Diagnostic technique for chronic viral hepatitis type c associated with opium addiction
SUBSTANCE: diagnostic technique for chronic viral hepatitis type C associated with opium addiction consisting in needle biopsy of a liver that is followed by optical microscopy to analyse the prepared biopsy materials for a basal, stimulated level of interleukine-10 (IL-10), and if observing the basal IL-10 within 218.5 pg/ml to 288.5 pg/ml, and the stimulated IL-10 within 468 pg/ml to 588 pg/ml, the presence of fibrosis of the central vein, then chronic viral hepatitis type C associated with opium addiction is diagnosed.
EFFECT: improved diagnostic accuracy.
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The invention relates to medicine, in particular to a gastroenterologist, and can be used for the diagnosis of chronic viral hepatitis C, due to opium addiction.
Despite the large number of factors influencing the course of viral hepatitis, today, undoubtedly, critical assessment of the role of a toxic agent - opiate-in cooperation with the virus in the liver, given that now formed the concept of drug addicts epidemiological process in chronic viral hepatitis C[1, 2, 3, 4]. Causing hepatitis viruses are detected, as a rule, drug addicts and often complex cause of chronic liver disease [5, 6]. Mainly in the younger age group (15-29 years), usually with markers concomitant viral infection HBV+HCV[7, 1, 8].
In a number of works identified the possibility of a more severe course of viral hepatitis among drug users due to direct toxic effects on hepatocyte input substances and immunosuppressive effect of the latter. Drugs enhance biochemical, microcirculation, immunological and morphological violations in viral hepatitis[1, 6, 2, 8].
Currently, there is extensive material, indicating grubigstein immunological reactivity in patients suffering from opium addiction. With the use of opioids is associated with more severe liver disease by reducing blood CD3+, CD4+, CD8+, a decrease in the ratio of CD4+/CD8+  note, immunological disorders with predominant disturbance of T-cell immunity, which is accompanied by a significant decrease of CD3+ and CD4+ lymphocytes .
In the analysis of the literature found only a few work with performance evaluation of cytokines in chronic viral hepatitis C in the background of opium addiction, in these studies were not conducted to study the morphological pattern of liver injury [11, 12, 13]. So, Dchkmrmy (2002) a characteristic feature of the immune status of patients, drug abusers with chronic viral hepatitis type b and b+C is an increase in the indicators of humoral immunity and increased concentrations of proinflammatory mediators and indicators of T-cell component differed little from the results obtained in patients who have not used drugs . In the work Vourable (2002), on the contrary, patients with opium addiction serum level of tumor necrosis factor-α were not significantly different from the healthy group, serum levels of interleukin-2 and interleukin-4 was increased in two and four and a half times, respectively, and in the induction of cytokine synthesis lippolis what heredom (LPS) that is coli LPS-induced synthesis of tumor necrosis factor-α oppressed by almost 2 times . According to Nvisual (2002) individual indicators of activity of IFN-γ were equally low in patients with isolated chronic viral hepatitis C, and on the background of opium addiction . However, not every work compared the performance of cytokine metabolism in patients with chronic viral hepatitis C with similar indicators for viral process on the background of opium addiction, the levels of anti-inflammatory interleukin-10 has not been studied. The authors of the works presented did not think the diagnosis of chronic viral hepatitis C, due to opium addiction.
The study, which presents the results of morphological patterns of chronic hepatitis C in patients receiving opiates are contradictory[6, 14, 15, 16]. Thus, according to Gstore (2001) drug use chronic viral hepatitis occur clinically heavier, while morphological changes characterized by increased signs of activity of the process and the degree of fibrosis . Higher activity of the pathological process in the liver, combined with the course of viral infection with drug addiction by pronounced necrosis and protein dystrophy of the liver, marked and close to the om other authors .
In other studies were predominantly observed latent oligosymptomatic the course of chronic viral hepatitis on the background of drug abuse, the absence of significant changes of immune status, identified mainly phase integration viruses and morphologically the predominance inactive and inactive forms of liver injury[17, 18, 2, 14, 15]. A number of studies revealed no effect of drugs on the liver, with the defeat of hepatocytes, according to the authors, due to extremely viral component . There are studies describing the morphological features characteristic of the combined effects of viruses and opiates in the form of sclerosis of the walls of the Central veins [17, 3], however, they are descriptive in nature, they contain no information about the frequency of occurrence of this feature in the control group. So, Aguzarovoy characteristic morphological patterns of biopsy specimens of patients with chronic viral hepatitis C in the background of opium addiction is sclerosis of the walls of the Central veins. However, this work did not compare the morphological picture of liver biopsy specimens obtained from patients, drug addicts, with a picture of isolated chronic viral hepatitis . According to Haematocele and others have also noted the specificity of fibrotic changes in the liver of drug addicts is of patients with HCV-infection with primary sclerosis Central veins, however, these data are not reliable and have not been compared with data obtained in patients with isolated chronic viral hepatitis C . The mention of the presence of significant sclerosis of the walls of the Central veins in patients with chronic viral hepatitis on the background of opium addiction is in the work of other authors , but not compared to the frequency of occurrence of this morphological characteristic in patients with chronic viral hepatitis, not suffering from opium addiction. Moreover, in these works we are talking about the sclerosis of the walls of the Central veins, while we are not talking about sclerosis, and fibrosis of the Central vein. Only in one work there is reliable evidence for the presence of fibrosis of the walls of the Central veins of the drug compared to those not used drugs, but the authors say it is on the walls of the Central veins. In addition, these data were obtained only on the results of autopsy studies , which can also distort the picture and, of course, reduces their value to the Clinician working with a live patient. Thus, in modern literature, there is no reliable information about the frequency of occurrence of fibrosis of the Central veins in patients with isolated chronic viral hepatitis C in comparison with during this process the sa when opium addiction in living patients.
Given the prevalence and severe outcomes of chronic viral hepatitis, young age of patients, it is really important search criteria for the diagnosis of chronic viral hepatitis C, caused by opium addiction, which is dedicated to our work.
New technical problem - a method for the diagnosis of chronic viral hepatitis C, due to opium addiction.
To solve the problem in the method for the diagnosis of chronic viral hepatitis C, caused by opium addiction in patients with chronic viral hepatitis To determine basal, stimulated the level of interleukin-10 (IL-10), spend a puncture biopsy of the liver followed by light microscopy biopsies obtained and when the basal level of IL-10 from 218,5 PG/ml to 288,5 PG/ml, stimulated IL-10 from 468 PG/ml to 588 PG/ml, the presence of fibrosis Central veins diagnosed with chronic viral hepatitis C, due to opium addiction.
The method is as follows. The proposed method is applicable for diagnosis in patients with no contraindications to biopsy of the liver. When referring a patient with diagnosed chronic viral hepatitis To determine the level of basal, stimulated IL-10 and conduct blind percutaneous needle b is opseu liver under local infiltration anesthesia 6 ml of 2% solution of novocaine.
Determining the level of IL-10 in supernatant cultured mononuclear performed using solid-phase immunoenzymatic "sandwich" method. The basis of this identification is the conjugation of one epitope of a molecule cytokine mouse monoclonal antibody, adsorbed on the solid phase of the microtiter plate. Procedure perform the immunoassay is performed according to the instructions offered by manufacturers of test systems ("Procon", Russia; "Cytimmune, USA).
To obtain supernatants selected mononuclear cells resuspended in complete culture medium consisting of 90% RPMI-1640, 10% inactivated (at 56°C for 30 min) fetal calf serum (ICN Biomedicals Inc., USA), 280 mg/l L-glutamine, 100 mg/l gentamycin and 2 mm/l HEPES ("Flow", UK). Standardizing the number of cells in suspension to 2.0×106/ml For stimulation of the secretory ability of lymphocytes in the sample contribute phytohemaglutinin (PHA) ("Difco, Germany) at a concentration of 0.01 mg/ml of culture. Cell suspension in 2 ml incubated at 37°C and 5% CO2within 24 hours After incubation, the tubes were shaken, centrifuged 10 min at 1500 rpm and the supernatant collected and used for the quantitative determination of the concentration of cytokines .
To assess IL-10 production by peripheral blood mononuclear automatic MIC the pipette into the appropriate wells of the microplate dobavlat 100 ál of "0 dose" and standards of IL-10 with known concentrations. In the remaining cells placed 100 μl of the supernatant and 25 µl of polyclonal antibodies and incubated tablet 3 h at room temperature. After the double rinse cycle to each well add 50 μl of goat anti-rabbit alkaline phosphatase and incubated 45 minutes After the regular wash cycle in the microtiter wells are injected with 200 µl of staining solution and incubated for 10 min at room temperature in a dark place. Upon completion of the final phase of incubation in the wells contribute 50 ál of stop solution. Analysis immunofermentnogo analysis is done using a microplate photometer for "Multiscan EX ("ThermoLabSystems, Finland) at a wavelength of 490 nm IL-10. The concentration of cytokines calculated by the calibration curve.
Blind percutaneous needle liver biopsy is performed under local infiltration anesthesia 6 ml of 2% solution of novocaine. Excluding the absolute and relative contraindications, after conducting a preliminary clinical examination of patients with the necessary control of the main indicators of hemostasis taking material. To obtain biopsies of the liver use disposable set Hepafix" by Braun (Germany) with an inner diameter of from 1.4 to 1.8 mm and a length of needle 88 mm Volume of biopsy specimens ranged from 20 to 35 mm3. Optical examination of biopsy specimens of the liver spend the trail the way. Taken to study the fragment of the liver tissue immediately placed in fixative liquid, consisting of 96% ethanol and formalin in the ratio of 1:4. The drugs are prepared by a standard method, pour in paraffin . Slices with a thickness of 5-6 microns examined using the following histological techniques: stained with hematoxilin and eosin, painting picrotoxinin by van gieson collagen fibers of the connective tissue of the liver, painting Pronina on the brush on RNA, CHIC-response Mac-Manus to glycogen and glycoproteins, painting lipids with Sudan black, reaction of Perls for verification of iron . The biopsy is considered informative when it detects four or more portal tracts with full triad (portal vein, hepatic artery, bile duct). The histology activity index (YOKE) hepatitis and fibrosis was calculated by V. J. Desmet et al. (1994): minimal activity from 1 to 3 points), and low (from 4 to 8 points, reasonable - from 9 to 12 points, high - from 13 to 18 points. In the presence of portal and periportal fibrosis it is considered weak, one or more of Porto-portal Sept - moderate, one or more Porto-Central Sept - heavy and in the presence of false lobules was diagnosed with cirrhosis of the liver [16, 22].
Study the qualitative and quantitative description of the state of hepatocytes as periportal and intralobular the nuclear biological chemical (NBC zones. Appreciate dystrophic processes, hydropic, fatty degeneration, the content of iron in hepatocytes. Pay attention to the necrosis of hepatocytes (monocellular, manual, bridges, focal intralobular), portal and intralobular cell infiltration, as well as the nature of fibrosis (portal, periportal, Porto-portal, Porto-Central and Porto-lobular), thus take into account perceptually fibrosis and fibrosis of the Central veins.
Quantitative characterization includes the analysis of the state of hepatocytes different parts of the hepatic lobules: the periportal zone, hepatocytes second and third zones. Assessment in periportal and intralobular areas subjected to degenerative, necrotic and regenerative processes. To assess the severity of dystrophic processes calculate the relative number of hepatocytes that contain in their cytoplasm fat vacuoles are small, medium and large caliber. Hepatocytes in nuclei was detected condensation of chromatin with marginal localization, refer to the hepatocytes with sand cores; count the relative number as intralobular and periportal. Determine the relative number of hepatocytes, the core of which was in a state of picknose. Hepatocytes expressed basophilia nuclei include the hyperchromic, and having in the cytoplasm of more than three nuclei - to multi-core. Hepatocytes with kernel differences in magnitude and tinctorial properties, consider cells with polimorfismo nuclei. The number of hepatocytes with the above changes calculated as a percentage of the number of cells in the periportal and intralobular zones.
Quantitative assessment of the subject and cellular infiltration of portal tracts and within the segments on sections stained with eosin and hematoxylin and brush. Calculate the absolute and relative number of lymphocytes, plasma cells, eosinophils, polymorphonuclear leukocytes, fibroblasts in all portal tracts biopsies. In addition, the Secretary of hepatocytes with mild, moderate and severe iron concentration.
For morphometric studies using computer image analysis, including light microscope with digital camera and software processing of the received images.
When the basal level of IL-10 from 218,5 PG/ml to 288,5 PG/ml, stimulated IL-10 from 468 PG/ml to 588 PG/ml and the presence of fibrosis Central veins diagnosed with chronic viral hepatitis C, due to opium addiction.
Proposed criteria selected on the basis of the analysis of the results of clinical observations of patients suffering from chronic viral hepatitis C and XP is clinical viral hepatitis C in the background of opium addiction. A survey of 49 patients with chronic viral hepatitis C, 16 of which came up with the main group and were opium addicts with a maximum duration of abstinence for 2 months. They all had positive PCR for HCV. Age of surveyed ranged from 18 to 48 years (mean age of surveyed to 28.2±5,1 years), men were 40 (81,6%), women - 9 (18.4 per cent). The time of inclusion in the study - verification in the hospital EDO Tomsk chronic viral hepatitis C and the fact that the system of regular intravenous use of opiates according to the anamnesis. Some patients (7 people), used drugs, when the survey was in a state of abstinence (with a maximum duration of 2 months), other (9 persons) studies were conducted in patients receiving opiates that were confirmed positive test for the presence of opiates in urine. In all cases, as the drug was used raw opium. All patients in the group of opium addicts enzyme-linked immunosorbent assay (ELISA) was determined markers of acute viral hepatitis in the form of isolated HB cor AT (IgG) and active HCV infection. The duration of drug use ranged from 2 to 14 years (average duration of opium addiction 6±3,4 years), duration of infection ranged from 2 to 14 years (mean duration of HCV infection of 7.3±3.6 years). From the study were excluding the obtained patients for which alcohol was isolated toxic factor in the presence of chronic viral hepatitis C patients with antiviral treatment in history, with severe comorbidity that could influence the severity of liver damage, prevent the abuse of drugs.
All patients were determined basal and stimulated levels of IL-10 and conducted blind percutaneous needle liver biopsy under local infiltration anesthesia 6 ml of 2% solution of novocaine. Healthy adult volunteers (15 men) were evaluated by basal and stimulated levels of IL-10.
To perform statistical processing of the actual material used statistical package SAS 8.0 (SAS Inc., USA). Check for the normality of distribution of the actual data was performed using criterion Shapiro-Fork. The results are presented as median, lower and upper quartiles (Me, Q1-Q3) and mean ± standard deviation (M±SD).
Comparisons of independent samples, when the number of groups =2 in the case of normal distribution and equal variances in the groups used the t-student test for independent observations or criterion Aspera-Walcha when unequal variances; when the deviation of the distribution from the normal criterion was used M the nna-Whitney. Statistically significant were considered differences at p<0,05. When analyzing the results (see table 1, 2). Table 1 presents the data of morphological examination of liver biopsy specimens in patients with isolated chronic viral hepatitis C and over viral process on the background of opium addiction. Table 2 presents data on the content of immunocytokines in supernatant (PG/ml) in patients with isolated chronic viral hepatitis C and over viral process on the background of opium addiction (Me; Q1:Q3). Received that patients with chronic viral hepatitis C, the level of IL-10 was higher than in healthy volunteers. However, the opium addicts was significantly higher IL-10 as compared with healthy volunteers, and compared with patients with isolated chronic viral hepatitis C. Fibrosis of the Central veins of the systemic drug met significantly more often than when isolated viral liver disease (50% and 18.2%, respectively), suggesting that the localization of fibrosis is a characteristic presence as an etiological factor of liver damage opiate. This allows you to see exactly fibrosis of the Central veins, as a diagnostic sign of toxic environmenta what I opiates.
Thus, with increasing basal levels of anti-inflammatory cytokine IL-10 from 218,5 PG/ml to 288,5 PG/ml, stimulated IL-10 from 468 PG/ml to 588 PG/ml and the presence of fibrosis Central veins diagnosed with chronic viral hepatitis C, due to opium addiction.
Examples of diagnostics
In the gastroenterology Department Bureau of Tomsk has been sick for 20 years with complaints of weakness, pain in the right upper quadrant of the pull of nature. The patient was an opium addict at the age of 17 years diagnosed with chronic viral hepatitis C. the Abuse of alcohol, drugs denied, antiviral treatment is not received. Objective examination revealed a body temperature rise up to 37,3°C, hepato - and splenomegaly. In the biochemical analysis of blood showed increased AST to 63 u/l, Alat to 52 u/l, increased bilirubin level to 28.3 Μmol/L. Diagnosed with kidney damage in the form of mixed forms of glomerulonephritis. In the serum was determined markers of viral hepatitis In and With, with the presence of active HCV infection in combination with serological markers of HBV in the form of "isolated" Hbcor AT (IgG). When examining immunity detected T-cell immunodeficiency (absolute lymphopenia, low content of Mature T-lymphocytes),reducing immunoregulatory index PNET is the nonspecific link protection (low number of natural killer cells), the increase in the number of lymphocytes with increased apoptotic readiness and high activity of humoral immunity (IgM 1.3 g/l), elevated amounts of circulating immune complexes (CIC) to 87 Units. The value of basal IL-10 was on the damage 218,5 PG/ml, stimulated - 468 PG/ml, the Patient is held needle biopsy of the liver. When carrying out light microscopy of biopsy material was revealed fibrosis of the Central veins and, if diagnosed, confirming the presence of as an etiological factor of liver damage opiate.
In the gastroenterology Department Bureau of Tomsk received patient 22 years old complaining of nagging pains in the right hypochondrium. Drug addiction, alcohol abuse, drugs denied, antiviral treatment is not received. According to the data of objective examination revealed hepatomegaly. In the biochemical analysis of blood revealed a twofold increase in Alat. Indicators of immunity testified to the presence of T-cell immunodeficiency, reducing immunoregulatory index, nonspecific inhibition of link protection, the increase in the number of lymphocytes with increased apoptotic readiness, increase the activity of humoral immunity (IgM 1.6 g/l) and complexation (level CEC 120 Ed). Basal levels of IL-10 was 288,5 PG/m is, stimulated - 588 PG/ml, the Patient is held needle biopsy of the liver. Results light microscopy of biopsy material was revealed fibrosis of the Central veins. Indicators of basal values-inflammatory cytokine in combination with the presence of fibrosis Central veins allowed to speak with confidence about the presence of opiates as an etiological factor of liver damage, which was confirmed by information obtained from relatives.
Asked the patient with chronic viral hepatitis With 28 years with complaints of fatigue, pain in the right upper quadrant of the pull of nature. From history revealed that the cause of the infection was blood transfusion carried out 6 years ago. Drug addiction, alcohol abuse, drugs denied, antiviral treatment is not received. Objective examination revealed hepatomegaly. In the biochemical analysis of blood revealed no pathology. The patient had a positive PCR for HCV.
When examining immunity revealed absolute lymphopenia, high activity of humoral immunity (IgG 12.5 g/l, IgM 2.3 g/l), increasing the number of CEC to 85 Units. Basal levels of IL-10 was 174 PG/ml, stimulated - 430 PG/ml According to the results of light microscopy of biopsy fibrosis of the Central veins are not identified. The values of basal and stimulating the consistent level of IL-10 in conjunction with the absence of fibrosis Central veins allowed to talk about isolated for viral process in the liver.
Thus, chronic viral hepatitis C, caused by opiate addiction, diagnosed in 16 patients, as confirmed in 8 patients the presence of fibrosis of the Central veins. The proposed method allows to diagnose presence as an etiological factor of liver damage opiate with a sufficient degree of accuracy.
Sources of information
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15. Tolokonsky I.E. Pato - and morphogenesis of hepatitis C. the Rationale for the strategy of the treatment of persistent infections: author. of thesis ... Dr. med. Sciences / Neptoona. - Novosibirsk, 1999. - 60 S.
16. Tomica G.S. Viral hepatitis coinfection etiology of drug abuse: author. of thesis ... Dr. med. Sciences / Gstore. - Moscow, 2001. - 39 S.
17. Histopathology and ultrastructure of the liver under the action of narcotic substances in combination with hepatitis C and / Higaonna, Neptoona, Aguzarova etc. // Bulletin of experimental biology. - 1999. - C, No. 9. - S-355.
18. Ilchenko LU liver damage in addicts and drug: author. of thesis ... Dr. med. Sciences / Lyulchenko. - Moscow, 1999. - 51 S.
19. Morphological markers of toxic affection of pecheni drug dependent patients with chronic HCV-infection / Haematocele etc. // Bulletin of the Siberian branch of the Russian Academy of medical Sciences: a quarterly theoretical journal. - 2003. No. 1. - P.28-31.
20. Drug-induced liver damage: universal structural markers / Ivashkin V.T., Nepomnyashchikh GI, Audiolava SV, Nepomnyashchikh DL, Dyubanova GA, Domnikov N.P., Maguskina H. // Russian journal of gastroenterology and proctology. - 2009. No. 2. - S-30.
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|Indicators||Hug-||The addict.||P pair|
|Fibrosis of the Central veins||abs.||6||8||P1-2=0,034|
|Indicators||Healthy donors||Hug-||Drug abuse||P porn.|
|IL-10 basal level||10||64,95||175,66||16||248,5||P1-2<0,001, P1-3<0,001, P2-3=0,034|
|IL-10||133,13||431,0||508,0||P1-2<0,001, P1-3<0,001, R2-3=0,001|
Method for the diagnosis of chronic viral hepatitis C, caused by opiate addiction, characterized in that the wire is changed needle biopsy of the liver, followed by light microscopy in biopsies obtained determine basal, stimulated levels of interleukin-10 (IL-10), and the basal level of IL-10 from 218,5 to 288,5 PG/ml, stimulated IL-10 from 468 to 588 PG/ml, the presence of fibrosis Central veins diagnosed with chronic viral hepatitis C, due to opium addiction.
SUBSTANCE: invention refers to medicine, namely to diagnosing malignant processes in a human body. A diagnostic technique for a malignant process in a human body consisting in sampling a patient's living tissue, grinding, mixing with physiologic saline to a state of suspension, keeping and agitating under certain conditions, then centrifuging, separating supernatant and detecting cancer-specific markers by an immunochemiluminisent assay.
EFFECT: method exhibits a simple and high degree of malignant process detection in the human body, both at the early, and following stages of disease.
SUBSTANCE: method of hepatic fibrosis assessment in patients with chronic viral hepatitis type C consisting in evaluating CB56+ phenotype in blood lymphocytes with the value range of blood CD3+/CD56+, CD3+/CD56+/CD4+, CD3+/CD56+/CD8+, CD56+/CD94+, CD56+/NKG2D+, CD56+/CD107a+ cell percentage showing third stage (pre-cirrhotic) or cirrhosis.
EFFECT: method allows higher diagnostic accuracy in fibrous hepatic changes.
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SUBSTANCE: determining individual genome sensitivity to radon exposure is ensured by genetic blood test to identify predisposing and protective genotypes: marker Arg280His of gene XRCC1 - predisposing genotype Arg/Arg, protective genotype Arg/His; marker Argl94Trp of gene XRCC1 - predisposing genotype Arg/Arg, protective genotype Arg/Trp; marker Asnl48Glu of gene APE1 - predisposing genotype Glu/Glu, protective genotypes Asn/Asn, Asn/Glu; marker A2455G of gene CYP1A1 - predisposing genotype A/G, protective genotypes A/A and G/G; a deletion marker in gene GSTM1 - predisposing genotype o/o, protective +. High individual sensitivity to high radon dose is stated by observing the quantitative prevalence of predisposing genotypes or the equal quantity of predisposing and protective genotypes. High individual resistance to high radon dose is determined by the quantitative prevalence of protective genotypes.
EFFECT: use of the method allows estimating genetically determined predisposition to formation of high level of chromosomal aberrations even before exposing to a radiating factor.
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SUBSTANCE: patient examination involves visual evaluation of a periodontal pocket depth, a tooth mobility degree, gum tissue state with using periodontal indexes. X-ray examination aims at evaluating an alveolar bone resorption degree. It is followed with biochemical blood analysis for the parathormone and calcitonin concentrations. If simultaneously observing the blood calcitonin concentration less than a lower physiological norm, while the parathormone concentration exceeding an upper physiological norm, the presence of chronic aggressive generalised periodontitis is concluded.
EFFECT: use of the technique allows advanced detection of chronic aggressive generalised periodontitis, early identification of said disease among the other inflammatory diseases of periodontium at initial morbidities.
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SUBSTANCE: biological tissue is crushed, processed twice for 30 minutes with portions of ethyl acetate, weight of each twice exceeding weight of a biological object; prepared extractions are combined, filtered through anhydrous sodium sulphate; a solvent is evaporated from the filtrate; the residue is dissolved in acetonitrile; the prepared solution is watered down in the volume ratio 1:4, extracted twice in portions of chloroform, volume of each being equal to volume of a hydrophilic layer; the chloroform extracts are combined, steamed to a dry residue; the residue is dissolved in mixed solvents hexane-dioxane-propanol-2, cleaned in a silica gel column L 40/100µ with using a mobile phase hexane-dioxane-propanol-2; eluate fractions containing an analysed substance are combined; the eluent is evaporated; the residue is dissolved in mixed solvents hexane-dioxane-propanol-2 and analysed by a HELC method in a column of dimensions 64×2 mm filled with the sorbent Silasorb 600 with using a mobile phase hexane-dioxane-propanol-2 and a UV detector.
EFFECT: invention allows higher selectivity, sensitivity and accuracy of biological material analysis for tetramethylthiuramdisulfide.
4 ex, 5 tbl
SUBSTANCE: conjunctival cell sample is collected by pressing a bulbar conjunctiva of an examined eyeball at 2-3 mm above a limb in the meridian 12 hours with a soft contact lens (SCL) placed with its concave side on a tonometre. Then, the SCL is removed from the tonometre and turned out so that the collected sample is at the bottom of the SCL which is fixed in 95% alcohol by single dip. Then, the SCL is washed with distilled water, then coloured with hematoxylin for 10-15 seconds, wash with distilled water and air-dried. Thereafter, the SCL is placed on a slide surface for cytological analysis.
EFFECT: invention allows simplifying the method for conjunctival cell preparation for cytological analysis, reduced analysis time and cost.
SUBSTANCE: heparinised blood, isotonic solution containing 0.1 % nitroblue tetrazolium are introduced in plate wells. The mixture is incubated for 30 minutes at 37°C, added with 3% acetic acids, heparinised blood preliminary dissolved in isotonic solution of BCG vaccine containing 0.1 % nitroblue tetrazolium. Further, the prepared mixture is incubated for 30 minutes at 37°C, added with 3 % acetic acids. It is followed with counting neutrophils with observed cytoplasm violet-blue formazan granule depositions in a Gorjaev's chamber with taking into account 100-200 neutrophils, and the percentage of positive NBT-cells is assessed.
EFFECT: use of the method enables higher accuracy and reliability of determining neutrophil capacity and reduced acquisition time.
3 tbl, 3 ex
FIELD: veterinary science.
SUBSTANCE: artificial gastric acid is composed according to prescription: distilled water of temperature 41-42°C - 1000.0 ml, concentrated hydrochloric acid with specific weight of 1.2 - 12.0-15.0 ml, porcine food pepsin 20-30 g. Incubation is carried out in "Gastros" device at the temperature of 41-42°C. Broth is settled, afterwards the residue is centrifuged. Then 8.0 ml of upper fluid level is aspirated from test tube, a drop is taken from lower level, and a smear is made on slide plate. Remaining part is applied with a dropper onto slide plates and dried, then made preparations are stained according to Romanovskiy-Giemsa for 10 minutes and are investigated under microscope with increase of ×400 and ×1250 with immersion.
EFFECT: method is convenient and simple to use, makes it possible to validly detect sarcocyst trophozoites.
SUBSTANCE: for an assay, 5-7 cm3 of blood is taken, and before extraction the sample is pre-treated with 2 cm3 of 60% sulphuric acid; the extraction process is executed with 30 cm3 of n-hexane once, and gas chromatography is preceded with single treatment of n-hexane extract with 10 cm3 of concentrated sulphuric acid.
EFFECT: invention provides higher reliability of α-HCCH, γ-HCCH test results and twofold reduced time of sample preparation.
1 ex, 1 tbl
SUBSTANCE: cystic bile recovered by fractional duodenal intubation is analysed for the concentration of cholic acid, phospholipids, cholesterol, bilirubin with calculating cholate- cholesterol (CCC) and phospholipid- cholesterol (FCC) coefficients. If observing decreasing cholic acid to 14.13±2.54 mmol/l, phospholipids to 1.62±0.27 mmol/l, bilirubin to 1.94±0.24 mmol/l, and increasing cholesterol to 9.04+1.35 mmol/l, decreasing the CCC to 1.59±0.22 and the FCC to 0.18+0.027, more severe clinical form of psoriasis, mainly erythrodermatitis developing is predicted.
EFFECT: invention provides exact prediction of transition of plaque psoriasis accompanied with chronic acalculous cholecystitis to erythrodermatitis.
4 tbl, 3 ex
FIELD: medicine, psychiatry.
SUBSTANCE: one should isolate DNA out of lymphocytes of peripheral venous blood, then due to the method of polymerase chain reaction of DNA synthesis one should amplify the fragments of hSERT locus of serotonin carrier gene and at detecting genotype 12/10 one should predict the risk for the development of hallucino-delirious forms of psychoses of cerebro-atherosclerotic genesis.
EFFECT: more objective prediction of disease development.
FIELD: medicine, urology.
SUBSTANCE: one should conduct subcutaneous prevocational tuberculin test and, additionally, both before the test and 48 h later it is necessary to perform the mapping of prostatic vessels and at decreased values of hemodynamics one should diagnose tuberculosis. The information obtained should be documented due to printing dopplerograms.
EFFECT: more reliable and objective information.
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.
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
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.
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.
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.