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INVENTION
Patent of the Russian Federation RU2156465

METHOD OF DIAGNOSTICS OF MICROSENSIAL SENSIBILIZATION

The name of the inventor: Ivanova LA; Izmerova N.I .; Pozdnyakova NV; Frolova Ya.A.
The name of the patent holder: Scientific Research Institute of Occupational Health of RAMS
Address for correspondence: 121359, Moscow, Marshal Timoshenko 21, UC, patent office, Evtukhov A.N.
Date of commencement of the patent: 2000.02.01

The invention relates to the field of medicine, namely, to dermatology and internal diseases, and can be used to diagnose sensitization in diseases of the skin and internal organs. Take 2 cone-shaped tubes, 0.1 ml of 0.1% aqueous solution of nitrosinium tetrazolium are added to each, 0.1 ml of blood is added to the tubes and 0.1 ml of blood is added to the test tubes with the addition of anticoagulant, 0 is added to the first tube, 3 ml of a 0.1% solution of the fungal antigen, and 0.3 ml of saline is added to the second tube and this tube is used as a control, both tubes are incubated at 37 ° C. for 30 minutes and then cooled to normal room temperature , And both tubes are centrifuged at 1000 rpm for 5 minutes, after which the strokes are prepared from the upper or middle layers of the cell suspension of both the first and second test tubes, fixed with methyl alcohol for 10 minutes, stained with dabs from the first and from the second tubes 1 % Solution of safranin for 5 min, colored neutrophilic leukocytes containing dark blue granules of formazan are considered and evaluated in the cytoplasm, NTS-positive neutrophils are considered, and in case of excess from 5 to 10% in smears from the first test tube in comparison with smears From the second, a conclusion is made about the presence of a weakly expressed, in the case of 10 to 20% - moderately pronounced and when exceeding from 20% or more - a pronounced mycogenic sensitization. The method ensures an increase in the reliability of the diagnosis.

DESCRIPTION OF THE INVENTION

The invention relates to medicine, viz., Dermatology and internal diseases, and can be used to diagnose sensitization in diseases of the skin and internal organs.

The essence of the invention is that two cone-shaped tubes are taken, 0.1 ml of a 0.1% aqueous solution of nitrous tetrazolium are added to each, blood is taken from the finger and 0.1 ml of blood is added to the tubes, with the addition of an anticoagulant; 0.3 ml of a 0.1% solution of a fungal antigen is introduced into the first tube and 0.3 ml of saline is added to the second tube and this tube is used as a control; Both tubes are incubated at 37 ^° C for 30 minutes and then cooled to normal room temperature; And both tubes are centrifuged at 1000 rpm for 5 minutes, after which dabs are prepared from the upper or middle layers of the cell suspension of both the first and second tubes, fixed with methyl alcohol for 10 minutes; Dye stains from the first and second tubes with 1% safranin solution for 5 minutes; Examine and evaluate stained neutrophilic leukocytes containing dark blue granules of formazan, consider NTS-positive neutrophils, and in the case of excess of 5 to 10% in smears from the first tube compared with smears from the second, it is concluded that there is a weakly expressed Case from 10 to 20% - moderately expressed and when exceeding from 20% or more - strongly pronounced mycogenic sensitization.

Methods for in vitro diagnostics of mycogenic sensitization [1, 2] are known in terms of the level of antibodies to the fungal antigen.

They have the following drawbacks. High trauma, as the blood is taken from the vein and in large quantities. Low informativity, since the allergic reaction to the fungus rarely proceeds by immediate type in the system of humoral immunity. The degree of expression of the level of sensitization is not revealed.

As a prototype, a method for diagnosing [3] sensitization has been chosen. He has the following drawbacks. The method is traumatic. There is not enough gradation to diagnose the level of sensitization.

In the proposed invention, the following tasks were solved: the traumatic nature of the sampling of the material for the study was reduced; The diagnostic reliability and correlation frequency with skin tests, the fungal antigen and the severity of the observed clinical picture were increased; The accuracy of three gradations of the degree of expression of the level of sensitization in vitro is increased.

This problem is solved by taking 2 cone-shaped tubes, 0.1 ml of a 0.1% solution of nitrous tetrazolium are injected into each, blood is taken from the finger and 0.1 ml of blood is added to these tubes with the addition of an anticoagulant; 0.3 ml of a 0.1% solution of a fungal antigen is introduced into the first tube and 0.3 ml of saline is added to the second tube and this tube is used as a control; Both tubes are incubated at 37 ^° C for 30 minutes and then cooled to normal room temperature; And both tubes are centrifuged at 1000 rpm for 5 minutes, after which the strokes are prepared from the upper or middle layers of the cell suspension of both the first and second tubes, fixed with methyl alcohol for 10 minutes, stained with dabs from the first and from the second tubes 1% Solution of safranin for 5 minutes; Examine and evaluate stained neutrophilic leukocytes containing dark blue granules of formazan, consider NTS-positive neutrophils, and in the case of excess of 5 to 10% in smears from the first tube compared with smears from the second, it is concluded that there is a weakly expressed Case from 10 to 20% - moderately expressed and when exceeding from 20% or more - strongly pronounced mycogenic sensitization. The method is carried out in the following sequence. Take 2 cone-shaped test tubes, 0.1 ml of a 0.1% aqueous solution of nitrosine tetrazolium is added to each. If less than 0.1% tetrazolium, then not all blood cells will undergo a reaction; If more than 0.1% tetrazolium, then part of the dye precipitates on the glass and decreases significantly and a sufficient number of gradations are not found to assess the level of sensitization.

Blood is taken from the finger in a volume of 0.3 ml. This volume does not exceed, because for further analysis, less blood is required.

Add 0.1 ml of blood with the addition of anticoagulant to the indicated 2 tubes. The value of 0.1 ml of blood is optimal for the mixing reaction with the previously introduced into the test tubes as a reagent and in a volume and 0.1 ml of nitrous tetrazolium.

In the first test tube, 0.3 ml of 0.1% fungal antigen is added. The volume of 0.3 ml is optimal for mixing with the previous ingredients. If the concentration of fungal antigen is less than 0.1%, then the manifestation of the cytochemical effect is hampered. If the concentration of this antigen is greater than 0.1%, the biochemical activity of the cell is suppressed in the reaction with nitrosine tetrazolium.

In the second tube add 0.3 ml of standard saline. This tube is subsequently used as a control.

Both tubes are incubated at 37 ^° C for 30 minutes. Conducting the incubation in less than 30 minutes does not ensure the detection of the reaction in the cells. Time more than 30 minutes does not improve the reaction rates, but leads to some cell damage.

After 30 minutes of incubation, both tubes are cooled to normal room temperature.

Centrifuge the two tubes at 1000 rpm for 5 minutes.

If the rotational speed is less than 1000 rpm, then insufficient fractionation is observed.

At a rotation speed of more than 1000 rpm, traumatism of the cells can be observed. The centrifugation time of less than 5 minutes results in inadequate cell separation. If centrifuged for more than 5 minutes, then the cells of the substrate are damaged.

Smears are prepared from the upper or middle layers of the suspensions obtained in the first and second test tubes. These smears, made from the middle layer, supplement the volume of the material under study. Smears prepared from the top layer serve as the main diagnostic material. These smears are fixed with methyl alcohol for 10 minutes. If the fixation time is less than 10 min, the cell structure will not be retained. Duration of fixation for more than 10 minutes leads to inhibition of biochemical free radical processes in cells.

Dye stains from the first and second test tubes with a 1% solution of safranin for 5 minutes. If the color is less than 5 minutes, then individual cell structures remain unpainted. A coloration time of more than 5 minutes results in repainting the smear.

They consider NST-positive neutrophils, that is, containing colored granules of formazan.

In the case of exceeding the content of HCT-positive neutrophils from 5 to 10% in smears from the first tube compared with smears from the second, conclude that there is a weakly expressed sensitization. In case of excess from 10 to 20% and in smears from the second test tube, in comparison with the first one, a conclusion is made about moderate expressed sensitization. In the case of exceeding the number of neutrophils by 20% or more, severe mycogenic sensitization is found.

Example 1 . Patient S., 45 years old. The process is localized in folds between 4 and 3, 4 and 5 toes, characterized by a slight erythematous-scaly foci, accompanied by a weak itch. An immuno-cytochemical study was performed to detect mycogenic sensitization. For this, 2 cone-shaped tubes are taken, 0.1 ml of a 0.1% aqueous solution of nitrous tetrazolium is added to each, blood is taken from the finger and 0.1 ml of blood is added to the tubes, with the addition of an anticoagulant; 0.3 ml of a 0.1% solution of a fungal antigen is introduced into the first tube and 0.3 ml of saline is added to the second tube and this tube is used as a control; Both tubes are incubated at 37 ^° C for 30 minutes and then cooled to normal room temperature; And both tubes are centrifuged at 1000 rpm for 5 minutes, after which dabs are prepared from the upper or middle layers of the cell suspension of both the first and second tubes, fixed with methyl alcohol for 10 minutes; Dye stains from the first and second tubes with 1% safranin solution for 5 minutes; Examine and evaluate stained neutrophilic leukocytes containing dark blue granules of formazan in the cytoplasm, HCT-positive neutrophils in blood smears from the first and second tubes. In smears from the first tube, the content of HCT-positive neutrophils is 19%. In smears from the second tube, these neutrophils are 11%. The difference of HCT-positive neutrophils in the smears and controls tested for allergic reaction is 8%. They conclude that there is poorly expressed mycogenic sensitization.

Example 2 . Patient N., 35 years old. Nail plates 5 toes stop yellowish, deformed, crumble, subungual hyperkeratosis noted. On the skin of the 4th interdigital fold of the feet, there is edematous erythema with maceration, microvegetation, accompanied by sensations of itching and burning. Take 2 cone-shaped test tubes, 0.1 ml of a 0.1% aqueous solution of nitrous tetrazolium is added to each, blood is taken from the finger and 0.1 ml of blood is added to these tubes with the addition of an anticoagulant; 0.3 ml of a 0.1% solution of a fungal antigen is introduced into the first tube and 0.3 ml of saline is added to the second tube and this tube is used as a control; Both tubes are incubated at 37 ^° C for 30 minutes and then cooled to normal room temperature; And both tubes are centrifuged at 1000 rpm for 5 minutes, after which dabs are prepared from the upper or middle layers of the cell suspension of both the first and second tubes, fixed with methyl alcohol for 10 minutes; Dye stains from the first and second tubes with 1% safranin solution for 5 minutes; Examine and evaluate stained neutrophilic leukocytes containing dark blue granules of formazan in the cytoplasm. Consider NST-positive neutrophils in blood smears from the first and second test tubes. In smears from the first tube, the activity of HCT-positive neutrophils is 29%. In smears from the second test tube, the activity is 12%, respectively. The difference in activity indicators is 17%. Diagnose moderately expressed mycogenic sensitization.

Example 3 . Patient M., 47 years old. The nail plates of all toes are deformed, thickened, and grayish, dull in color. In interdigital folds, cracks with a clear peeling around. The skin of the soles is dry, against the background of stagnant erythema, there are folds with mucous peeling. Subjectively, itchy. The patient was assigned a cytochemical study of the NST-test with a fungal antigen. Take 2 cone-shaped test tubes, 0.1 ml of a 0.1% aqueous solution of nitrous tetrazolium is added to each, blood is taken from the finger and 0.1 ml of blood is added to these tubes with the addition of an anticoagulant; 0.3 ml of a 0.1% solution of a fungal antigen is introduced into the first tube and 0.3 ml of saline is added to the second tube and this tube is used as a control; Both tubes are incubated at 37 ^° C for 30 minutes and then cooled to normal room temperature; And both tubes are centrifuged at 1000 rpm for 5 minutes, after which dabs are prepared from the upper or middle layers of the cell suspension of both the first and second tubes, fixed with methyl alcohol for 10 minutes; Dye stains from the first and second tubes with 1% safranin solution for 5 minutes; Examine and evaluate stained neutrophilic leukocytes containing dark blue granules of formazan in the cytoplasm. HST-positive neutrophils of the smear are considered. 41% of these neutrophils were detected in smears from the first tube; In smears from the second - a control tube, the calculated data is 15%. The difference in the number of stained neutrophils in smears from the first and second tubes is 26%. Diagnose strongly pronounced mycogenic sensitization. The diagnosis was confirmed by microscopic examination, in which the mycelia of a pathogenic fungus was revealed in scrapings of nail plates.

The efficiency of the method

1. The traumatic nature of the sampling of the material for the study has been reduced. Take only 0.1 ml of blood from the finger. In all existing methods, blood is taken from the vein, which is traumatic. There is a risk of infectious complications.

2. The reliability of the analysis was increased, as the degree of severity of the level of sensitization in three grades was differentiated.

3. The results of the treatment (see table) confirm the effectiveness.

USED ​​BOOKS

1. Kishkin P.N. Dermatomycosis. L. 1954.

2. Kassirsky I.A. Alekseev G.A. Clinical hematology. M. 1970.

3. Ado A.D. General allergology. M. 1970.

CLAIM

A method for diagnosing mycogenic sensitization by carrying out a cytochemical reaction, characterized in that 2 cone-shaped tubes are taken, 0.1 ml of a 0.1% aqueous solution of nitrosinium tetrazolium is added to each, blood is taken from the finger and the tubes are introduced into 0, 1 ml of blood with the addition of anticoagulant, 0.3 ml of 0.1% solution of fungal antigen is added to the first tube and 0.3 ml of saline is added to the second tube, and this tube is used as a control, both tubes are incubated at 37 ^° C for 30 minutes and then cooled to normal room temperature, and both tubes are centrifuged at 10,000 rpm for 5 minutes, after which dabs are prepared from the upper or middle layers of the cell suspension, both first and second tubes, fixed Methyl alcohol 10 minutes, dye stains from the first and second tubes with 1% safranin solution for 5 minutes, treated and evaluated stained neutrophilic leukocytes containing dark blue granules of formazan in the cytoplasm, NTS-positive neutrophils are considered, and in case of excess From 5 to 10% in smears from the first tube in comparison with smears from the second make a conclusion about the presence of poorly expressed, in the case of 10 to 20% - moderately expressed and when exceeding from 20% or more - severe mycogenic sensitization.

print version
Date of publication 01.04.2007гг

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