RU2156465C1 - Method for diagnosing mycogenous sensitization - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves taking blood sample from finger. Two conic test tubes are taken and 0.1 ml of 0.1% aqueous solution of nitro blue tetrazolium. 0.1 ml of the taken blood is placed into each test tube with anticoagulant agent added. 0.3 ml of 0.1% solution of fungous antigen is added to the first test tube. 0.3 ml of physiological salt solution is added to the second test tube and the latter is used as a control one. Both test tubes are incubated at 37 C during 30 min and then cooled to normal room temperature. Both tubes are centrifuged at 1000 rpm during 5 min. Smears are prepared from the upper or middle layers of cell suspension taken both from the first and the second test tube. The smears are fixed with methyl alcohol during 10 min. The smears both from the first and the second test tube are repeatedly stained to finished state with 1% Safranin solution during 5 min. The stained neutrophil leukocytes containing dark blue formazan molecules in cytoplasm are examined and evaluated. NBT- positive leukocytes are counted. The smears from the first test tube showing 5 to 10% excess when compared to the smears from the second test tube, conclusion is made that weakly marked mycogenous sensitization is the case. 10 to 20% being the case, moderate degree mycogenous sensitization is considered to take place. 20% and higher being the case, highly marked mycogenous sensitization is considered to take place. EFFECT: enhanced reliability of diagnosis. 1 tbl

Description

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

The essence of the invention is that they take 2 cone-shaped tubes, 0.1 ml of a 0.1% aqueous solution of nitro-blue tetrazolium are introduced into each, blood is drawn from the finger and 0.1 ml of blood is added to the test tubes with the addition of an anticoagulant; 0.3 ml of a 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; both tubes are centrifuged at 1000 rpm for 5 minutes, after which smears 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; stain smears from the first and second tubes with 1% safranin solution for 5 min; stained neutrophilic leukocytes containing dark blue formazan granules in the cytoplasm are examined and evaluated, NTS are considered positive neutrophils, and in the case of excess from 5 to 10% in smears from the first tube in comparison with smears from the second, they conclude that there is a weak case from 10 to 20% - moderately expressed and in excess of 20% or more - strongly expressed mycogenous sensitization.

 Known methods for diagnosing in vitro mycogenous sensitization [1,2] by the level of determination of antibodies to fungal antigen.

 They have the following disadvantages. High invasiveness, as blood is taken from a vein and in large quantities. Low information content, since an allergic reaction to a fungus rarely proceeds according to an immediate type in the system of humoral immunity. The severity of the level of sensitization is not detected.

 As a prototype of the selected diagnostic method [3] sensitization. It also has the following disadvantages. The method is traumatic. There are not enough gradations to diagnose the level of sensitization.

 In the proposed invention, the following tasks are solved: the invasiveness of the sampling of material for research is reduced; the reliability of diagnosis and the frequency of correlation with skin samples, with fungal antigen and the severity of the observed clinical picture are increased; the accuracy of three gradations of the severity level of sensitization in vitro was increased.

This problem is solved by taking 2 cone-shaped tubes, 0.1 ml of a 0.1% solution of nitro-blue tetrazolium is added to each, blood is drawn from the finger and 0.1 ml of blood is added to the indicated tubes with the addition of an anticoagulant; 0.3 ml of a 0.1% fungal antigen solution 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; both tubes are centrifuged at 1000 rpm for 5 minutes, after which smears 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, the smears are repainted from the first and second tubes 1% - with safranin solution for 5 min; stained neutrophilic leukocytes containing dark blue formazan granules in the cytoplasm are examined and evaluated, NTS are considered positive neutrophils, and in the case of excess from 5 to 10% in smears from the first tube in comparison with smears from the second, they conclude that there is a weak case from 10 to 20% - moderately expressed and in excess of 20% or more - strongly expressed mycogenous sensitization. The method is performed in the following sequence. Take 2 cone-shaped tubes, in each add 0.1 ml of a 0.1% aqueous solution of nitrosine tetrazolium. 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 glass and decreases significantly and not enough gradations are 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, since a smaller amount of blood is required for further analysis.

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

 0.3 ml of 0.1% fungal antigen is added to the first tube. A 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 a cytochemical effect is difficult. If the concentration of the indicated antigen is more than 0.1%, then the biochemical activity of the cell is suppressed in the reaction with nitro-blue tetrazolium.

 0.3 ml of standard saline is added to the second tube. This tube is further used as a control.

Both tubes are incubated at 37 ^° C. for 30 minutes. Conducting incubation in less than 30 minutes does not provide a detection of the reaction in the cells. A time of more than 30 minutes does not improve the response, but leads to some damage to the cells.

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

 Centrifuge these two tubes at 1000 rpm for 5 minutes.

 If the rotation speed is less than 1000 rpm, then there is insufficient separation into fractions.

 At rotational speeds of more than 1000 rpm, trauma to the cells may occur. Centrifugation time of less than 5 min leads to insufficient cell separation. If centrifuged for more than 5 min, the substrate cells are damaged.

 From the upper or middle layers of the suspensions obtained in the first and second test tubes, smears are prepared. These smears made from the middle layer supplement the volume of the test material. 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 minutes, then the cell structure will not be preserved. Duration of fixation of more than 10 min leads to inhibition of biochemical free radical processes in cells.

 Smear the smears from the first and second tubes with a 1% safranin solution for 5 minutes. If stained for less than 5 min, individual cell structures remain unpainted. A staining time of more than 5 minutes leads to repainting of the smear.

 HCT-positive neutrophils, that is, containing stained formazan granules, are considered.

 If the content of HCT-positive neutrophils is exceeded from 5 to 10% in smears from the first tube in comparison with smears from the second, a conclusion is drawn on the presence of mild sensitization. In case of excess from 10 to 20% also in smears from the second test tube, in comparison with the first one, a conclusion is drawn about moderately pronounced sensitization. If the number of neutrophils is exceeded by 20% or more, strongly pronounced mycogenic sensitization is found.

Example 1. Patient S., 45 years old. The process is localized in the folds between the 4 and 3, 4 and 5 toes, is characterized by minor erythematous-scaly foci, accompanied by mild itching. An immuno-cytochemical study is performed to detect mycogenous sensitization. To do this, take 2 cone-shaped tubes, 0.1 ml of a 0.1% aqueous solution of nitro-blue 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% fungal antigen solution 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; both tubes are centrifuged at 1000 rpm for 5 minutes, after which smears 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; stain smears from the first and second tubes with 1% safranin solution for 5 min; stained neutrophilic leukocytes containing dark blue formazan granules, HCT-positive neutrophils in blood smears from the first and second test tubes are examined and evaluated. In smears from the first tube, the content of HCT-positive neutrophils is 19%. In smears from the second tube, these neutrophils make up 11%. The difference between HCT-positive neutrophils in the smears tested for the allergic reaction and in the control is 8%. Make a conclusion about the presence of mild mycogenic sensitization.

Example 2. Patient N., 35 years old. The nail plates of the 5 toes are yellowish, deformed, crumble, subungual hyperkeratosis is noted. On the skin of the 4th interdigital fold of the feet, there is edematous erythema with maceration, microvesiculation, accompanied by sensations of itching and burning. Take 2 cone-shaped tubes, 0.1 ml of a 0.1% aqueous solution of nitro-blue 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% fungal antigen solution 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; both tubes are centrifuged at 1000 rpm for 5 minutes, after which smears 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; stain smears from the first and second tubes with 1% safranin solution for 5 min; stained neutrophilic leukocytes containing dark blue formazan granules in the cytoplasm are examined and evaluated. NST-positive neutrophils in blood smears from the first and second test tubes are considered. In smears from the first tube, the activity of HCT-positive neutrophils is 29%. In smears from the second tube, the activity is 12%, respectively. The difference in activity indicators is 17%. Diagnose moderately expressed mycogenous sensitization.

Example 3. Patient M., 47 years old. Nail plates of all toes are deformed, thickened, grayish dull color. In interdigital folds cracks with a clear peeling around. The skin of the soles is dry, against the background of congestive erythema, folds with flour-like peeling are noted. Subjectively, itching. The patient was assigned a cytochemical study of the HCT test with a fungal antigen. Take 2 cone-shaped tubes, 0.1 ml of a 0.1% aqueous solution of nitro-blue 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% fungal antigen solution 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; both tubes are centrifuged at 1000 rpm for 5 minutes, after which smears 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; stain smears from the first and second tubes with 1% safranin solution for 5 min; stained neutrophilic leukocytes containing dark blue formazan granules in the cytoplasm are examined and evaluated. NST-positive smear neutrophils are considered. 41% of these neutrophils are determined in smears from the first tube; in smears from the second 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 expressed mycogenous sensitization. The diagnosis was confirmed by microscopic examination, in which the mycelium of a pathogenic fungus was revealed in scrapings of the nail plates.

The effectiveness of the method
1. The morbidity of the sampling material for the study is reduced. Take only 0.1 ml of blood from the finger. In all existing methods, blood is drawn from a vein, which is traumatic. There is a risk of infectious complications.

 2. The reliability of the analysis is increased, since the differentiation of the degree of severity of the level of sensitization by three gradations is carried out.

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

Literature
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.

Claims (1)

A method for diagnosing mycogenous sensitization by carrying out a cytochemical reaction, characterized in that they take 2 cone-shaped tubes, add 0.1 ml of a 0.1% aqueous solution of nitro-blue tetrazolium to each, draw blood from the finger and bring into these tubes at 0, 1 ml of blood with the addition of an anticoagulant, 0.3 ml of a 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 ^o C for 30 minutes then cooled to normal room temperature, also both tubes are centrifuged at 10,000 rpm for 5 minutes, after which smears are prepared from the upper or middle layers of the cell suspension, both the first and second tubes, fixed with methyl alcohol for 10 minutes, the smears are repainted stained neutrophilic leukocytes containing dark blue formazan granules in the cytoplasm are examined and evaluated from the first and second test tubes with a 1% solution of safranin for 5 min, NTS are considered positive neutrophils, and in case of excess from 5 to 10% in m In the first test tube, in comparison with the second smear, they conclude that there is a mild, in the case of 10 to 20%, moderate and, if exceeded from 20% or more, severe mycogen sensitization.

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