RU2009491C1 - Method of diagnosing mucoviscidosis - Google Patents

Abstract

FIELD: medicine; pediatrics. SUBSTANCE: researchers studied patient's saliva before and after exposure to mucosalvine. Saliva and mucosalvine were taken in ratio 4 : 1. Resultant preparation was kept in darkness during 24 hours at temperatures of +18 C to +25 C following which liquid-crystalline structure was studied morphometrically. When anisotropic textures diminished to 10 m and no increase in number thereof was observed relative to initial level, researchers diagnosed mucoviscidosis. EFFECT: increased accuracy of diagnosis. 4 dwg

Description

 The invention relates to medicine, in particular to a laboratory study of saliva for the diagnosis of cystic fibrosis in children.

 Widely known methods for the diagnosis of cystic fibrosis, based on the determination of the content of Na and Cl ions in various biological environments of the body (in sweat, nail plates).

 Among the diagnostic tests for cystic fibrosis, the most important is the exercise sweat test, which is based on an increase in the release of Na and Cl followed by pilocarpine electrophoresis.

 However, the sample has several disadvantages. It was found that the amount of sodium and chlorine depends on the salt in the food, the physical activity of the child, the time of the year, and the rate of sweating (fast sweating gives false positive results, and slow sweat gives false negative results). According to known data, it was found that a positive sweat test was observed in only half of patients with cystic fibrosis. In addition, the sample requires at least 100 mg of sweat, and the procedure requires about 2 hours, which is difficult for sick children.

 Closest to the proposed method are methods for studying solid crystalline structures in saliva, used as diagnostic tests in gastroenterology and allergology.

 This is due to the fact that with cystic fibrosis, the salivary glands are involved in the process, in which mucus secretes, mainly mucoproteins. This makes it possible to use saliva as a biological fluid that can characterize a pathological process.

 The feasibility of using saliva as an object of study for cystic fibrosis is explained by its huge physiological role in the body: participation in the digestion process, in protecting the digestive system, in regulating the constancy of the internal environment of the body. The validity of the assessment of the functional state of the digestive system by changing the biochemical composition of saliva is proved by the presence of anatomical and functional connections between the mucous membrane of the oral cavity and various sections of the gastrointestinal tract, as well as by the uniformity of changes in them.

 However, the interpretation of the solid crystalline structures of saliva is only of a qualitative nature, the crystallogram pattern depends on the use of various crystal-forming substances, which is often difficult to interpret, and the features of the dynamics of the structuring of a biological fluid from a state of a micellar solution are not taken into account - a liquid crystal-solid crystal due to the rapid crystallization of an open drop . The revealed changes are not specific, the technique is not accurate, since it does not give quantitative parameters.

 The aim of the invention is to improve the accuracy of diagnosis of cystic fibrosis by polarizing optical crystallogram of saliva.

 This goal is achieved by the fact that the method takes samples of saliva before and after exposure to mucosalvin, conducts a polarization-optical study of their liquid crystal structures in saliva, taking into account the light refraction intensity of birefringent (anisotropic) structures and changes in the size of textures by their morphometry. When lysing textures or reducing their thickness to 10 microns, in combination with the absence of an increase in the optical indicator of their number, cystic fibrosis is diagnosed.

 In FIG. 1 shows a crystallographic picture of a conglomerate of the central part of the textures in cystic fibrosis in native saliva (B. B.); in FIG. 2 - a crystallographic picture of textures in saliva after adding mucosalvin for cystic fibrosis (B. B.); in FIG. 3 - a crystallographic picture of textures in native saliva in pneumonia (b-th M.); in FIG. 4 - crystallographic picture of the textures in saliva after adding mucosalvin for pneumonia (b. M.).

 A significant difference of the proposed method is that for the first time a study was made of the forming crystals of saliva between crossed polarizers on a spectrophotometer and an optical microscope with an eyepiece-micrometer nozzle depending on the nature of the pathological process in children (hereditary or acquired diseases).

 For the first time, a diagnostic test test was developed and carried out with mucosalvin, a specific mucolytic drug used to treat cystic fibrosis. The drug is able to liquefy (lyse) viscous substrate abundantly secreted by various organs.

 As criteria for evaluating the results of a sample, the proposed polarization-optical method of crystallographic study of saliva is used, which is a new sign that improves the accuracy of diagnosis.

 Viscous saliva secretion (mucoproteins) in cystic fibrosis according to the data obtained after 24 hours in an unpressurized microchamber crystallizes in the form of a branched system from bands of confocal domains ("grooves" non-birefringent dendrites).

 The conglomerate of the central part of these liquid crystal textures has a thickness of 20-50 microns and a length of 60-120 microns, the length of the branched part is 80-300 microns. This is a specific sign of the secreted secretion in cystic fibrosis, since in saliva with another pathology (bronchopulmonary, gastroenterological), only scraps of "grooves" can be found, not exceeding a thickness of 10 microns and a length of 20-40 microns.

 A significant difference is also in the number of anisotropic structures. In cystic fibrosis, photometric indicators characterizing the number of structures are in the range of 0.07-0.18 conventional optical units, while in diseases of the bronchopulmonary system (obstructive bronchitis, pneumonia) 0.28-0.42 conv. opt. units.

 A positive result for the diagnosis of cystic fibrosis is the presence of branched "grooves" textures with a thickness in the center of more than 15 microns and a length of more than 50 microns, while the number of anisotropic structures is low (0.07-0.18 conventional optical units).

 After the use of mucosalvin, lysing or a decrease in the thickness of morphostructures to 1-8 microns, the presence of liquid crystal lines are noted. In this case, the content of anisotropic structures remains at the initial low level.

 In the comparison group (patients with obstructive bronchitis, pneumonia, with chronic eating disorder, patients suspicious of cystic fibrosis), a test with mucosalvin leads to an increase in saliva by 1.5-2.0 times the number of anisotropic structures.

 The effect of mucosalvin on saliva in the method can be achieved in two ways: by inhalation of the drug or by direct introduction of saliva into the sample. The result of crystallographic research after these effects is similar.

 The positive effect is that the proposed method allowed to increase the detection of cystic fibrosis up to 90%.

 The method is as follows. For polarization-optical studies of saliva, 3 preparations are prepared that are placed in microchambers formed between a solid-state substrate and a coverslip: 1) 0.20 ml of saliva; 2) 0.02 ml of saliva is combined on a substrate with 0.005 ml of mucosalvin; 3) control (0.02 ml of saliva combined with 0.005 ml of distilled water). As a solid-state substrate, new, glass-free alcohol-free slides are used. Coverslips are processed as well. Saliva is collected on an empty stomach in small plastic tubes. It is centrifuged, the supernatant in the amount of 0.02 ml is applied to a glass slide and covered with a coverslip. Thus, an unpressurized crystallization cell with a sample of saliva is formed.

 The preparations are aged 24 hours at room temperature in the dark to undergo the structuring process. The study of drugs is carried out in crossed polarizers by photometry on an SF-46 spectrophotometer and by the optical method.

 The number of anisotropic structures is determined in arbitrary optical units at a wavelength of 520 nm, the sizes of morphological textures are measured by an eyepiece micrometer.

 PRI me R 1. Patient B. 2 years old (and / b N 14800, 1990). A child from 1.5 months has a persistent cough and repeated pneumonia. The condition of the girl upon admission to the department is difficult. Pneumonia was confirmed by laboratory x-ray examination. Based on the medical history, clinical picture of the disease, laboratory and X-ray examination data, a chronic bronchopulmonary process (cystic fibrosis) was suspected. A crystallographic study of saliva was carried out. Saliva collected on an empty stomach, preparations were prepared: 1) 0.02 ml of saliva was applied drop on a glass slide and covered with a coverslip; 2) 0.02 ml of saliva was applied to a glass slide, 0.05 ml of mucosalvin was added thereto, covered with a coverslip, and the preparations were kept for 24 hours in the dark at room temperature. In 2 preparations, a morphometric study was then performed on a polarization-optical microscope using an eyepiece nozzle and polarization photometry on an SF-46 spectrophotometer with mounted polarizers.

 In the preparation of native saliva (see Fig. 1), the presence of conglomerates-textures with dimensions of: thickness 95 μm, length 100 μm, the absence of liquid crystal lines (lcd lines), an indicator of light transmission, reflecting the number of anisotropic textures, was 0.12 srvc. opt. units In the preparation, after combining saliva and mucosalvin, the presence of liquid crystal lines, single conglomerates of grooves of the following sizes was noted: thickness 2 microns, length 85 microns, the number of anisotropic textures decreased to 0.09 conv. opt. units Based on a crystallographic study, the diagnosis of cystic fibrosis was clarified. The same child underwent a crystallographic study after inhalation with mucosalvin (according to the generally accepted method). After inhalation, a decrease in conglomerate textures to a thickness of 8 microns, the appearance of lcd lines, the number of anisotropic textures decreased from 0.16 to 0.12 conv. opt. units From this it follows that the nature of the change in the indicators is identical both in the direct connection of saliva with mucosalvin, and after its inhalation by the physiotherapeutic method.

 PRI me R 2. Patient M., 2.5 years old (and / b N 2552). In a child from 6 months of age, repeated pneumonia, bronchitis. Persistent cough with discharge of serous-purulent sputum. He was repeatedly in the hospital for examination and treatment with a diagnosis of repeated pneumonia. He entered the clinic in serious condition. During the examination, cystic fibrosis was suspected. On the chest x-ray: signs of exacerbation of the bronchopulmonary process.

 A crystallographic study of saliva was carried out with the following results: in the native saliva preparation (see Fig. 3), the presence of single "grooves" of 10 x 20 microliters of lcd lines, spherocytes, an indicator of light transmittance of 0.24 conv. opt. units

 In the preparation, after combining the saliva of 0.02 and 0.05 ml of mucosalon (see Fig. 4), the presence of lcd lines of single "grooves", spherocytes, the quantitative content of anisotropic textures increased to 0.36 conv. opt. units

 On the basis of crystallography data in a sample with mucosalvin, the diagnosis of cystic fibrosis was removed, which was then confirmed by a comprehensive clinical, laboratory and instrumental study.

 Thus, the proposed method allowed to increase the accuracy of diagnosis of cystic fibrosis up to 91%, while the availability of the methodology in children's medical institutions. (56) B. Bratanova. Clinical Pediatrics. Sofia. 1983. Medicine and physical education. T. 8, p. 368-373.

Claims (1)

METHOD FOR DIAGNOSTIC OF MUKOVISCIDOSIS by examining a biological fluid, characterized in that, in order to improve the accuracy of the method, the patient’s saliva is examined as a biological fluid before and after exposure to mucosalvin at a ratio of saliva: mucosalvin 4: 1 by volume, the resulting preparation is kept in the dark in for 24 hours at 18 - 25 ^o C, after which a morphometric polarization-optical study of liquid crystal structures is carried out and when the anisotropic textures are reduced to 10 microns in combination with the absence of bunker When their number relative to the initial level is diagnosed, cystic fibrosis.

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