RU2225984C1 - Method for determining functional activity of phagocytes in the cases of allergy - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves combining labeled corpuscle-type allergen-specific diagnosticum preparations with whole blood. The produced mixture is divided into two portions. The first one is incubated for 20 min and the second one for 120 min at 37 C. The incubation times being over, formalin is added to each laboratory glass content. Smears are produced on microscopic slide glass, dried on the air and scrutinized using immersion objective of luminescent microscope. The number of cells, having swallowed and not swallowed the labeled corpuscle-type allergen-specific diagnosticum, is counted. Functional (swallowing and not swallowing) activity of phagocytes and digestive ability of phagocytes is calculated from formula X= (Y-Z)/Y*100%, where X is the digestive ability index value, Y is the phagocytosis efficiency in 20 min, Z is the phagocytosis efficiency in 120 min. X being less than 50±3%, functional activity phagocytes disorder is diagnosed in allergy cases. EFFECT: high accuracy and sensitivity of method. 3 tbl

Description

The invention relates to medicine, namely allergology and immunology, and can be used in the practice of clinical allergological and immunological laboratories in assessing the immune response (phagocytosis) to allergens in adults and children.

In medical practice, the assessment of the cellular immune response (phagocytosis) to allergens is carried out using different methods and various bioreagents.

A known method for evaluating the functional activity of phagocytes of human peripheral blood using staphylococci labeled with fluorescein isothiocyanate (FITC-labeled staphylococci).

Mazurov D.V., Khamidullina K.F., Pinegin B.V. A comprehensive method for evaluating the functional activity of peripheral blood phagocytic cells. // Medical immunology, 1999, v. 1, No. 3 and 4, p. 74 and 75.

Mazurov D.V., Pinegin B.V. The use of flow cytometry to assess the absorption and bactericidal functions of granulocytes and peripheral blood monocytes // Allergy, Asthma and Clinical Immunology, 1999, No. 9, p.154-156.

Disadvantages: the method includes the use of chemical reagents, centrifugation of leukocytes, which leads to cell damage, and their fractionation by flow cytometry promotes the activation of phagocytic cells and, consequently, changes in the results and reliability of the method.

The closest analogue is a method for determining phagocytosis using formalized ram erythrocytes (FEB).

Kaplin V.N. Unconventional immunology of infections. - Perm: Perm state. honey. Academy, 1966, 163 p.

Kaplin V.N., Kuznetsov V.F., Obernesova T.P. Methodological aspects of the study of phagocytosis // I Congress of Russian Immunologists: Abstracts. Doc. (June 23-25, 1992). - Novosibirsk, 1992, p.200 and 201.

Shilov Yu.I., Vladykina V.P., Atnaguzina A.T. Some methodological approaches to assessing indicators of general and differentiated phagocytosis of peripheral blood leukocytes. Characterization of differences in healthy people // Perm Medical Journal, 1998, No. 2 and 3, vol. XV, p. 3-9.

Disadvantages: the method using the FEB does not allow to register the digesting ability of phagocytes, but only the absorption, which leads to an incomplete assessment of phagocytosis, in addition, the color of the smears according to Romanovsky-Giemsa reduces the sensitivity of the method.

The invention is aimed at solving problems: increasing the sensitivity of the method and the ability to assess the digesting ability of phagocytes.

These tasks are achieved by combining the object of phagocytosis - the phobic cell with whole blood and calculating the absorption capacity of phagocytes. The method is characterized in that labeled corpuscular allergen-specific diagnostics (MKAD) are used as an object of phagocytosis. Labeled corpuscular allergen diagnostics are formalized, tanized sheep erythrocytes sensitized with a certain food, household, pollen allergen labeled with a fluorescent dye (fluorescein isothioyanate - FITC or tetrarodamine isothiocyanate). The resulting mixture is divided into two portions of 100 μl, the first is incubated for 20 minutes, and the second for 120 minutes at 37 ° C, then, immediately after the incubation period, 10 μl of formalin is added to each tube, smears are prepared on a glass slide, dried in air, viewed under an immersion lens of a luminescent microscope, at least 100 phagocytes that absorbed and did not absorb MKAD were counted. The indicators of total phagocytosis are calculated: the percentage of phagocytosis, phagocytic number, phagocytic index, phagocytosis efficiency and the index of digesting ability of phagocytes is calculated by the formula

where X is the index of digesting ability of phagocytes;

Y is the efficiency of phagocytosis after 20 minutes;

Z - the effectiveness of phagocytosis after 120 minutes,

and with a value of X less than (50 ± 3)%, damage to the functional activity of phagocytes in case of allergy is determined.

The method is as follows: the object of phagocytosis - MKAD is combined with whole blood, the resulting mixture is divided into two parts of 100 μl, the first is incubated for 20 minutes; and the second - for 120 minutes at a temperature of 37 ° C, then, immediately after the incubation period, 10 μl of formalin are added to each tube, smears are prepared on a glass slide, dried in air for 5-7 minutes, and viewed under immersion lens of a luminescent microscope and count at least 100 of all phagocytes that have absorbed and not absorbed MKAD. The indicators of total phagocytosis are calculated: the percentage of phagocytosis, phagocytic number, phagocytic index, the effectiveness of phagocytosis.

The functional (absorption) activity of all types of phagocytes is determined by counting 100 phagocytized and non-phagocytized cells in both portions (20 min and 120 min) of the studied suspension and calculating the following indicators.

(Bakhmetyev B.A., Shirshov S.V., Kevorkov N.N. Main indicators of the immunogram of children and adults of the Perm region. Perm, 2002, 44 pp.)

Percentage of phagocytosis is the number of cells that have captured the MKAD from 100 counted phagocytes.

Phagocytic number - the amount of MKAD which on average is in 1 out of 100 counted phagocytes.

Phagocytic index - the amount of MKAD that falls on one phagocytic cell.

The efficiency of phagocytosis is the absolute number of phagocytes absorbed by the MKAD per 1 mm ^{3 of} blood.

Calculation of the functional (digesting) activity of phagocytosis:

The number of digested MKAD - the difference in the effectiveness of phagocytosis after 20 minutes and 120 minutes of exposure of portions of the test mixture of blood.

The index of functional (digesting) ability of phagocytes is the quotient of dividing the difference in the efficiency of phagocytosis of the 20-minute and 120-minute servings of the studied blood mixtures by the efficiency of phagocytosis at 20 minutes and is expressed as a percentage.

The formula for calculating the index of digestion ability of phagocytes

where X is the index of digesting ability of phagocytes;

Y is the efficiency of phagocytosis after 20 minutes;

Z - the effectiveness of phagocytosis after 120 minutes,

and with a value of X less than (50 ± 3)%, damage to the functional activity of phagocytes in case of allergy is determined.

Examples of specific performance.

Example 1

Child A. 12 years old. He complained of skin rashes on the face after eating sausages, cow's milk.

Anamnesis of the disease: manifestations of allergic diathesis in the form of rashes, manifested by mild hyperemia, exudation, desquamation and the appearance of papules, accompanied by itching of the skin, notes from 3 years on dairy products. Heredity for allergic diseases is burdened: the mother has atopic dermatitis. Exacerbations occur 1-2 times a year in violation of the diet. The duration of remission is 6-8 months.

Objective examination data: hyperemia on the skin of the face, presence of papules, hemorrhagic crusts, scratches.

The pharynx is calm, the tongue is covered with a white coating. Single submandibular lymph nodes are palpated. In the lungs, vesicular breathing. Heart sounds are muffled, rhythmic. The abdomen is soft, painless. A blood test was performed: leukocytes - 7100, stab neutrophils - 1, segmented neutrophils - 41, eosinophils - 10, monocytes - 9, lymphocytes - 39, immunoglobulins of classes: Ig G (g / l) - 7.7, IgA (g / l ) - 2.8, IgM (g / l) - 1.1, IgE (ME / ml) - 800.

Diagnosis. Limited atopic dermatitis, mild course, period of manifestation. Sensitive sensitization (allergy).

The result of the study: the absorption capacity of phagocytes within normal limits, the digesting ability of phagocytes is reduced and amounts to 29%.

Example 2

Patient P. 60 years.

Complaints of sneezing, itchy nose, eyes, difficulty breathing after visiting a circus, zoo, contact with a cat, working with animals.

During the inspection, the condition is satisfactory. The pharynx is calm, the lymph nodes are not enlarged. Hard breathing, no wheezing. Heart sounds are muffled, rhythmic. The abdomen is soft, painless.

Survey 1. Spirography. There is a risk of decreasing FVC at the border of the conditional norm. Exhalation time is increased. The test with a bronchodilator is positive.

2. Index of local eosinophilia 4, 3

3. Total IgE 285 ME / ml

4. Specific IgE: CC 3+,

5. Scarification skin tests: TUK neg., Histamine 2+, cat hair 2+, horse dander 2+.

6. Intracutaneous tests TKZH neg., Histamine 3+, cat hair 2+, house dust 2+, library dust 2+.

7. Indirect method of fluorescent antibodies: cat hair 1-2 +, house dust 2+.

A blood test was performed: leukocytes - 5600, stab neutrophils - 3, segmented neutrophils - 43, eosinophils - 11, monocytes - 7, lymphocytes - 36, immunoglobulins of classes: log G (g / l) - 15.6, IgA (g / l ) - 2.39, IgM (g / l) - 2.14, IgE (ME / ml) - 285.

Diagnosis. Household, epidermal sensitization (allergy).

Atonic bronchial asthma, submission.

Allergic rhinitis, intermittent, remission.

Allergic conjunctivitis, intermittent, remission.

The result of the study: the absorptive capacity of phagocytes within normal limits, the digesting ability of phagocytes is reduced and amounts to 33.6%.

Example 3

Patient Eve., Born in 1989

No complaints. A medical examination is being carried out in connection with the entry to work.

At the time of inspection, the condition is satisfactory. By internal organs without deviations.

A blood test was performed: leukocytes - 6800, stab neutrophils - 2, segmented neutrophils - 48, eosinophils - 2, monocytes - 5, lymphocytes - 43, immunoglobulins of classes: Ig G (g / l) - 16.9, IgA (g / l ) - 2.6, IgM (g / l) - 1.28, IgE (ME / ml) - 80.

The result of the study: the functional (absorbing and digesting) ability of phagocytes within normal limits.

Diagnosis. Almost healthy. Suitable for work in the laboratory.

Positive effect. The method allows to increase the sensitivity of the analysis through the use of MKAD, and also to evaluate the digesting ability of phagocytes, which is important for searching for violations of the main stages of phagocytosis both at the diagnostic stage and at the stage of treatment of allergic diseases.

Claims (1)

A method for determining the functional activity of phagocytes in allergies by combining a phagocytosis object with whole blood, characterized in that labeled corpuscular allergen-specific diagnostics (MKAD) are used as a phagocytosis object, combined with whole blood, the resulting mixture is divided into two parts, the first is incubated for 20 minutes and the second for 120 minutes at a temperature of 37 ° C, then, after the expiration of the incubation period, formalin is added to each tube, smears are prepared on a glass slide, dried in air, viewed under a fluorescence microscope immersion objective, count the number of cells engulfing Moscow, calculates the absorption and digestive activity of phagocytes and calculate index digestive ability of phagocytes formula

where X is the index of digesting ability of phagocytes; Y is the efficiency of phagocytosis after 20 minutes; Z - the effectiveness of phagocytosis after 120 minutes, and with an X value of less than 50 ± 3%, damage to the functional activity of phagocytes in allergies is determined.