Method of quantitative estimation of histological activity in case of chronic diffuse liver diseases

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, in particular hepatology. Method of quantitative estimation of histological activity in liver biopsy materials in case of chronic diffuse liver diseases results in protocol of counting in absolute numbers, percent and points of pathological signs, identified in the course of morphological analysis of liver biopsy materials from patients with chronic diffuse liver diseases, by sections: necrosis of hepatocytes, globular and atomised fatty hydropic degeneration of hepatocytes, small cell dysplasia of hepatocytes, portal, periportal, intralobular and central infiltration. After that conversion of absolute values into point system is performed by means of said protocol, and histological activity is determined by number of points.

EFFECT: method makes it possible to create quantitative unification of obtained results in case of said diseases.

 

The invention relates to medicine, in particular Hepatology, and can be used for the diagnosis of chronic diffuse liver diseases.

Liver biopsy plays an important role in determining inflammatory activity and degree of fibrosis in various forms of chronic hepatitis [5]. In 1981 there was a breakthrough in this area, as was proposed so-called index of histological activity (IGA) [4]. YOKE is a way of ranking assessment of the severity of inflammation, necrosis and fibrosis developed by R. G. Knodell estimated et al. This method allows to assess the dynamics necrotic-inflammatory process in liver tissue during the natural course of chronic hepatitis and on the etiological background (antiviral) hepatoprotective and pathogenetic therapy. System R. G. Knodell estimated underwent a number of changes, most widely known are systems J. V. Desmet, K. G. Ishak, P. J. Sheuer, METAVIR [2, 3, 6]. However, the most commonly used, including in the Russian Federation remains the system R. G. Knodell estimated. The principle of this semi-quantitative ranking system is scoring of histological changes in biopsy specimens of the liver: periportal and bridge necrosis, which are valued from 1 to 10 points, parenchymal damage in the form of focal intralobular necrosis and di�of lake trophy hepatocytes from 1 to 4 points, portal inflammation from 1 to 4 points, the assessment of the level of fibrosis from 1 to 4 points. Points 1-3 corresponds to the minimal activity of hepatitis, 4-8 - mild, 9-12 is moderate, 13-22 - severe hepatitis activity. The disadvantage of this system is a substantial element of subjectivity in the assessment of morphological changes, blurring of boundaries, the paucity of morphological parameters, as well as the fact that the 4th component of the system - fibrosis - reflects not so much the activity, and stage of the disease. The inclusion of fibrosis in the counting system may lead to an overestimation of the level of activity of hepatitis. In this connection a number of authors [2, 6] were proposed separately to assess speed and partienlarly necrosis, as prognostically more serious, and the 4th component of the system R. G. Knodell estimated to allocate separately, designating it as a histological index of multiple sclerosis or fibrosis of varying degrees 1-4.

In 1996, Vladimir Serov, O. L. Severiana proposed a method for evaluation of chronic hepatitis, which is based on the classification of J. V. Desmet [1]. The new system introduces the term histological index of the degree of chronicity (GISH), expanding the scoring system of fibrosis. These same authors more detail the degree of activity and offers several different term of GIS histological index of the degree of activity instead of a YOKE. Higashiura�scopic, that YOKE was developed for chronic hepatitis b, and since there are significant differences between chronic hepatitis C introduced new histological markers of this infection. A feature of this system is the absence of chronic hepatitis minimal activity and assess the activity of hepatitis B and C in different quantity, which makes their quantitative characteristics.

Thus, the proposed semi-quantitative ranking system for the evaluation of morphological changes in chronic diffuse liver diseases need improvement. The technique should be simple to use, affordable, succinct content, and one for hepatitis of various etiologies for the possibility of comparative analysis.

The technical result achieved in the proposed method, quantitative assessment of histological activity in chronic diffuse liver diseases, is that can improve the quality of assessing the degree of activity in chronic diffuse liver diseases, to predict the course of hepatitis and to monitor the effectiveness of therapy.

The technical result is achieved in that reveal parenchymal damage and quantitatively evaluate its components - hydropic, globular and atomized fatty distro�Oia, further define the signs of small cell dysplasia of hepatocytes (liver cells with increased nuclear-cytoplasmic ratio, a reduced amount of cytoplasm, creating the effect of compression cores), intralobular inflammatory infiltration, Central infiltration, then the absolute value of the obtained morphological data converted into scores using the proposed Protocol for the quantitative assessment, which include determining at magnification × 150 in 10 fields of view of necrosis of hepatocytes: 1 point - less than 5 cells per field of view, 2 points - 5-10 cells per field of view, 3 points - more than 10 cells per field of view; globular fatty dystrophy of hepatocytes: 1 point - less than 20 cells per field of view, 2 points - 20-50 cells per field of view, 3 points - 51-100 cells in field of view, 4 points - more than 100 cells per field of view; atomized fatty hepatocytes: 1 point - less than 5 cells per field of view, 2 points is between 5 and 25 cells per field of view, 3 points - 26-50 cells in field of view, 4 points - more than 50 cells per field of view; hydropic dystrophy: 1 point - less than 10 cells per field of view, 2 points - 10-20 cells per field of view, 3 points - 21 to 30 cells per field of view, 4 points - more than 30 cells per field of view; portal infiltration: 1 point - minor inflammatory infiltration single portal tracts, 2 points - moderate inflammatory infiltration Bo�of esista portal tracts, 3 points - severe inflammatory infiltration of most portal tracts, 4 points - enhanced inflammatory infiltration of all portal tracts with the presence of lymphoid aggregates and follicles; stepped necrosis and periportal infiltration: 1 point - stage necrosis is less than 10% of the circumference of the portal tracts, 2 points - speed necrosis take 10-20% of the circumference of the portal tracts, 3 points - speed necrosis occupy 20-30% of the circumference of the portal tracts, score 4 - speed automatic necrosis occupy 30-40% of the circumference of the portal tracts, 5 points - stepped necrosis occupy 40-50% of the circumference of the portal tracts, 6 - speed automatic necrosis occupy 50-60% of the circumference of the portal tracts with penetration into slices, 7 - speed automatic necrosis occupy 60-70% of the circumference of the portal tracts with penetration into slices with a single bridge portcontroller and/or portaportal necrosis, 8 points - speed necrosis occupy 70-80% of the circumference of the portal tracts with the formation of a single portcontroller and/or portaportal necrosis, 9 points - speed necrosis occupy 80-90% of the circumference of the portal tracts with the formation of a single portcontroller and portaportal necrosis, 10 points - speed necrosis is 90-100% from circle p�ntalnyh paths with the formation of many portcontroller and portaportal necrosis; intralobular inflammatory infiltration with necrosis of hepatocytes: 1 point inflammatory infiltration is less than 5% of the area of the liver parenchyma in the field of view, 2 points - inflammatory infiltration is less than 5-10% of the parenchyma in the field of view, 3 points-inflammatory infiltration is less than 10-20% of the parenchyma in the field of view, 4 points - inflammatory infiltration occupies more than 20% of the parenchyma in the field of view; Central inflammatory infiltration with necrosis of hepatocytes: 1 point inflammatory infiltration is less than 10% of the circumference of the Central vein, 2 points - inflammatory infiltration takes 10-20% from the circumference of the Central vein, 3 points - inflammatory infiltration takes 20-30% of the circumference of the Central vein, 4 points - inflammatory infiltration takes about 30-40% of the circumference of the Central vein, 5 points - inflammatory infiltration takes 40-50% of the circumference of the Central vein and slightly penetrates into slices, 6 points - inflammatory infiltration takes 50-60% of the circumference of the Central vein and slightly penetrates into slices, 7 points - inflammatory infiltration occupies 60-70% of the circumference of the Central vein and into the inside of the cloves to 1/4 of the distance from the Central vein to the portal tract, 8 points - inflammatory infiltration occupies 70-80% of the okra�ness of the Central vein and penetrates into slices about 1/3 of the distance from the Central vein to the portal tract, 9 points - inflammatory infiltration takes 80-90% of the circumference of the Central vein and into the inside of the slices more than 1/3 of the distance from the Central vein to the portal tract, 10 points - inflammatory infiltration occupies 90-100% of the circumference of the Central vein and into the inside of the slices more than 1/3 of the distance from Central vein to portal tract; and small cell dysplasia of hepatocytes in 10 fields of view at magnification × 600: 1 point - less than 3 cells per field of view, 2 points - 3-5 cells per field of view, 3 points from 6 to 10 cells per field of view, 4 points - more than 10 cells per field of view, and then on the basis of the scoring results of changes in liver biopsy specimens with values from 1 to 11 points define chronic hepatitis with minimal activity, with values from 12 to 20 points - mild chronic hepatitis activity, from 21 to 30 with chronic hepatitis of moderate activity, from 31 to 47 - chronic hepatitis high activity.

Method of quantitative evaluation of histological activity in chronic diffuse liver diseases includes the following operations: performing a liver biopsy (blind percutaneous puncture laparoscopic or sighting), the fixation of liver tissue in 10% formalin, fill in paraffin to prepare sections with a thickness of 3-4 microns, coloring preparations stained with hematoxylin and eosin, the study of Ki - �teologicheskih preparations with a microscope with counting in absolute numbers and percentage scores of the pathological signs of necrosis and degeneration of hepatocytes, portal and periportal inflammatory infiltration, stepped necrosis of hepatocytes, characterized in that reveal parenchymal damage and quantitatively evaluate its components - hydropic, globular and atomized fatty degeneration, further comprising determining the indication of small cell dysplasia of hepatocytes, intralobular inflammatory infiltration, Central infiltration. The next operation is the creation of the Protocol.

Protocol

At magnification × 150 in 10 fields of view are determined by the following morphological parameters: the average number of necrotic hepatocytes, the average number of cells in the globular state of fatty degeneration, atomized fatty and hydropic degeneration, portal infiltration, stepped necrosis and periportal infiltration, intralobular inflammatory infiltration with necrosis of hepatocytes, inflammatory infiltration around the Central vein with necrosis of hepatocytes.

10 fields of view at magnification × 600 is determined by the average number of hepatocytes with signs of small cell dysplasia. Cells small cell dysplasia is defined as hepatocytes with increased nuclear-cytoplasmic ratio, reduced Koli�the degree of cytoplasm, creating the effect of compression cores.

Necrosis of hepatocytes:

1 point - less than 5 cells in field of view

2 points - 5-10 cells in field of view

3 points - more than 10 cells per field of view Globular fatty degeneration of hepatocytes:

1 point - less than 20 cells per field of view

2 points - 20-50 cells in field of view

3 points - 51-100 cells in field of view

4 points - more than 100 cells in field of view

Atomized fatty degeneration of hepatocytes:

1 point - less than 5 cells in field of view

2 points - 5-25 cells in field of view

3 points - 26-50 cells in field of view

4 points - more than 50 cells in field of view

Hydropic dystrophy:

1 point - less than 10 cells per field of view

2 points - 10-20 cells in field of view

3 points 21-30 cells in field of view

4 points - more than 30 cells per field of view

Small cell dysplasia of hepatocytes:

1 point - less than 3 cells in field of view

2 points - 3-5 cells in field of view

3 points - 6-10 cells in field of view

4 points - more than 10 cells per field of view

Portal infiltration:

1 point - minor inflammatory infiltration single portal tracts

2 points - moderate inflammatory infiltration of most portal tracts

3 points - severe inflammatory infiltration of most portal tracts

4 points - enhanced inflammatory infiltration of all portal� paths with the presence of lymphoid aggregates and follicles

Stepped necrosis and periportal infiltration:

1 point - stage necrosis is less than 10% of the circumference of the portal tracts

2 points - speed necrosis take 10-20% of the circumference of the portal tracts

3 points - speed necrosis occupy 20-30% of the circumference of the portal tracts

4 points - speed necrosis occupy 30-40% of the circumference of the portal tracts

5 points - stepped necrosis occupy 40-50% of the circumference of the portal tracts

6 - speed automatic necrosis occupy 50-60% of the circumference of the portal tracts with penetration into slices

7 points - stepped necrosis occupy 60-70% of the circumference of the portal tracts with penetration into slices with a single bridge portcontroller and/or portaportal necrosis

8 points - speed necrosis occupy 70-80% of the circumference of the portal tracts with the formation of a single portcontroller and/or portaportal necrosis

9 points - speed necrosis occupy 80-90% of the circumference of the portal tracts with the formation of a single portcontroller and portaportal necrosis

10 points - speed necrosis is 90-100% of the circumference of the portal tracts with the formation of many portcontroller and portaportal necrosis

Intralobular inflammatory infiltration with necrosis of hepatic�tov:

1 point - the inflammatory infiltration is less than 5% of the area of the liver parenchyma in the field of view

2 points - inflammatory infiltration is less than 5-10% of the parenchyma in the field of view

3 points - inflammatory infiltration is less than 10-20% of the parenchyma in the field of view

4 points - inflammatory infiltration occupies more than 20% of the parenchyma in the field of view

Central inflammatory infiltration with necrosis of hepatocytes:

1 point - the inflammatory infiltration is less than 10% of the circumference of the Central vein

2 points - inflammatory infiltration takes 10-20% of the circumference of the Central vein

3 points - inflammatory infiltration takes 20-30% of the circumference of the Central vein

4 points - inflammatory infiltration takes about 30-40% of the circumference of the Central vein

5 points - inflammatory infiltration takes 40-50% of the circumference of the Central vein and slightly penetrates into slices

6 points - inflammatory infiltration takes 50-60% of the circumference of the Central vein and slightly penetrates into slices

7 points - inflammatory infiltration occupies 60-70% of the circumference of the Central vein and into the inside of the cloves to 1/4 of the distance from Central vein to portal tract

8 points - inflammatory infiltration occupies 70-80% of the circumference of the Central ve�s and penetrates into slices about 1/3 of the distance from Central vein to portal tract

9 points - inflammatory infiltration takes 80-90% of the circumference of the Central vein and into the inside of the slices more than 1/3 of the distance from Central vein to portal tract

10 points - inflammatory infiltration occupies 90-100% of the circumference of the Central vein and into the inside of the slices more than 1/3 of the distance from Central vein to portal tract

Estimating the degree of activity depending on the number of points that can be delivered the following morphologic diagnosis:

1-11 points - chronic hepatitis minimal activity

12-20 points - mild chronic hepatitis activity

21-30 points - chronic hepatitis of moderate activity

31-47 points - chronic hepatitis high activity

The proposed method of quantitative evaluation of histological activity in liver biopsy specimens in chronic diffuse liver diseases represents a number of the tasks listed above and the evaluation of drugs using a standardized scoring system. The maximum score for the evaluation of parenchymal damage, inflammatory processes in liver tissue - 47, more than R. G. Knodell estimated and J. V. Desmet, which is associated with a more advanced examination of histological samples. Assessment of parenchymal damage to the liver tissue Knodell estimated by the method is performed on a 4-point system,in which no attention is paid to mind dystrophy of hepatocytes, its prevalence. Also in the assessment of histological activity, we have introduced the definition of the level of small cell dysplasia of hepatocytes. This indicator is considered as an additional marker of inflammatory processes of the liver tissue. Descendants of hepatocytes in the state of small cell dysplasia are progenitor cells, activation of which occurs with an increase in peroxidation of lipids, which is associated with inflammatory processes in the liver parenchyma. It should be noted that in the conventional system, Knodell estimated accounting is not performed intralobular inflammatory infiltration and infiltration around the Central vein (Central infiltration).

The effectiveness of the proposed method is determined by the quality and reliability of diagnostic criteria, which correspond to modern international standards of morphological diagnosis of chronic hepatitis. Analysis and filling the Protocol for assessing histological activity of process may be made on the basis of standard methods of staining with hematoxylin and eosin.

Method of quantitative evaluation of histological activity in chronic diffuse liver diseases was tested in Departmental clinical hospital at the station Petrozavodsk JSC "RZD" on 191 cases of chronic hepatitis differnt� etiology. 53 patients had chronic hepatitis b (CHB), 49 - chronic viral hepatitis C (CHC), 46 patients with chronic viral hepatitis B+C, 43 - steatohepatitis. The diagnosis was established on the basis of traditional clinical, laboratory, serological criteria, as well as using polymerase chain reaction on DNA of hepatitis B virus and RNA of hepatitis C virus in blood and liver tissue.

Method of quantitative evaluation of histological activity allows you to extract the maximum possible information from histological material, using the most basic technique is coloring with hematoxylin and eosin.

In 82% of our patients have noted a discrepancy between the activity of the process in liver tissue and indicators alanine aminotransferase (ALT) - a traditional marker of cytolysis and indicators mesenchimal-inflammatory syndrome (ESR, the level of gamma-globulin). So, chronic hepatitis weak activity, established on histological examination, the ALT level was above normal (40 U/l) in 75% of patients, while moderate and high activity level of ALT was above normal only in 1,5-2 times, which once again emphasized the importance of histological examination.

Thus, using a tested method we were able to obtain more accurate and precise diagnostic information about the degree of activity n�kriticheski-inflammatory process in the liver not only in comparison with clinical data, but compared with histological evaluation performed Knodell estimated by the method [4].

Besides the visualization of morphological changes in the presence of periportal and bridge necrosis at a preclinical stage allows you to verify which began the transformation of chronic hepatitis b cirrhosis of the liver, that is, to predict an unfavorable course of chronic diffuse liver diseases.

Treatment of chronic diffuse liver diseases including chronic viral hepatitis) is expensive preparations of interferon and nucleoside analogues with a sufficiently high frequency of adverse reactions and job performance. Medications, the medication ursodeoxycholic acid, is also essential in treating this group of patients, are of high value. Therefore, of particular importance is the possibility of monitoring the effectiveness of treatment not only through traditional laboratory, molecular genetic and serological criteria, but also morphological method with repeated liver biopsies. The proposed method allows us to detect even minimal dynamics of the histological picture, hepatocellular inflammation in chronic diffuse liver diseases, improving control of the disease.

Thus, the method of kolichestvo�th assessing histological activity in liver biopsy specimens in chronic diffuse liver diseases allows to evaluate the degree of histological activity, and also to monitor the effectiveness of therapy and to predict the risk of an unfavorable course of the disease, the probability of transformation of chronic hepatitis to cirrhosis.

Sources of information

1. Serov V. V., Severiana L. O. Morphological criteria for evaluating the etiology, the degree of activity and stage of the process in chronic viral hepatitis b and C // Archives of pathology. 1996. No. 4. Pp. 61-64.

2. Desmet J. V., Gerber m, Hoofnagle J. H. et al. Classification of chronic hepatitis: Diagnosis, Grading and Staging // Hepatology. 1994. No. 6. P. 1513-1520.

3. Ishak K. G., A. Baptista, L. Bianchi et al. Histalogical Grading and Staging of chronic hepatitis // J. Hepatology. 1995. Vol.22. P. 696-699.

4. R. G. Knodell estimated, K. G. Ishak, W. C. Black et al. Formulation and application of numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis // Hepatology. 1981. Vol.1. P. 431-435.

5. Schaff Z. The value of liver biopsy in chronic hepatitis // Orv. Hetil. 2011. Vol.22. P. 856-858.

6. Scheuer P. J. Classification of chronic viral hepatitis: a need for reassessment // J. Hepatology. 1991. Vol.13. P. 372-374.

Method of quantitative evaluation of histological activity in chronic diffuse liver diseases, including performing a liver biopsy, fixation of liver tissue in 10% formalin solution, fill the tissue in paraffin, preparation of tissue sections with a thickness of 3-4 microns, stained with hematoxylin and eosin, the study of tissue sections using a microscope with counting in absolute numbers and percentage points of the pathological signs of necrosis and degeneration of hepatocytes, portal and Periyar�sentatives of inflammatory infiltration, stepped necrosis of hepatocytes, characterized in that reveal parenchymal damage and quantitatively evaluate its components - hydropic, globular and atomized fatty degeneration, further comprising determining the indication of small cell dysplasia of hepatocytes (liver cells with increased nuclear-cytoplasmic ratio, a reduced amount of cytoplasm, creating the effect of compression cores), intralobular inflammatory infiltration, Central infiltration, then the absolute value of the obtained morphological data converted into scores using the proposed Protocol for the quantitative assessment, which include determining at magnification × 150 in 10 fields of view of necrosis of hepatocytes: 1 point - less than 5 cells per field of view, 2 points - 5-10 cells per field of view, 3 points - more than 10 cells per field of view; globular fatty hepatocytes: 1 point - less than 20 cells per field of view, 2 points - 20-50 cells per field of view, 3 points - 51-100 cells in field of view, 4 points - more than 100 cells per field of view; atomized fatty hepatocytes: 1 point - less than 5 cells per field of view, 2 points is between 5 and 25 cells per field of view, 3 points - 26-50 cells in field of view, 4 points - more than 50 cells per field of view; hydropic dystrophy: 1 point - less than 10 cells per field of view, 2 points 10 to 20 cells per field of view, 3 points 21-30 cells�to the field of view, 4 points - more than 30 cells per field of view; portal infiltration: 1 point - minor inflammatory infiltration single portal tracts, 2 points - moderate inflammatory infiltration of most portal tracts, 3 points - severe inflammatory infiltration of most portal tracts, 4 points - enhanced inflammatory infiltration of all portal tracts with the presence of lymphoid aggregates and follicles; stepped necrosis and periportal infiltration: 1 point - stage necrosis is less than 10% of the circumference of the portal tracts, 2 points - speed necrosis take 10-20% of the circumference of the portal tracts, 3 points - speed necrosis take 20-30% from the circumference of the portal tracts, score 4 - speed automatic necrosis occupy 30-40% of the circumference of the portal tracts, 5 points - stepped necrosis occupy 40-50% of the circumference of the portal tracts, 6 - speed automatic necrosis occupy 50-60% of the circumference of the portal tracts with penetration into slices, 7 - speed automatic necrosis occupy 60-70% of the circumference of the portal tracts with penetration into slices with a single bridge portcontroller and/or portaportal necrosis, 8 points - speed necrosis occupy 70-80% of the circumference of the portal tracts with the formation of a single portcontroller and/or portaportal�x necrosis, 9 points - speed necrosis occupy 80-90% of the circumference of the portal tracts with the formation of a single portcontroller and portaportal necrosis, 10 points - speed necrosis is 90-100% of the circumference of the portal tracts with the formation of many portcontroller and portaportal necrosis; intralobular inflammatory infiltration with necrosis of hepatocytes: 1 point inflammatory infiltration is less than 5% of the area of the liver parenchyma in the field of view, 2 points - inflammatory infiltration is less than 5-10% of the parenchyma in the field of view, 3 points - inflammatory infiltration is less than 10-20% of the parenchyma in the field of view, 4 points - inflammatory infiltration occupies more than 20% of the parenchyma in the field of view; Central inflammatory infiltration with necrosis of hepatocytes: 1 point inflammatory infiltration is less than 10% of the circumference of the Central vein, 2 points - inflammatory infiltration takes 10-20% of the circumference of the Central vein, 3 points - inflammatory infiltration takes 20-30% of the circumference of the Central vein, 4 points - inflammatory infiltration takes about 30-40% of the circumference of the Central vein, 5 points - inflammatory infiltration takes 40-50% of the circumference of the Central vein and slightly penetrates into slices, 6 points - inflammatory infiltration takes 50-60% of� the circumference of the Central vein and slightly penetrates into slices, 7 points - inflammatory infiltration occupies 60-70% of the circumference of the Central vein and into the inside of the cloves to 1/4 of the distance from the Central vein to the portal tract, 8 points - inflammatory infiltration occupies 70-80% of the circumference of the Central vein and penetrates into slices about 1/3 of the distance from the Central vein to the portal tract, 9 points - inflammatory infiltration takes 80-90% of the circumference of the Central vein and into the inside of the slices more than 1/3 of the distance from the Central vein to the portal tract, 10 points - inflammatory infiltration is 90-100% from the circumference of the Central vein and into the inside of the slices more than 1/3 of the distance from Central vein to portal tract; and small cell dysplasia of hepatocytes in 10 fields of view at magnification × 600: 1 point - less than 3 cells per field of view, 2 points - 3-5 cells per field of view, 3 points from 6 to 10 cells per field of view, 4 points - more than 10 cells per field of view, and then on the basis of the scoring results of changes in liver biopsy specimens with values from 1 to 11 points define chronic hepatitis with minimal activity, at values of from 12 to 20 points - mild chronic hepatitis activity, from 21 to 30 with chronic hepatitis of moderate activity, from 31 to 47 - chronic hepatitis high activity.



 

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1 ex, 1 tbl

FIELD: molecular biology.

SUBSTANCE: the suggested innovation deals with the fact that nucleic acids should be isolated directly out of the sample without pipetting stage but with the help of interconnected reservoirs being prepared beforehand. The above-mentioned vessels should be applied either separately or being interconnected according to standard microtitrating format. The sample should be mixed with a lyzing buffer and nucleic acids are bound with matrix in closed system including, at least, two interconnected reservoirs. Forced movement of sample's mixture and buffer back and forth from one reservoir into another one for several times through narrow passage provides their thorough intermixing. The method provides quick and safe isolation of nucleic acids.

EFFECT: higher efficiency.

44 cl, 4 dwg, 1 ex

FIELD: medicine, phthisiology, microbiology.

SUBSTANCE: diagnostic material is poured preliminary with chlorohexidine bigluconium solution, homogenized, kept at room temperature for 10-12 h and centrifuged. Precipitate is poured with Shkolnikova's liquid medium, incubated at 37oC for 3 days, supernatant part of Shkolnokova's medium is removed, fresh Shkolnikova's medium is added, and precipitate is stirred and inoculated on the dense cellular egg media. Sensitivity of the strain is determined in 3 weeks by the presence of growth in the control tube only. Invention provides enhancing precision and reducing time for assay. Invention can be used in assay for medicinal sensitivity of tuberculosis mycobacterium.

EFFECT: improved assay method.

3 ex

FIELD: medicine, biotechnology, pharmacy.

SUBSTANCE: invention relates to agents used for treatment of pathological states associated with disorder of synthesis of neuromediating substances. Method involves the development of pharmaceutical composition and a method for it preparing. Pharmaceutical composition represents subcellular synaptosomal fractions: synaptic membranes, "light" synaptosomes and "heavy" synaptosomes prepared from gray matter of cerebral hemispheres from experimental animals based on the goal-seeking modification of humoral mediators of nerve endings transformed to synaptosomes in development and regression of malignant processes. The composition provides inhibiting the growth of tumor cells, to elevate span-life of patients with ascite Ehrlich's sarcoma, breast adenocarcinoma Ca-755, Wolker's carcinosarcoma-256.

EFFECT: valuable medicinal and anti-tumor properties of composition.

12 cl, 3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: method involves carrying out microscopic examination of blood serum samples taken from femoral vein and cubital vein. Femoral vein sample is taken on injured side. The examination is carried out before and after treatment. The blood serum samples are placed on fat-free glass slide in the amount of 0.01-0.02 ml as drops, dried at 18-30°C for 18-24 h. The set of pathological symptoms becoming larger or not changed after the treatment in comparison to sample taken before treatment, and morphological picture of samples under comparison taken from the cubital vein showing no changes or being changed to worse, the treatment is considered to be effective.

EFFECT: enabled medicamentous treatment evaluation in course of treatment to allow the treatment mode to be changed in due time; avoided surgical intervention (amputation); retained active life-style of aged patients.

4 dwg

FIELD: medicine, clinical toxicology.

SUBSTANCE: at patient's hospitalization one should gather the data of clinical and laboratory values: on the type of chemical substance, patient's age, data of clinical survey and laboratory values: body temperature, the presence or absence of dysphonia, oliguria being below 30 ml/h, hemoglobinuria, erythrocytic hemolysis, exotoxic shock, glucose level in blood, fibrinogen and creatinine concentration in blood serum, general bilirubin, prothrombin index (PTI), Ph-plasma, the state of blood clotting system. The state of every sign should be evaluated in points to be then summed up and at exceeding the sum of points being above "+20" one should predict unfavorable result. At the sum of "-13" prediction should be stated upon as favorable and at "-13" up to "+20" - prediction is considered to be doubtful.

EFFECT: higher accuracy of prediction.

2 ex, 3 tbl

FIELD: medicine, juvenile clinical nephrology.

SUBSTANCE: disease duration in case of obstructive pyelonephritis should be detected by two ways: either by detecting the value of NADPH-diaphorase activity, as the marker of nitroxide synthase activity in different renal department and comparing it to established norm, or by detecting clinico-laboratory values, such as: hemoglobin, leukocytes, eosinophils, urea, beta-lipoproteides, lymphocytes, neutrophils, the level of glomerular filtration, that of canalicular reabsorption, urinary specific weight, daily excretion of oxalates, arterial pressure, and estimating their deviation against average statistical values by taking into account a child's age.

EFFECT: higher efficiency of detection.

7 dwg, 1 ex, 6 tbl

FIELD: medicine, urology.

SUBSTANCE: the present innovation deals with differential diagnostics of prostatic cancer and other prostatic diseases at the stage of primary inspection. The method includes the detection of PCA and calculation of probability coefficient for prostatic cancer (PCC) by the following formula: where e - the foundation of natural logarithm (e=2.718…), PCA - the level of total blood PCA in ng/ml, V - patient's age in years. At PCC value being above 0.2 one should diagnose prostatic cancer and to establish final diagnosis one should perform polyfocal prostatic biopsy. The method enables to increase accuracy of diagnostics at decreased number of unjustified prostatic biopsies.

EFFECT: higher efficiency of diagnostics.

2 ex

FIELD: medicine, biology.

SUBSTANCE: invention relates to nutrient medium used for accumulation of cells for the following cytological and/or immunocytochemical analysis carrying out. Invention relates to medium containing salts NaCl, KCl, anhydrous CaCl2, MgSO4 x 6 H2O, MgCl2 x 6 H2O, Na2HPO4 x 2 H2O, KHPO4, NaHCO3, and also glucose and Henx's solution, 10% albumin solution and polyglucin taken in the ratio 1:1:1. Invention provides enhancing the preservation of cells.

EFFECT: improved an valuable properties of nutrient medium.

3 ex

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