematology analyser obtained from Toa also generates a histogram (see, for example, Figures 2, 4 and 6 of the Drawings hereto) which shows the relative numbers of white blood cells counted/calculated in each cell population. The food antigen-free control sample was supplied to the haematology analyser, and the relative concentrations of the respective leucocyte populations in the control are set out in Table 1 below. TABLE 1 LEUCOCYTE PERCENT (%) neutrophi1 45.2 lymphocyte 44.7 monocyte 8.0 eosinophil 1.6 basiphil 0.5 The corresponding scattergram and histogram for the control blood sample are shown in Figures 1 and 2 respectively. Each blood/food antigen mixture in turn was then supplied to the haematology analyser, and the results for two of the food antigens are shown in Tables 2 and 3 below corresponding to Figures 3 and 4 and Figures 5 and 6 respectively. TABLE 2 LEUCOCYTE PERCENTAGE (%) neutrophil 41.1 lymphocyte 42.2 monocyte 14.5 eosinophil 1.7 basiphil 0.5 TABLE 3 LEUCOCYTE PERCENTAGE (%) neutrophil 16.2 lymphocyte 56.6 monocyte 18.5 eosinophil 3.5 basiphil 5.2 On comparing Tables 1 and Figures 1 and 2 with Table 2 and Figures 3 and 4 respectively, it can be seen that there is little apparent change in the relative concentrations of the respective leucocyte populations. In Table 3 and Figures 5 and 6, however, a considerable apparent decrease in the neutrophil population and an apparent increase in the monocyte population can be detected. Without being bound by theory, it is thought that the apparent increase in the number of monocytes and decrease in the number of neutrophils, and the concomitant apparent shift of white blood cells from the granulocyte region of the scattergram to the monocyte region (box 12) is caused by degranulation of the granulocytes, including the neutrophils, so that the ruptured granulocytes are counted on aspirating the first aliquot, and appear in the scattergram, as monocytes or lymphocytes which do not contain any granules; the granules themselves being released into the blood serum where, in vivo, they are transported around the body and may give rise to allergic symptoms such, for example, as migraine, rheumatism etc. The results in Table 2 and Figures 3 and 4, on the other hand, indicate an apparent non-reaction, indicating that the patient is not significantly sensitive to that particular food antigen. It will be appreciated that the various leucocyte populations in blood vary naturally with time. Thus, it is important that the blood/food antigen mixtures and the control mixture are probed using the haematology analyser at as close to the same time as possible. Furthermore, depending on the concentration of the food antigens and suspension media used, small changes in the granulocyte/monocyte populations may be detected with any of the food antigens, and it is important, therefore, to determine which of the changes indicates a positive reaction to a particular food antigen. The results obtained for a patient, therefore, are compared with the mean leucocyte population changes for a particular food-antigen derived from a plurality of blood samples taken from different patients. For each food-antigen, a marked change in one of the leucocyte populations as compared with the mean may indicate an intolerance to the food antigen. Thus, a statistically significant decline in, for example, the neutrophil population for a patient in response to a given food antigen as compared with the average for that antigen may indicate an intolerance to the same. Where an intolerance to a given food antigen is indicated using the method outlined above, the patient may be encouraged to adopt a diet which includes only foods to which a definite non-reaction is indicated. Foods which are not included in the test may be restored to the patient's diet one by one over a period of time. Using a haematology analyser such, for example, as that available under the trade mark SYSMEX NE 8000, a patient may be tested for intolerance to 99 food substances in about 45 minutes plus the required incubation time of about one hour. The method of the present invention may, therefore, provide a considerably faster method for testing the allergenic response of a sample of blood to a plurality of food antigens as compared with methods known in the prior art such, for example as the "skin-prick" test. It will be appreciated by a person skilled in the art that the output of data from the haematology analyser may, in some embodiments of the invention be processed by computer to automate further the analysis procedure.

Claims: CLAIMS 1. A method of determining the allergenic response of a sample of mammalian blood to an antigen; which method comprises the steps of:- a) adding the antigen to the blood cell to challenge the white blood cells in the blood sample; b) allowing the blood sample/antigen mixture to incubate; c) thereafter probing the mixture to determine the amount of degranulation of one or more of the granulocyte populations in the blood sample relative to an incubated antigen-free control blood sample; and d) comparing the amount of degranulation with a statistical mean degranulation derived from testing a plurality of other blood samples, thereby to determine whether the amount of degranulation is significant as compared with said mean degranulation. 2. A method as claimed in Claim 1 wherein the blood sample/antigen mixture is probed to determine the amount of degranulation in the neutrophil population of the sample. 3. A method as claimed in Claim 1 or Claim 2 wherein the amount of degranulation is determined by directly measuring the number of granules leaked by the granulocytes in the sample into the blood plasma. 4. A method as claimed in Claim 1 or Claim 2 wherein the amount of degranulation is determined indirectly by measuring the loss of granules in the granulocytes in the sample. 5. A method as claimed in any preceding claim wherein the amount of degranulation is determined using a haematology analyser which probes the white blood cell populations of the blood sample using a combination of radio frequency alternating current and direct current. 6. A method as claimed in Claim 5 wherein a haematology analyser of the type commercially available from Messrs. Toa of Japan under the trade name SYSMEX NE 8000 is used. 7. A method as claimed in any preceding claim wherein the amount of degranulation is deterrnined by calculating the apparent increase or decrease respectively in the monocyte/lymphocyte and granulocyte populations of the blood sample relative to the control. 8. A method as claimed in any preceding claim wherein a plurality of blood samples are tested, each with a different antigen. 9. A method as claimed in any preceding claim wherein the antigen is a food antigen. 10. A method as claimed in any preceding claim wherein a plurality of food antigens are selected which include sufficient foods to constitute a complete and balanced diet. 11. An in vitro method of diagnosing an allergy in a patient, the method comprising taking a blood sample from the patient and determining the allergenic response of the sample to an antigen utilising a method as claimed in any preceding claim. 12. Apparatus for determining the allergenic response of a sample of mammalian blood to an antigen; which apparatus comprises:- means for adding the antigen to the blood sample; means for probing the blood sample/antigen mixture to determine the amount of degranulation of one or more of the granulocyte populations in the blood sample relative to an antigen-free control blood sample; and means for comparing the amount of degranulation with a statistical mean degranulation derived from testing a plurality of other blood samples thereby to determine whether the amount of degranulation is significant as compared with the said mean degranulation.

Inventors: PRICE THOMAS GAIRDNER, STOAKES IAN, PRICE, THOMAS, GAIRDNER, STOAKES, IAN
Applicants: NUTRON LIMITED, PRICE THOMAS GAIRDNER, STOAKES IAN, PRICE, THOMAS, GAIRDNER
Application year: 1993
Main classification: G01N33
IPC codes: G01N33