Method of hepatic fibrosis assessment in patients with chronic viral hepatitis type c
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.
7 dwg, 3 tbl, 3 ex
The invention relates to medicine, in particular, Hepatology and infectious diseases, and can be used to assess liver fibrosis on the background of chronic viral hepatitis C for registration pretsirroticheskie stage of fibrosis and its transition to cirrhosis of the liver by the study sample of venous blood.
More than 175 million inhabitants of our planet (3% of the population) are infected with hepatitis C virus (HCV), which allows to ascribe it to the rank of a global pandemic agents. Every year, the incidence of HCV infection increases by 3-4 million, in Russia, the annual number of detectable HCV-infected subjects is approximately 220 thousand people [1, 2]. In 70-90% of cases the disease takes a chronic course, which is accompanied by a progressive liver disease with the development of cirrhosis or hepatocellular carcinoma. Thus, the transition frequency of the disease in cirrhosis of the liver is after 1-2 decades about 20% with the development of complications, incompatible with life .
There are certain difficulties in predicting progressive course of fibrosis and cirrhosis of the liver. Currently, the gold standard for determining the degree of fibrotic changes in the liver is needle biopsy followed by histological stage of fibrosis [4, 5], however this method, the relative is present among highly invasive, not always possible to accurately obtain the fabric material from a plot of the maximum severity of fibrotic changes, and sometimes leads to serious complications that threaten the life of the patient. In response to the latter, needle biopsy is recommended to produce not more than 1 time in 5 years, what makes you continue the search for less invasive ways of defining criteria of cirrhosis, in order to monitor the progression of fibrotic changes in the liver on the background of chronic viral hepatitis C (hugs) on circumstantial evidence .
Due to the fact that the decisive role in the development and progression of chronic hepatitis C plays the immune system, and HCV-induced liver damage largely mediated immune mechanisms than direct cytopathic effects of the virus itself , one of the criteria for progression of fibrosis and eventually cirrhosis of the liver can serve as the nature of the immune response to HCV involving natural killer cells (EC) and the PROJECT - CD56+ cells of the immune system, which is widely represented in the hepatic tissue and which is now recognized the leading role in the elimination of HCV [8, 9].
The closest analogue of the claimed invention is a method of monitoring liver fibrosis in patients with chronic hepatitis C (CHC) [RF Patent 230906, publ. 2007]. The method is based on the definition in the serum content of the cytokines IL-4, IL-10, IL-l, TNF-alpha. When IL-4 above and 5.6 PG/ml, IL-10 above 35 PG/ml, TNF-alpha above 14 PG/ml and IL-R above 8.5 PG/ml established the presence of pronounced liver fibrosis. The invention allows for efficient diagnosis of liver fibrosis in patients with chronic hepatitis C.
The main disadvantage of analog should be the inaccuracy of the result, which does not allow to define the indications for changing medical tactics and transition to intensive therapeutic intervention for the prevention of cirrhosis of the liver.
The present invention is to develop an efficient method for the assessment of liver fibrosis stages precirrhotic and cirrhotic changes and, thus, the prognosis of cirrhosis of the liver, with hugs using phenotype CD56+ lymphocytes.
The problem is solved as follows.
A method for assessment of liver fibrosis in patients with chronic viral hepatitis C, including studies of the phenotype CD56+ lymphocytes in the blood, thus determine the percentage of CD3+/CD56+, CD3+/CD56+/CD4+, CD3+/CD56+/CD8+, CD56+/CD94+, CD56+/NKG2D+, CD56+/CD107a+ cells and modify content in the blood CD3+/CD56+ cells above the 3.7%, CD3+/CD56+/CD4+ cells above 23,8%, CD3+/CD56+/CD8+ cells below 47,6%, CD56+/CD94+ cells below to 46.7%, CD56+/NKG2D+ cells in the above 98,4%, and CD56+/CD107a+ cells below the level 98.2% establish if the third stage (pretsirroticheskie) fibrosis of the liver and changes in blood 5 out of 6 parameters: CD3+/CD56+ cells below the 4.3%, CD3+/CD56+/CD4+ cells below 24%, CD3+/CD56+/CD8+ cells above 27,7%, CD56+/CD94+ cells below 31,6% or higher, 41.6%of CD56+/NKG2D+ cells below to 98.5%, and CD56+/CD107a+ cells above the 58.9% regarded as the transition of the disease to cirrhosis of the liver.
Patients hugs were divided into 4 subgroups according to the stage fibrotic changes in the liver according to the METAVIR score: 1-I subgroup (stage fibrosis 1) - 16; 2nd subgroup (stage fibrosis 2) 6; 3-I subgroup (stage fibrosis 3) - 8 people; 4-I subgroup (stage fibrosis 4) - 11 people. All patients in the latter subgroup was diagnosed with cirrhosis of the liver on the basis of history, clinical manifestations, results of biochemical and physical studies, data, ultrasound examination of abdominal cavity organs and esophagogastroduodenoscopy.
The research material was venous blood. Blood sampling was carried out in patients in the morning on an empty stomach in an amount of 5 ml in a test tube Vacuum Tube EDTA.K3 (whole blood). Blood examination was carried out within 2 hours after collection. The study of the cellular immune system factors was conducted using a flow cytometer BD FACSCanto II (Becton Dickinson, USA) after automated sample preparation whole the blood with the help of automatic sample preparation BD FACS Sample Prep Assistant II (Becton Dickinson, USA) in accordance with the instructions for use of the devices and monoclonal antibodies and included typical phenotypic testing of blood lymphocytes, as well as the study subpopulation composition and functional activity of CD56+ cells in the blood.
When the model fesoterodine lymphocytes was used a standardized set of monoclonal antibodies (MCAT) BD Multitest 6-Color TBNK Reagent (BD Biosciences)containing labeled with PerCP-A anti-CD45, MCAT, FITC labeled anti-CD3, MCAT labeled with PE-A anti-CD4, MCAT marked ARS-S anti-CD8, MCAT labeled with APC anti-CD19, MCAT labeled with PE anti-CD 16/anti-CD56, MCAT. When this was recorded the following parameters: absolute and relative number of CD3+ cells; absolute and relative number of CD3+/CD4+/CD8+ cells; absolute and relative number of CD3+/CD4+ cells; absolute and relative number of CD3+/CD8+ cells; immunoregulatory index (IRI); the absolute and relative number of CD3+/CD16+/CD56+ cells (KNOWN); the absolute and relative number of CD19+cells; absolute and relative number of CD3-/CD16+/CD56+ cells (EC).
For the PROJECT was determined by the proportion of individual subpopulations among the total number of KNOWN kit, MCAT BD Multitest 6-Color TBNK Reagent (BD Biosciences): the relative number of CD3+/CD56+/CD4+ cells; the relative number of CD3+/CD56+/CD8+ cells; the relative number of CD3+/CD56+/CD8-/CD4- (DN) cells. For natural killer cells was carried out to calculate the ratio of two subpopulations of EC blood CD56bright and CD56dim histogram based on the peaks of the "bright" and "dim glow of samples labeled with PE-A anti-CD56, MCAT (IOTest, Beckman Coulter).
To analyze the functional activity of CD56+ cells was recorded levels of expression on the membrane of such markers as: 1) challengingbecause receptors (KIR) of the superfamily of immunoglobulins (CD158a,h); 2) inhibiting receptor superfamily lectins C-type (CD94); 3) activating receptor superfamily, pectin-type (NKG2D); 4) glycoproteins associated with the membrane of lysosomes and reflecting the state of a granular cell apparatus (CD107a); 5) receptors natural cytotoxicity (NKp46); 6) CD16 receptors, mediating the mechanism of antibody-dependent cytotoxicity (ASCCT). Determining the relative number of CD56+/CD158a,h, CD56+/CD94+, CD56+/NKG2D+, CD56+/NKp46+CD56+/CD16+ cells (from all CD56+ cells) was carried out in a separate blood sample; in each case, we used the set of two monoclonal antibodies: labeled RE-A anti-CD56 MKAT (IOTest, Beckman Coulter) and labeled with PE anti-CD158a,h MCAT (IOTest, Beckman Coulter); labeled RE-A anti-CD56 MKAT (IOTest, Beckman Coulter) and labeled with PE anti-CD94 MKAT (Immunotech); labeled RE-A anti-CD56 MKAT (IOTest, Beckman Coulter) and labeled with PE anti-NKG2D MKAT (IOTest, Beckman Coulter); FITC labeled anti-NKp46 MKAT (BD Biosciences) and labeled with PE-A anti-CD56 MKAT (IOTest, Beckman Coulter); FITC labeled anti-CD 16 MKAT (IOTest, Beckman Coulter), and labeled RE-A anti-CD56 MKAT (IOTest, Beckman Coulter). To register CD56+ cells carrying the marker CD107a were produced intracellular definition of this receptor using permeabilizer the existing components of the company Beckman Coulter and the set of two monoclonal antibodies: FITC labeled anti-CD107a MKAT (BD Biosciences) and labeled with PerCP-SW-5 anti-CD56 MKAT (BD Biosciences) (Fig 1).
For statistical data processing methods were applied nonparametric statistics using criteria Mann-Whitney and Kolmogorov-Smirnov (data not given a normal distribution). On the basis of laboratory data was conducted discriminant analysis, which was used for selection of the most informative criteria values. Statistical data processing was performed using the statistical software package SPSS 17.0 (allowable error E=5%).
After testing the cellular composition in each sub-stages of liver fibrosis using statistical comparisons of mean values, ranges of values (minimum-maximum), discriminant analysis was defined set of the most informative indicators and identified the ranges of their values, respectively, each fibrosis in the liver.
It was found that in a set of informative indicators included only 6 parameters characterizing the phenotype of subpopulation structure and functional state of CD56+ cells. The ranges of values of the six informative indicators in accordance with the stage of liver fibrosis in patients hugs and in the control group are presented in table 1.
The first and second stages of fibrotic changes in the liver, with hugs deviation ranges informative the performance indicators from the previous stage or indicators of healthy people were not unique, while the transition from the second to the third stage of liver fibrosis was accompanied by a characteristic shift ranges of values for all 6 of informative parameters of all patients hugs. This fact is very important prognostically, as the third stage is the stage preconditions changes in the liver, when using the expansion of the range of therapeutic measures is still possible to influence the intensity of disease progression and delay the formation of cirrhosis of the liver.
During the transition from the third stage of fibrosis in liver cirrhosis patients hugs set of descriptive indicators again changes the range of values in 72,7-100% of cases, which allows us to state the transition to cirrhosis of the liver.
Control of specificity of the selected criteria for stage 3 liver fibrosis and cirrhosis in patients hugs was carried out by comparison of the results obtained by selected characteristics in the groups matching in healthy people and in patients with chronic viral hepatitis C. Summary data on the diagnostic value of the selected criterion of signs at the third stage fibrotic changes are presented in table 2, in the fourth stage in table 3 and in General - in the drawings (figure 2-7).
It can be stated that at the third stage of fibrotic changes in the liver (pretsirroticheskie) of 6 set of criterial features in our the x studies in all patients hugs recorded all 6 signs. Nor when HUGW, neither in healthy people more than 3 signs never were noted. Thus, in the process of monitoring the patient's blood with six characteristics of the six selected criteria, the patient hugs you can define the third stage of liver fibrosis, which serves him as a prognostic criterion threats of transition of chronic viral hepatitis With cirrhosis of the liver.
With regard to the development of liver cirrhosis on the background hugs, the presence of 5 of the 6 selected criterial features allows you to make a diagnosis of cirrhosis of the liver, but this result is non-specific for hugs, as may be logged when the cirrhosis of other etiology (alcohol, amid HVHV). Indicated criteria expressed acquire diagnostic value only immunological monitoring of patients when changes criterial attributes characteristic of fibrosis stage 3, change the range of values to that inherent in stage 4. Since in our research ranges of values of the performance characteristic of stage 4, were never registered at stage 3, the change of the range of values suggests cirrhosis of the liver on the background of chronic viral hepatitis C.
Thus, the use for monitoring patients set of criterial features that characterize the subpopulation composition is KNOWN and the functional state of CD56+ cells blood on their phenotype and in certain ranges of values, it allows for a clear and specific to hugs to set the stage preconditions changes in the liver and to predict the risk of developing cirrhosis of the liver, referring the patient at risk, and to determine the transition of the disease to the stage of cirrhosis of the liver.
The method is illustrated by the following drawings.
Figure 1. Determining the relative number of CD56+/CD107a+ cells in blood by the method of flow cytofluorimetry.
Figure 2. The range of individual values of the percentage of CD3+/CD56+ cells in blood of patients hugs dynamics, patients HUGW at the stage of cirrhosis and alcoholic cirrhosis.
Figure 3. The range of individual values of the percentage of CD3+/CD56+/CD4+ PROJECT in the blood of patients hugs dynamics, patients HUGW at the stage of cirrhosis and in patients with alcoholic cirrhosis.
Figure 4. The range of individual values of the percentage of CD3+/CD56+/CD8+ PROJECT in the blood of patients hugs dynamics, patients HUGW at the stage of cirrhosis and in patients with alcoholic cirrhosis.
Figure 5. The range of individual values of the percentage of CD56+/CD94+ cells in blood of patients hugs dynamics, patients HUGW at the stage of cirrhosis and in patients with alcoholic cirrhosis.
6. The range of individual values of the percentage of CD56+/NKG2D+ cells in blood of patients hugs dynamics, patients HUGW at the stage of cirrhosis and in patients with alcoholic cirrhosis.
7. The range of individualmente the percentage of CD56+/CD107a+ cells in blood of patients hugs dynamics, patients HUGW at the stage of cirrhosis and in patients with alcoholic cirrhosis.
The method is confirmed by the following clinical examples.
Example 1. Patient C., 51, is observed in outpatient Hepatology center for Infectious clinical hospital №1 with the diagnosis of Chronic viral hepatitis With moderate activity, severe fibrosis. Concomitant diagnosis: chronic prostatitis, hypertension".
From the anamnesis it is known that for the first time hyperferritinemia identified in 1998. Antibodies to hepatitis C virus was detected in 2002. Antiviral therapy was not given. In December 2008 made the first needle biopsy of the liver, histological examination which was revealed liver fibrosis stage 3 according to the METAVIR score.
Passed a comprehensive examination in February 2009. During the physical examination drew attention to the enlargement of the liver, which even royalty to 1 cm below the edge of the costal arch, but otherwise unremarkable. Complete blood count: erythrocytes - 5,77·1012/l; hemoglobin - 186 g/l; hematocrit - 52,4%; platelet - 126·109/l; leukocytes to 7.4·109/l; monocytes - 5% (0,37·109/l); granulocyte - 73,9% (5,47·109/l; lymphocytes to 21.1% (1,56·109/l). Biochemical blood test: whole protein - 71 g/l; albumin - 62,2%; alpha1-globulins - 3,2%; alpha2-globulins and 10.8%; beta-globulin - 9,4%; DIN the a-globulins - 14,4%; aspartate aminotransferase - 99 ME; alanine aminotransferase - 67 ME.
Criteria immunological parameters: CD3+/CD56+, and 9.6%; CD3+/CD56+/CD4+ - 24%; CD3+/CD56+/CD8+ - 27,7%; CD56+/CD94+ 31.6 per cent; CD56+/NKG2D+ 99.1 per cent; CD56+/CD107a+ at 46.6%.
Thus, the patient determined according the values of all 6 criteria parameters as for stage 3, which fully coincides with the data of histological studies and can be attributed to a patient at risk for development of cirrhosis of the liver.
Example 2. Sick Hours, 59 years, is registered in the Hepatology consultative center for Infectious clinical hospital №1 with the diagnosis of Chronic viral hepatitis C, cirrhosis, portal hypertension. Concomitant diagnosis: coxarthrosis, hypertension".
From the anamnesis it is known that for the first time, antibodies to HCV and HCV RNA detected in 1995 during routine examination. In 1996 he received interferon therapy for years with no effect. In 1995, was first produced by needle liver biopsy, the histological pattern of biopsy consistent with chronic hepatitis of moderate activity, with the formation of liver cirrhosis. In 2000, he was made repeated liver biopsy, which was identified hepatitis of moderate activity, severe fibrosis (formation manoloblahnik cirrhosis).
The results of the last inst is amentally research: esophagogastroduodenoscopy (27.05.2009) - the expansion of varicose veins of the esophagus stage 1; ultrasound examination of abdominal cavity organs (09.06.2009) - liver: the path is clear, uneven, sometimes wavy; size increased liver: left lobe - 126 mm, a thickness of 52 mm, caudate lobe - 25 mm, the right share - 170 mm, the thickness is 89 mm; the echo is slightly increased; vascular pattern moderately depleted; the structure of the parenchyma is heterogeneous, determined cirrhotic changes; intrahepatic ducts are not expanded; portal vein 13,5 mm; the gall bladder is increased, the detected polyp walls 4×5 mm; choledoch 5 mm; pancreas - diffuse changes; the spleen is not enlarged, the splenic vein is not expanded - 8.2 mm; free fluid in the abdominal cavity no. Biochemical analysis of blood (30.04.2009): total protein - 88 g/l; albumin - 41,8%; alpha1- globulin - 3%; alpha2-globulins - 14,5%; beta-globulin - 13,5%; gamma-globulins is 27.3%.
In June 2009, Including patient gave consent to participate in research. Upon physical examination draws attention to the enlargement of the liver, which acts from under the costal arch 2 cm, but otherwise unremarkable. Complete blood count: erythrocytes - 4,06·1012/l; hemoglobin - 132 g/l; hematocrit was 37.1%; platelet - 117·109/l; leucocytes - 4,3·109/l; monocytes - 8,1% (0,35·109/l); granulocyte - 58% (2,5·109/l; lymphocytes - 33,9% (1,46·109/sup> /l). Biochemical blood test: aspartate aminotransferase - 301 ME; alanine aminotransferase - 275 ME; bilirubin total - 16 µmol/l; direct bilirubin - 4 µmol/l; bilirubin indirect - 12 µmol/L.
Criteria immunological parameters: CD3+/CD56+ - 0,4%; CD3+/CD56+/CD4+ 44,4%; CD3+/CD56+/CD8+ - 55,6%; CD56+/CD94+ - 52,1%; CD56+/NKG2D+ - 98,1%; CD56+/CD107a+ - 82%.
Thus, according to a study in the criteria of immunological parameters defined 5 signs 6, characteristic of cirrhosis of the liver, which is consistent with the diagnosis and other clinical and laboratory signs of cirrhosis of the liver in this patient.
Example 3. Patient K., 52 years, is registered in the EMC Hepatology center for Infectious clinical hospital No. 1 since 2000 with a diagnosis of Chronic viral hepatitis C. Concomitant diagnosis: hyperthyroidism, peptic ulcer".
From the anamnesis it is known that in 1992 first screened for HBsAg negative result, the biochemical analysis of a blood - norm. In the same year, was treated in hospital with the diagnosis of Diffuse toxic goiter stage III", which was held Subtotal resection of the thyroid gland. In 1993 the first marked increase in alanine aminotransferase, was diagnosed with toxic hepatitis. In 1998, for the first time examined for antibodies to HCV and the result is positive. In the same year was conducted therapy is stronom And 3 mined 3 times a week, for 6 months. After treatment remained hyperfeminine to 90 ME. Repeated courses etiotropic therapy was not performed due to severe comorbidity. In 2001, for the first time was produced by needle biopsy of the liver, which was defined by 1 stage of fibrosis according to METAVIR. Repeat liver biopsy was done in December 2007, the results of which revealed stage 1 fibrosis according to METAVIR (sampling visited twice in two independent laboratories).
Because constantly the question of re-course etiotropic therapy, in October 2007, the patient was examined by indirect transient elastography liver on the device "FibroScan", which showed the result corresponding 3 fibrosis METAVIR liver. Also in December 2008, the patient was asked to conduct a study of venous blood for specific markers of tissue injury in the liver. The result FibroTest showed stage 4 liver fibrosis, a ActiTest revealed the third degree of activity nitrosomorpholine process in the liver tissue.
Viral load (13.01.2009): HCV RNA - 3,1·106IU/ml, 1.2 x 107copies/ml Genotype 1b. Ultrasound examination of abdominal cavity organs (22.01.2009): increase in liver size (+0,4; +0,7), diffuse changes. Elastohydrodynamic (26.01.2009): extending ariozna veins of the esophagus stage 1. The study of humoral immunity (13.01.2009): rheumatoid factor - negative; C3 of complementa - 174 mg/DL; C4 complement - 31 mg/DL; IgA at 2.59 g/l; IgG - 18 g/l; IgM - 0.8 g/l In late January 2009, the patient K. gave consent to participate in research. The patient was not included in the analyzed group due to severe comorbidity and ambiguity of results to determine the degree of liver fibrosis. Complete blood count: erythrocytes - 4,78·1012/l; hemoglobin - 157 g/l; hematocrit - 43,5%; platelet - 207·109/l; leucocytes - 5,8·109/l; monocytes - 6,8% (0,39·109/l); granulocyte - 55,3% (3,21·109/l; lymphocytes to 37.9% (2,2·109/l). Biochemical blood test: whole protein - 70 g/l; albumin - 61,8%; alpha1-globulins - 2,6%; alpha2-globulins - 9,4%; beta-globulins was 8.8%; gamma-globulins was 17.4%; aspartate aminotransferase - 45 ME; alanine aminotransferase - 86 ME; ratio of 0.5; bilirubin less than 9 mmol/l; α-fetoprotein - to 3.02.
Criteria immunological parameters: CD3+/CD56+ - 3.7%; and CD3+/CD56+/CD4+ - 0%; CD3+/CD56+/CD8+ - 56,9%; CD56+/CD94+ from 65.1%; CD56+/NKG2D+ - 98,4%; CD56+/CD107a+ is 98.2%.
Thus, on the basis of a puncture biopsy performed 2 years ago, was defined stage of fibrosis 1, the following year instrumental methods of analysis allowed us to identify the stage of fibrosis 3, and biochemical (FibroTest) - stage 4. This inconsistency of data created difficulties diagnostics is in communication with a low probability combinations results obtained by different methods. After another year at the time of immunological examination of the patient were registered some signs of cirrhosis of the liver, in particular the expansion of varicose veins of the esophagus. The study, completed with the definition of immunological criteria, allows us to state the stage of cirrhosis of the liver (4) 6 characteristics. In other words, needle biopsy showed an inaccurate result, while immunological study gave more accurate information, because it was combined with the results of other (refunctioned) methods.
This method allows you to identify a clear line of immunological changes in the blood of the third and fourth stage fibrotic changes in the liver. Furthermore, the method allows to determine pronounced fibrotic changes in the liver at concentrations of cytokines in the serum and register precirrhotic and cirrhotic stage of fibrosis in liver content in the blood lymphocytes of a particular phenotype as a predictor of real threat of cirrhosis of the liver.
Table 1 presents the range of values of informative parameters of CD56+ cells in control and patients hugs at different stages of liver fibrosis.
|A control group of healthy people (stage fibrosis 0), n=26|
|CD3+/CD56+, %||0,40 to 10.7||-||-|
|CD56+/NRG2D+cells, %||of 83.4-99,4||-||-|
|Patients hugs, stage of fibrosis 1, n=16|
|CD3+/CD56+, %||0,20-14,2||2 people-|
|CD3+/CD56+/CD4+, %||0-66,7||>13,4%||5 people-|
|CD3+/CD56+/CD8+, %||33,3-br93.1||>87,5%||1 person-|
|CD56+/CD107a+cells, %||28,7-100||<40,6%||5 people-|
|Patients hugs, stage of fibrosis 2, n=6|
|CD56+/CD94+cells, %||46,7 from 65.1||-||-|
|CD56+/CD107a+cells, %||of 98.2-100||-||-|
|Patients hugs, stage of fibrosis 3, n=8|
|CD3+/CD56+, %||4,30-9,60*||>3,7%*||8 persons-|
|CD3+/CD56+/CD4+, %||24,0-78,1*||>23,8%*||8 persons-|
|CD3+/CD56+/CD8+, %||the 15.6-27,7*||<47,6%*||8 persons-|
|CD56+/CD94+cells, %||31,6 of 41.6*||<46,7%*||8 persons-|
|CD56+/NRG2D+cells, %||98,5-99,1*||>98,4%*||8 persons-|
|CD56+/CD107a+cells, %||46,6 to 58.9*||<level 98.2%*||8 persons-|
|Patients hugs, stage of fibrosis 4 (cirrhosis), n=11|
|CD3+/CD56+, %||0,50-9,10*||<4,3%*||9 people.-|
|CD3+/CD56+/CD4+, %||0-37,5*||<24,0% *||9 people.-|
|CD3+/CD56+/CD8+, %||31,2-90,2*||>27,7% *||10 persons-|
|CD56+/CD94+cells, %||27,1-70,7*||<31,6%* or||8 persons-|
|CD56+/NRG2D+cells, %||89,9 reached 98.9*||<98,5%*||9 people.-|
|CD56+/CD107a+cells, %||82,0-99,5*||>58,9%*||10 people of-0.9%|
|Note: * marked deviation criterion of signs within the designated range from the previous stage of liver fibrosis more than 70% of cases.|
Table 2 presents a summary table of information on how immunological indication fibrosis stage 3 in patients with chronic hepatitis C
Table 2 Method display (criteria parameters) Criterial value Frequency registration The frequency of combinations of different criteria CD3+/CD56+cells >3,7% 100% When hugs, stage 3: 6 signs - 100%
When hugs, stage 2: 0 features - 100%
When hugs, stage 4: 0 features - 100%
CD3+/CD56+/CD4+cells >23,8% 100% CD3+/CD56+/CD8+cell <47,6% 100% CD56+/CD94+cells <46,7% 100% CD56+/NKG2D+cells >98,4% 100% CD56+/CD107a+cells <level 98.2% 100%
Table 3 presents a summary table of information on how immunological indication fibrosis stage 4 patients with chronic hepatitis C.
|Method display (criteria parameters)||Criterial value||Frequency registration||The frequency of combinations of different criteria|
|CD3+/CD56+cells||<a 4.3%||81,8%||When hugs, stage 4: 6 signs is 27.3% 5 signs - 72,7%|
When hugs, 3 phase: 0 features - 100% At HUGW, 4 stage:
|CD56+/CD94+cells||<31,6% or >41,68%||72,7%|
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Method of assessment of liver fibrosis in patients with chronic viral hepatitis C, including studies of the phenotype CD56+lymphocytes, characterized in that to determine the percentage of CD3+/CD56+, CD3+/CD56+/CD4+, CD3+/CD56+/CD8+, CD56+/CD94+, CD56+/NKG2D+, D56+/D107+cells and modify content in the blood D3+/D56+cells above the 3.7%, CD3+/CD56+/CD4+cells above 23,8%, CD3+/CD56+/CD8+cells below 47,6%, CD56+/CD94+ cells below to 46.7%, CD56+/NKG2D+ cells above to 98.4%, and CD56+/CD107a+ cells below the level 98.2% establish if the third stage (pretsirroticheskie) fibrosis of the liver and changes in blood 5 out of 6 parameters: CD3+/CD56+ cells below the 4.3%, CD3+/CD56+/CD4+ cells below 24%, CD3+/CD56+/CD8+ cells above 27,7%, CD56+/CD94+ cells below 31,6% or higher, 41.6%of CD56+/NKG2D+ cells below to 98.5%, and CD56+/CD107a+ cells above the 58.9% are regarded as the transition of the disease to cirrhosis.
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.
3 dwg, 2 tbl, 2 ex
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
SUBSTANCE: diagnostic technique for latent radiation sickness consisting in biopsy procedure of externally unaffected leg skin followed with histological study of a biopsy material, and if certain morphological signs of inflammatory changes of microvasculature vessels presented, latent radiation sickness is diagnosed.
EFFECT: technique allows diagnosing radiation sickness at early stages of development.
1 tbl, 1 ex
SUBSTANCE: method of early prediction of septic complications in newborns with a respiratory pathology, by examining venous blood by direct immunofluorescence on a flow cytometre, a relative monocyte Annexine V concentration is evaluated, and if observing the value equal to 17.4 % and more, septic complications are diagnosed.
EFFECT: method exhibits high sensitivity and specificity and refers to near-patient testing.
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.