RU2214599C2 - Method of revealing persons belonging to risk group related to development of allergic diseases - Google Patents

Abstract

FIELD: allergology and immunology. SUBSTANCE: blood level of adrenaline and serotonine and summary coefficient of amine content in big granular lymphocytes in loading test with biogenic amines before and after hyperventilation of sample carried out during 20 min with volumes 40 l/min and when combination of following features is available: blood adrenaline level after hyperventilation remains stable; summary coefficient of amine content in big granular lymphocytes before and/or after hyperventilation stays within a range of 2.1 to 3.6; percentage of big granular lymphocytes bearing serotonine granules after hyperventilation rises; and blood serotonine level after hyperventilation falls - clinically healthy person is qualified as belonging to risk group related to development of allergic diseases. EFFECT: facilitated revealing persons potentially affected by allergic diseases. 5 ex

Description

 The invention relates to medicine, namely to the field of allergology and immunology, and can be used to identify people at risk for the development of allergic diseases among practically healthy people.

 The problem of timely diagnosis of allergic diseases, especially early forms of allergopathology, is one of the most important problems of modern medicine. It is especially important to identify among practically healthy people at-risk groups for the development of allergopathology, since it allows the timely implementation of the necessary preventive measures and more effective treatment of allergic diseases. Currently, early allergic diagnosis is carried out by various methods. There is, for example, a method for predicting the development of an allergic disease in a child (RF patent 2108582, class G 01 N 33/53, bull. 10 of 04/10/98) by identifying the clinical form of allergy in which risk factors are determined: environmental pollution, presence specific occupational hazards in parents, the presence of a congenital hereditary pathology, as well as the pathology of pregnancy of the mother or the child at birth, the presence of potential allergens in the house, take into account the age, gender of the child, blood type, his living conditions, fed set, then the data is carried on specific prognostic calculation coefficients by the computer and when the threshold value amounts predictive coefficients over 13 predict the likelihood of developing a particular form of allergy in children.

 The disadvantage of this method is its complexity and complexity, since it is necessary to take into account more than 100 different indicators.

 Methods for diagnosing allergic diseases using various biochemical blood tests are also known. For example, a method for diagnosing allergic diseases (RF patent 2098826 according to class G 01 N 33/53, bull. 34 dated 12/10/97) by setting up a stress test with theophylline provides for the simultaneous reaction of spontaneous rosette formation with sheep erythrocytes and when the number of rosetting neutrophils is equal to 1067 in 1 ml of blood and below, and theophylline-resistant T-lymphocytes - 27% and below - are diagnosed with an allergic disease.

 The disadvantage of this method is its limited use (only for employees of the microbiological industry) and the inability to identify among practically healthy people people at risk for the development of allergic diseases.

Closest to the proposed invention are non-dosed in depth and duration non-specific load hyperventilation test (Malkin V.B., Gora E.L. Hyperventilation. - M .: Hauka, 1990, p. 184) and a method for determining the functional activity of blood cells of donors ( according to ed. St. USSR 1686361 according to class G 01 N 33/50, bull. 39 from 10.23.91) by examining large granular lymphocytes, in which lymphocytes are incubated in parallel in environments containing adrenaline, serotonin, histamine and not containing them, for 40 minutes at 37 ^o C. further samples tsentrifu they are smeared, a smear is prepared from each precipitate, processed according to the Sevka method, the number of large granular lymphocytes containing adrenaline, serotonin and histamine is determined in each, the percentage of cells containing monoamines in all the smears studied is calculated, the total amino content coefficient is calculated and, with its value from 2, 1 to 3.6 determine the cells are functionally active.

 A disadvantage of the known methods is the limited diagnostic capabilities, in particular, in allergology and immunology - only to identify an existing pathology (non-specific load hyperventilation test - for the diagnosis of predominantly bronchial asthma with a deterioration in respiratory function by more than 20%, and the method according to AS 1686361 - to determine the functional activity of blood cells during transfusion). To identify among practically healthy people people belonging to the risk group for the development of allergic diseases, the methods described do not allow.

 The objective of the invention is to create a relatively simple and reliable way to identify people at risk for the development of allergic diseases among healthy people.

The problem is solved in the proposed method for identifying persons at risk for the development of allergic diseases, including a hyperventilation test, as well as a biochemical blood test for adrenaline and serotonin and histochemical studies of large granular lymphocytes (BHL) in a load test with biogenic amines with the calculation of the total amino content coefficient (SKA), due to the fact that the indicated biochemical and histochemical blood tests are carried out before and after dosed over time and hyperventilation and volume of the sample, and the presence of all of the following characteristics:
- after hyperventilation, the level of adrenaline in the blood remains stable,
- before and (or) after hyperventilation, the total coefficient of nutrient amines (CAS) in large granular blood lymphocytes (BHL) is in the range from 2.1 to 3.6,
- after hyperventilation, the percentage of large granular lymphocytes carrying granules of serotonin increases,
- after hyperventilation, the level of blood serotonin decreases
include a clinically healthy person at risk for the development of allergic diseases. At the same time, a dosed hyperventilation test is carried out for 20 minutes with volumes of 40 l / min.

 The proposed method is as follows. In the initial state (resting state), 10-12 ml of venous blood from the cubital vein are taken from the patient under examination for the necessary biochemical and histochemical studies. Then a dosed hyperventilation test is carried out for 20 minutes with volumes of 40 l / min. Breathing is carried out by ordinary room (atmospheric) air. Hyperventilation is achieved thanks to a voluntary (voluntary) increase in the depth of breathing using a gas clock that records the depth of breathing in liters and minute ventilation in liters per minute. The respiratory rate is set by the metronome.

After a hyperventilation test, venous blood is retrieved again. Biochemical blood tests include determining the concentration of adrenaline and serotonin in the blood (see, for example, Laboratory research methods in the clinic, a guide edited by V. V. Menshikov - M .: Medicine, 1987, p. 368). The histochemical (chromafin) method of Sevka is used to detect adrenaline, histamine, serotonin in granules of large granular lymphocytes (BGL) - natural killers: in the original test system and after incubation with 10 ^-4 M of each of these amines.

From blood taken from the ulnar vein, a lymphocyte / macrophage fraction is obtained by centrifugation in a density gradient of ficoll-verographin. The number of cells was adjusted to a concentration of 2x10 ⁶ cells in 1 ml of 199 medium. Lymphoid-macrophage suspension was poured into 0.2 ml in 4 tubes. 0.2 ml of medium 199 was added to the first test tube - this is the initial test system (control), to the second 0.2 ml of 10 ^-4 M adrenaline hydrochloride solution, to the third 0.2 ml of 10 ^-4 M creatine sulfate solution serotonin, in the fourth - 0.2 ml of a 10 ^-4 M histamine hydrochloride solution. Samples were incubated for 40 minutes at 37 ^° C. After incubation, all samples were centrifuged at 1,500 rpm for 5 minutes. Sevka smears were prepared from pellet tubes. For this, the preparations were fixed in Sevka's reagent (40% formaldehyde - 12 ml, 5% potassium dichromate solution - 50 ml, 1 M sodium acetate solution - 20 ml, distilled water - up to 100 ml) for 6-8 hours . The fixing mixture was prepared immediately before use. After fixation, the preparations were washed for 1 hour in distilled water, and then stained with Giemsa solution for 1 hour. In the finished preparations, the number of cells carrying granules of serotonin, adrenaline, histamine in at least 10 fields of view was separately counted, and the relative (%) amount of amino-bearing BGL was determined. Then calculated the total coefficient of nutrient amines (CAS) according to the formula
SKA = Frame + Ksep + Kgist (1),
where K is a coefficient characterizing the quantitative ratio of BGL for each incubation method in the presence of a test biogenic amine.

где А - % БГЛ, несущих гранулы адреналина,
С - % БГЛ, несущих гранулы серотонина,
Г - % БГЛ, несущих гранулы гистамина,
в числителе (с индексом "и") - результаты определения для лимфоцитов, инкубированных с моноамином, для которого рассчитывается коэффициент, в знаменателе (с индексом "к") - показатели для интактных лимфоцитов (контроль).In turn

where A -% BGL bearing granules of adrenaline,
With -% BGL bearing granules of serotonin,
G -% BGL bearing histamine granules,
in the numerator (with the "and" index) - the results of determination for lymphocytes incubated with monoamine, for which the coefficient is calculated, in the denominator (with the "k" index) - indicators for intact lymphocytes (control).

Thus, the following indicators were determined:
- concentration of blood adrenaline,
- concentration of blood serotonin,
- the total number of BGL (in %%),
- the percentage of BGL carrying granules of adrenaline, serotonin, histamine,
- SKA value.

As a result of the research, the following risk criteria for the development of allergic diseases in clinically healthy people were identified - this is a combination of the following symptoms:
- after hyperventilation, the level of adrenaline in the blood remains stable,
- before and (or) after hyperventilation, SKA in BGL blood is in the average range of values (2.1 <SKA <3.6),
- after hyperventilation, the percentage of BGL carrying granules of serotonin increases,
- after hyperventilation, the level of blood serotonin decreases.

 The presence of a combination of all these symptoms allows us to attribute a clinically healthy person to a risk group for the development of allergic diseases.

 The proposed method for identifying persons at risk for the development of allergic diseases passed clinical and laboratory tests at the Reaviz Samara Medical Institute in a group of clinically healthy volunteer students. The value of SKA in all cases was calculated using formulas (1) and (2).

 Clinical examples.

 Example 1. Student K., born in 1975, weight 76 kg, height 177 cm, athlete (swimming), practically healthy. Allergopathology is not revealed. Examined in 1998. Before the hyperventilation test: adrenaline - 1.13 nM / L, serotonin - 32.5 μg%, BGL carrying serotonin - 4%, SKA - 3.37. After the hyperventilation test: adrenaline - 1.13 nM / L, serotonin - 27.6 μg%, BGL containing serotonin in granules - 6%, SKA - 3.37. Thus, the examinee identified all the criteria that can be attributed to the risk group for the development of allergic diseases. A follow-up examination in 2001 revealed an allergic reaction to drugs (broad-spectrum antibiotics) in the form of urticaria.

 Example 2

 Student Z., born in 1980, weight 70 kg, height 180 cm, practically healthy, no allergic diseases. Surveyed in 1998. Before the hyperventilation test: adrenaline - 6.7 nM / L, serotonin - 68 μg%, BGL, containing serotonin in granules - 3%, SKA - 2.54. After the hyperventilation test: adrenaline - 6.7 nM / L, serotonin - 46 μg%, BGL, bearing granules of serotonin - 4%, SKA - 4.56. All risk criteria are present. It is classified as a risk group for the development of allergic diseases. In 2000, signs of seasonal rhinitis and conjunctivitis (pollinosis) appeared.

 Example 3

 Student F., born in 1976, weight 59 kg, height 175 cm, practically healthy. There are no allergic diseases. Surveyed in 1998. Before the hyperventilation test: adrenaline - 3.38 nM / L, serotonin - 23.0 μg%, BGL, containing serotonin in granules - 4%, SKA - 4.08. After the hyperventilation test: adrenaline - 3.38 nM / L, serotonin - 15.95 μg%, BGL, containing serotonin in granules - 9%, SKA - 2.49. All risk criteria are present. It is classified as a risk group for the development of allergic diseases. In 2000 - 2001, urticaria was observed for penicillin and its analogues.

 Example 4

 Student M., born in 1980, weight 70 kg, height 175 cm, practically healthy. There are no allergic diseases. Surveyed in 1998. Before the hyperventilation test: adrenaline - 1.02 nM / L, serotonin - 50 μg%, BGL containing serotonin in granules - 2%, SKA - 3.88. After the hyperventilation test: adrenaline - 1.016 nM / L, serotonin - 25 μg%, BGL, containing serotonin in granules - 4%, SKA - 3.03. All risk criteria are present. It is classified as a risk group for the development of allergic diseases. In 2001, an allergic reaction to food products (citrus fruits) in the form of urticaria was identified.

 Example 5

 Student F., born in 1979, weight 71 kg, height 182 cm, practically healthy. There are no allergic diseases. Surveyed in 1998. Before the hyperventilation test: adrenaline - 1.13 nM / L, serotonin - 24.4 μg%, BGL, carrying serotonin - 6%, SKA - 4.49. After the hyperventilation test: adrenaline - 5.64 nM / L, serotonin - 27.63 μg%, BGL, carrying serotonin - 8%, SKA - 3.86. The set of criteria to classify it as a risk group for the development of allergic diseases is absent. In 2001, he passed a control examination. Diagnosis: almost healthy.

 A total of 92 clinically healthy volunteers were examined. According to the specified criteria, 35 people were assigned to the risk group (38% of all examined), of which after 3 years signs of allergic diseases were recorded in 16 people (45.7% of the number of examined allocated to risk group). In 57 patients not assigned to the risk group, repeated studies of allergic diseases were not detected. The control group included 20 patients with allergic diseases, in whom both during remission and at the time of exacerbation, a set of indicators was selected that were selected as criteria for allergopathology risk. Thus, the presence of a combination of all risk criteria allows us to attribute a clinically healthy patient to a risk group for the development of allergic diseases. The absence of at least one of the criteria means that this patient is not at risk for the development of allergic diseases.

 The present invention provides reliable identification of persons at risk for the development of allergic diseases among clinically healthy people and due to early diagnosis and a course of preventive treatment, reducing the incidence of various forms of allergies, especially dangerous ones such as bronchial asthma and anaphylactic shock, as well as increasing the effectiveness of the treatment of allergic diseases.

Claims (1)

 A method for identifying persons at risk for the development of allergic diseases, characterized in that they carry out a biochemical blood test for adrenaline and serotonin and a histochemical study of large granular lymphocytes in a load test with biogenic amines with the calculation of the total amino content coefficient before and after the hyperventilation test for 20 min with volumes of 40 l / min, and in the presence of a combination of the following symptoms: after hyperventilation, the level of blood adrenaline remains stable Before, and / or after hyperventilation, the total amino content coefficient for large granular blood lymphocytes is in the range from 2.1 to 3.6, after hyperventilation, the percentage of large granular lymphocytes carrying serotonin granules increases, after hyperventilation, the level of blood serotonin decreases - include a clinically healthy person at risk for the development of allergic diseases.