RU2433409C1 - Method of hystochemical detection of methacide in liver in case of poisoning with polymer-based surrogate alcohol - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: method includes preparation of paraffin cut, deparafination, staining with 2% solution of congo red in 10% alcohol, washing in water, analysis of micropreparations of liver by light microscope. Preliminarily review microscopy is performed, micropreparations with suspicion on toxic hepatitis with intralobular cholestasis are sorted out, staining with congo red is carried out during 10-15 minutes, after washing in water micropreparation of liver is placed into glycerol, after that it is analysed under microscope and in case if orange lining is visualised around bile inclusions and cylinders and irregular orange staining of cytoplasm of hepatocytes, presence in liver tissue of methacide (polyhexamethylene guanidine hydrochloride) is determined. ^ EFFECT: simplification, increase of toxicity and reliability of determining type of poisoning. ^ 3 ex, 2 dwg

Description

The invention relates to medicine, in particular to histochemistry (histology), and can be used in histological examination of liver micropreparations, namely for the qualitative determination of polymers with basic properties in liver tissues.

Recently, the use of polymer compounds in all spheres of human life has sharply increased, including they have found their application in medicine. Polymers of natural and synthetic origin (hormones, enzymes, blood protein preparations, polyvinylpyrrolidone, etc.) are used in medicine and biotechnology as medicines, plasma substitutes, and detoxifiers. The interest in the possibility of using polymers to combat microorganisms, the creation of antibacterial, antiviral and antifungal polymer preparations, preservatives for anatomical embalming (Gembitsky P.A., Vasiliev O.D., 2008) increased. A significant increase in the latest means of the chemical and pharmaceutical industries is combined with their undetermined effect on the human body.

The growth of the spectrum of toxic substances, the introduction of innovative technology products is ahead of technical capabilities and creates certain problems for the forensic chemical examination. A modern qualitative change in the composition of poisons is accompanied not only by the presence, but also by the appearance of features of structural tissue damage (Pigolkin Yu.I., Bogomolova I.N., 2006). Therefore, a morphological study becomes essential in the diagnosis of poisoning.

The results of a histomorphological study help to determine the damaging factor, reflect the histochemical features of pathological processes in the internal organs, establish leading syndromes and conduct a comprehensive clinical and anatomical analysis of the available data. The pathomorphological picture reflects the main mechanisms of dying, participates in the justification of the causal relationship between poisoning and death, in the exclusion of other possible causes of death of victims, which allows you to combine them into one group by the type of damaging effects on humans. In 2006, with mass poisoning by alcohol-containing liquids, it was found that they necessarily included the Metacid polymer additive (polyhexamethylene guanidine hydrochloride - PHMG-GC). It was not possible to establish the cause of toxic hepatitis with cholestasis, expressed by jaundice and damage to all internal organs, by the method of forensic chemical examination of the organs of the victims, experimental methods of investigation.

Within the framework of the anti-alcohol concept of the Russian Federation, the development of scientifically based criteria for forensic diagnostics and expert assessment of polymer-based surrogate alcohol poisoning to destroy the markets for these products and ensure administrative and legal norms aimed at protecting the population from chemical trauma and promoting a healthy lifestyle has become a necessity .

Known methods for the determination of substances based on polymers having the property of accumulating in the tissues of human or animal organs. These are compounds such as, for example, dextran, polyvinylpyrrolidone (PVP).

In the complex of methods of morphological research, a mandatory technique is the staining of the analyzed tissue.

A known method for detecting dextran in tissues, in which fresh preparations fixed with methanol, was stained with a solution of Niagara sky blue in 90% methanol for 15-20 minutes (Yankso-Gabor, 1955). It is known that to detect dextran sulfate, a series of alcoholic solutions of decreasing strength containing toluidine blue (Mowry) is used. This dye forms a precipitate insoluble in water with dextran sulfate, which makes it possible to switch from alcoholic solutions of toluidine blue to aqueous, in which intense coloring is observed.

A known method for the detection of polyvinylpyrrolidone accumulated in human tissues, with a strong pink chlorozol (E. Piers. Theoretical and applied histochemistry (translated from English). - M.: Publishing House of Foreign Literature. - 1962. - P.888). When implementing the method, it is necessary: - to bring the slices to water; - stain with a 1% solution of strong chlorozol pink in 50% alcohol at 60 ° for 10-30 minutes; rinse in water; additionally stain the nucleus with Mayer hemalown for 3 min; rinse, differentiate in 1% acidified alcohol; rinse until the core turns blue; dehydrate, enlighten and encapsulate in synthetic resin. Result: polyvinylpyrrolidone is colored pink or cherry red, and the nuclei are blue.

A known method of detecting polyvinylpyrrolidone accumulated in human tissues with Congo red (E. Piers. Histochemistry theoretical and applied (translated from English). - M .: Publishing House. Foreign Literature. - 1962. - P.887) - prototype . The method includes the following: - bring the slices to water; - stain with a 2% solution of Congo red in 10% alcohol for 30 minutes; - rinse in water; - additionally stain with a 1% aqueous solution of light green SF for 2 minutes; - rinse quickly; - get wet with filter paper and put into Apati gum-syrup; - examine liver micropreparations under a light-optical microscope. Result: polyvinylpyrrolidone stains orange or cherry red, elastic fabrics brown, kernels brown to green, and the rest of the structures green.

The disadvantages of the method: staining of PVP Congo red in the specified method is unstable, since the paint quickly fades, and staining the slice with light green lubricates the histological picture due to the green staining of bile pigments in toxic hepatitis with cholestasis, which does not allow to detect the presence of other polymers in the tissues, for example such as metacid (PHMG-GC).

The basis of the invention is the task to expand the range of polymers with the basic properties that accumulate in the tissues of the body, to identify metacid in the liver to argue the clinical and morphological features of toxic hepatitis with cholestasis and the possibility of differential diagnosis of polymer-based surrogate alcohol poisoning.

This problem is solved by the fact that the method of histochemical detection of liver metacide in case of polymer-based surrogate alcohol poisoning, including the preparation of a paraffin slice, dewaxing, staining with a 2% solution of Congo red in 10% alcohol, washing in water, studying micropreparations of the liver using a light microscope, is characterized preliminary microscopy is performed, micropreparations with suspected toxic hepatitis with intralobular cholestasis are selected, and Congo staining is performed. it is carried out for 10-15 minutes, after washing in water, the liver micropreparation is placed in glycerol, then it is examined under a microscope and, when visualizing an orange rim around bile inclusions and cylinders and uneven orange staining of hepatocyte cytoplasm, the presence of metacide (polyhexamethylene guanidine hydrochloride) in the liver tissue .

The proposed method, unlike the prototype, makes it possible to identify such a polymer with basic properties as metacid (PHMG-GC) in the liver tissue, which causes the development of slowly resolving cholestasis in toxic hepatitis, distinguish poisoning from surrogate alcohol from alcoholic liver disease, poisoning by other alcohol-containing liquids.

Metacid or polyhexamethylene guanidine hydrochloride (P. Gembitsky, 1984) - a patented disinfectant for external use - a linear or branched polymer, a transparent glassy mass, soluble in water, is not detected at the present stage from biological tissues by gas and liquid chromatography. At room temperature in aqueous 10%, higher concentrations of solutions, its gelation occurs due to the intensive formation of hydrogen bonds. The polymer, according to the author, passes through the wall of the stomach and accumulates in the liver tissue, giving it a characteristic green tint when examined with the naked eye before and / or after fixing the material.

The fact of accumulation of metacid (PHMG-GC) in the liver has not yet been established. The etiology of toxic hepatitis with cholestasis is debated. In 2008, an experimental work was published on rats of the Federal State Institution of Toxicology of the Institute of Toxicology of the FMBA of Russia (St. Petersburg). Given the inconsistency of information on the cumulative properties of metacid (PHMG-GC), the cumulative effect was estimated using the “subchronic toxicity” test in three series of experiments. The obtained average cumulation coefficient of 1.83 (according to the classification of L.I. Medvedya) indicated a pronounced cumulation of this substance (Bonitenko E.Yu., Petrov A.N., Shevchuk M.K. Effect of polyhexamethylene guanidine hydrochloride on the toxicity of ethyl alcohol. / Problems standardization and implementation of modern diagnostic and therapeutic technologies in practical toxicological assistance to victims of acute chemical influences: abstracts of the Russian Scientific Conference - Ekaterinburg, 2008. - P.100-103).

According to other leading experts of the Russian Federation, the role of the toxicant is played by a mixture of alcohols (ethanol, industrial alcohols), mutually reinforcing the hepatotoxic effect. Mono-poisoning of polyhexamethylene guanidine with hydrochloride can only be discussed hypothetically, since reliable signs confirming its direct hepatotoxic effect have not yet been established. (V.V. Benemansky, Yu.V. Solodun, G.G. Yushkov. Comparative morphological characteristics of changes in the liver in humans during poisoning by alcohol-containing liquids and in animals after subacute exposure to ethyl, propyl alcohol, ethylene glycol and their mixture. // Forensic -medical expertise. - 2010. - No. 3. - P.14-16).

Based on the proposed method for the histochemical detection of metacide (PHMG-GC) in the liver during polymer-based surrogate alcohol poisoning, it was established that this compound has the role of a biological matrix for the sedimentation of bile salts, hemoglobinogenic pigments, attaches endogenous iron as a complexing agent. Slow resolving cholestasis causes hepatonecrosis, the development of toxic hepatitis with hyperbilirubinemia and jaundice. This in turn leads to the formation of a characteristic clinical and morphological picture of poisoning accompanying toxic hepatitis. Thus, the version about methacid poisoning (polyhexamethylene guanidine hydrochloride) found in alcohol-containing liquids and having a direct toxic effect on the liver is confirmed.

The proposed method is carried out generally accepted in the conditions of histological laboratories, practical health care institutions by the method (Bureau of forensic medical examination, pathology departments and bureaus).

For a histological examination, an autopsy takes a piece of liver 1 × 1 × 1 cm in size from a deceased patient with suspected poisoning with hepatotropic poison, toxic hepatitis, alcoholic liver disease. A piece of tissue for 24 hours is fixed in 10% formalin, washed in running water and processed according to the following scheme. Liver tissue is cut out (taking into account the size of the molds for pouring into paraffin) and a thickness of not more than 0.5 cm, marking pieces. The material enters the wiring, where it is repeatedly fixed in 10% formalin during the day. Then it is washed with running water for at least 20 hours and dehydrated in up-concentration alcohols (in the histological battery). Illuminated in two xylene shifts. It is impregnated with alcohol and chloroform, chloroform, chloroform and paraffin, paraffin for at least 30-45 minutes. For this, modern automatic laboratory assistants are used. Then follows pouring in molten paraffin, placed in special molds (for 4-6 hours) and cooling. Fabric slices are manufactured on microtomes of the toboggan and rotor types. Spreading of tissue sections occurs in a water bath and glass slides. This is followed by drying the slices on a heating table. Tissue sections dewax - pass through a paraffin solvent (xylene), alcohols of a descending concentration. Washed in three shifts of distilled water. Blot a glass slide around the cut with filter paper.

A 2% solution of Congo red in 10% alcohol is prepared. Apply to the slices 2-3 drops of a coloring solution related to acidic dyes. Incubate for 10-15 minutes. The remaining dye is drained. Washed by immersing the slice 8-10 times in a laboratory glass with distilled water. Blot water with filter paper and enclose the micropreparation in glycerin.

A histological examination of liver micropreparations is performed under a light-optical microscope. The redox reaction, aimed at the properties of chemical compounds with the basic properties of binding acidic fabric dyes, reveals the basic properties of a substrate stained with Congo red. Around the inclusions in the composition of the bile cylinders, an orange rim forms, orange staining acquires the cytoplasm of Kupffer cells, hepatocytes, which allows us to conclude that there is a metacide in the analyzed preparation (PHMG-GC).

The proposed method, unlike the known ones, allows one to identify the main compounds (biopolymers with basic properties) in the liver tissue, which cause the development of slowly resolving cholestasis in toxic hepatitis, and distinguish polymer poisoning from surrogate alcohol from other poisonings.

Previously, similar properties to accumulate in tissues were established for polyvinylpyrrolidone preparations, the histological staining of which was developed by Pierce (1962). Unlike the Pierce method, the proposed method does not stain cells with light green. Interpretation of the result of painting according to Pierce varies somewhat. Metacid is colored in orange. Bile deposits - in green. Elastic fibers and other structures, cell nuclei are not stained.

The results of the proposed method are illustrated by drawings, where Fig. 1 shows a picture of Congo red metacid staining; orange staining is observed around bile cylinders and intracellular bile pigments; figure 2 - gives a picture of the accumulation of material of moderate electron density (metabolites PHMG-GC) in increased in size and number of lysosomes (phagolysosomes) of Kupffer cells.

Example 1. The liver tissue of a deceased in hospital A., 46 years old, from toxic hepatitis was examined. The histomorphological picture of liver changes was characterized by hepatonecrosis in the centers of the hepatic lobules. Around the foci of liver cell alteration, segmented white blood cells, lymphocytes, macrophages accumulated, and histiogenic cellular elements proliferated. Intralobular cholestasis was accompanied by the formation of pigment cylinders in the bile capillaries, intracellular accumulation of bile. When stained with an alcoholic solution of Congo red in the liver tissue, deposits of bile and bile cylinders were surrounded by an orange rim.

Example 2. The liver tissue of a deceased at home citizen V., 41, from poisoning by unidentified hepatotropic poison was examined. In the liver tissue - hepatonecrosis, fat-protein detritus with reactive leukostasis. Against the background of the foci of destruction are pigment cylinders with a clear border, irregular and rounded in shape. Around the bile pigment there is an orange rim.

Example 3. The liver tissue of citizen S., who died at home from hepatotropic poisoning, was examined. Hepatonecrosis is combined with intraductal cholestasis, pigment cylinders lie freely in fat-protein detritus. When electron microscopy revealed changes in Kupffer cells - hepatic macrophages, presented in figure 2.

In basic research, such changes are interpreted as a macrophage reaction to foreign material (Mayansky D.N., 1991). An increase in the number and size of macrophages, the formation of macrophage nodules in the liver was detected by the applicant during an immunohistochemical reaction using monoclonal antibodies to SD68.

The proposed method can be used for differential diagnosis of polymer-based surrogate alcohol poisoning. The method is more simple, convenient and cost-effective to perform it in the conditions of histological laboratories, since only one dye is used, the exposure of the color is reduced. In the proposed method, staining with light green (like Pierce) is not desirable, since it “lubricates” the histological picture due to green staining of bile pigments in toxic hepatitis, which reduces the information content of the study. Apati Gum Syrup has not been supplied to chemical companies and laboratories for the past decade. The instability of Congo red staining, the acidic reaction of balms makes it unnecessary to conclude in resins (balms), which are laborious to use and designed for long-term storage of histological micropreparations. In the proposed method, glycerin is used, and microphotography is used to fix the results.

Claims (1)

A method for histochemical detection of methacide in the liver in case of polymer-based surrogate alcohol poisoning, including preparation of a paraffin slice, dewaxing, staining with a 2% solution of Congo red in 10% alcohol, washing in water, examination of liver micropreparations using a light microscope, characterized in that preliminary carry out survey microscopy, select micropreparations with suspected toxic hepatitis with intralobular cholestasis, and Congo red staining is carried out for 10-15 m in, after washing in water, the liver micropreparation is placed in glycerol, then it is examined under a microscope and when visualizing an orange rim around the bile inclusions and cylinders and uneven orange staining of the hepatocyte cytoplasm, the presence of metacide (polyhexamethylene guanidine hydrochloride) in the liver tissue is determined.

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