Noninvasive method for diagnosing hepatic fibrosis grade in patients suffering from chronic viral hepatitis

FIELD: medicine.

SUBSTANCE: method involves determining spontaneous blood platelets aggregation and one induced by adrenalin and collagen, thrombocytospecific peptides activity of β-thromboglobulin and thrombocytic factor 4 in blood plasma.

EFFECT: high accuracy of diagnosis.

2 tbl

 

The invention is used in medicine, namely in the clinic of internal medicine (gastroenterology).

Determining the expression of fibrosis in patients with chronic viral hepatitis is an important task, as the rapid progression of fibrosis leads to cirrhosis or liver cancer in one-third of patients within 20 years [1, 2]. On the other hand, determining the severity of fibrosis (stage hepatitis) is important to determine the indication for antiviral therapy.

Determining progression of liver fibrosis is possible without holding a needle biopsy on the basis of clinical and ultrasonographic parameters [3, 4].

However, the precise predictors, indicating some degree of fibrosis compared with histological results obtained by biopsy of the liver, currently no. Thus, according to S.A.Coverdale and G.Borroni [2, 5], revealed insignificant correlation between routine hepatic biochemical and ultrasonographic tests and fibrational stage of chronic hepatitis. .Mattiello and .Penz [3, 4] using these data revealed sufficient correlation for determining the stage of fibrosis. With the rapid development of fibrosis associated male gender, disease duration, 1 genotype of the virus, the gene polymorphism factor V clotting, R-village of the tin [6].

The platelets in the peripheral blood [7, 8, 9] in combination with age may be a predictor of the development of significant histological changes in chronic viral hepatitis [10]. According to S.A.Coverdale [2], the clearance of antipyrine and the level of platelets for detection fibrational progression of viral hepatitis are more sensitive than albumin and other biochemical tests. According to G.Borroni [5] of 3 clinical and 6 biochemical factors, the level of platelets and the concentration of immunoglobulins significantly and independently correlated with the presence of cirrhosis in chronic liver pathology with infection with hepatitis C virus, and the level of platelets less than 134×109/l had 100% specificity.

Based on the above, you should characterize the relationship between the level of platelets and structural changes in the liver and to define the role of platelets in the progression of chronic viral liver disease.

Under the influence of lipopolysaccharide, interleukin-1, tumor necrosis factor-α platelets pass through the hepatic sinusoidal space and come into contact with hepatocytes [11], which leads to their activation, the release of ATP and ADP [12]. Activation of platelets is a potential trigger remodeling of the liver tissue with chronic viral hepatitis [13] secreted in excessive amounts of OS-granules platelet-derived growth factor [14], transforming growth factor - β and thrombin stimulate the proliferation of fibroblasts and the development of liver fibrosis, disruption of the architecture of the liver even after cessation of exposure to the initiating factor[15, 16, 17, 18, 19]. Thus, there is a clear link between inflammation and collagenopathy in the liver and the functional activity of platelets.

However, the reduced level of platelets in the peripheral blood in chronic viral liver pathology may be associated not only with fibrosis of the liver, but also due to the direct influence of viruses on trombozitopoez, to reduce the level of messenger RNA of thrombopoetin, low production of hepatic tissue, increasing the level of antiplatelet IgG. Therefore, to more accurately assess the degree of fibrosis on the basis of the criterion of platelets is necessary to use indicators of their functional activity, including the reaction products release components α-granules.

Reliable method to determine the degree of liver fibrosis (stage hepatitis) is a histological method for the study of the liver obtained by biopsy of the liver performed percutaneous or laparoscopic by [1].

The applied method has numerous disadvantages:

1. The invasiveness of the procedure, which reduces the compliance of the patient.

2. The need to conduct is of ultrasonographic studies before the procedure to exclude focal liver formations.

3. Numerous contraindications to needle biopsy of liver lesions (cysts, hemangioma), reduced level of platelets in the peripheral blood, increasing the duration of bleeding, clotting time, decreased prothrombin activity and other

4. The need of the patient's stay in hospital overnight after the procedure.

5. Complications of biopsy: abdominal and thoracic bleeding, pneumothorax, injury to the gall bladder and ducts, cysts, peritonitis.

6. The frequent inability of the procedure to dynamically assess the severity of fibrosis.

7. The need for a doctor's certificate (surgical specialties) to conduct research on therapeutic doctor (gastroenterology) Department.

While laparoscopic biopsy in fact equivalent to a small surgery on the organs of abdominal cavity.

The task is non-invasive diagnosis of the severity of liver fibrosis in patients with chronic viral hepatitis.

This object is achieved by defining indicators of spontaneous and induced (adrenaline, collagen) platelet aggregation and activity thrombocytopathies peptides (β-thromboglobulin, platelet factor 4) in the plasma.

The method implemented is aetsa as follows.

The criterion for inclusion is the availability of proven chronic viral hepatitis. To determine fibrosis indicators are used induced platelet aggregation (inductors: adrenaline, collagen), the level of spontaneous platelet aggregation, activity thrombocytopathies peptides in plasma (β-thromboglobulin, platelet factor 4). Exclusion criteria are related pathology (acute or chronic diseases in the acute stage), the medication within 7 days prior to the survey.

The blood produced from the cubital vein in the morning, on an empty stomach, a needle with a wide opening without tourniquet is applied and massage of the forearm with the further use of citrate plasma rich or poor (depending on the designated parameter) platelets. Platelet aggregation is logged on aggregometry AR (SOLAR, Belarus), paired with an IBM-compatible computer. To exclude the contact activation of platelets is only used plastic utensils. Spontaneous platelet aggregation register since the beginning of the mixing platelet-rich plasma (no inductor) for 10 minutes to determine the maximum extent of aggregation. Normal spontaneous aggregation is 0.67±0,07%. Induced aggregation studied by adding the value of the inductors to platelet-rich plasma: adrenaline (5 μm/ml, "Aoede Richter, Hungary), collagen (0.2 mg/ml, "NGOs to Rena", Russia) in the indicated final concentrations in the test system with the assessment of the maximum aggregation level in %in healthy equal 64,70±1,94% 60,01±1.35%, respectively.

Activity β-thromboglobulin in the plasma is determined using standard test kits Roche ELISA. Blood (4.5 ml) are placed in plastic tubes with addition of 0.5 ml of 3.8% solution translesanas of sodium citrate, 2 mg/ml of theophylline and dipyridamole, centrifuged 30 minutes (4000 rpm, 4° (C), the plasma is selected and stored at a temperature of -25°C. the Method for determining the activity of 4 platelet factor-based actions estramboticos heated plasma containing thermostable factor 4, thrombin-heparin clotting time control estramboticos plasma. The degree of shortening of the thrombin-heparin clotting time is a measure of the activity of the 4 factors. In healthy people the plasma level β-thromboglobulin is 146,12±8,13 IU/ml, activity 4 platelet factor - 3,02±0,67 C. Data of platelet aggregation and activity thrombocytopathies peptides in healthy individuals, and patients are presented in table 1. The indicators of the functional activity of platelets with the purpose of further evaluation of fibrosis was performed with the plot is that the results of histological examination of the liver tissue, obtained by needle biopsy.

/tr>
Table 1
IndicatorsHealthySick
index of fibrosis
0-1 points2-3 points4 points
Adrenalinen=32n=13n=30n=10
aggregation64,70±1,9476,92±2,9240,35±4,4320,261±5,95
platelets (%)**/***/**/***
Collagenn=32n=13n=30n=10
aggregation60,01±1,3573,68±2,1243,58±3,5417,92±6,66
platelets (%)**/***/**/***
Spontaneousn=10n=17n=21n=6
aggregation0,67±0,071,64±0,252,30±0,171,18±0,21
platelets (%)**/*****
β-thromboglobulinn=10n=17n=21n=6
(IU/ml)146,12±8,13176,82±6,91206,10±7,68168,22±6,04
**/*****
Plateletn=10n=17n=21n=6
factor 4(seconds)3,02±0,575,16±0,657,30±0,514,45±0,35
**/*****
Note that n is the number of patients. * - p<0.05 compared with control, ** p<0.05 compared with minimal fibrosis, *** p<0.05 compared with moderate fibrosis.

Chronic viral hepatitis with minimal signs of fibrosis according to Desmet (0-1 point) characterized by an increase (compared to control) performance of spontaneous platelet aggregation induced by epinephrine and collagen platelet aggregation, levels β-thromboglobulin and platelet factor 4. Unidirectional changes in the direction of increasing values of the functional activity of platelets are the difference between chronic viral hepatitis with minimal about what areas of fibrosis from chronic viral hepatitis with moderate to severe fibrosis. While the absolute values of these parameters are as follows:

- adrenaline platelet aggregation >76%,

- collagen platelet aggregation >68%,

spontaneous platelet aggregation >0,9%,

- β-thromboglobulin >170 IU/ml,

- 4 platelet factor >4,7 sec.

Chronic viral hepatitis with moderate to severe liver fibrosis (2-3 points) characterized by a reduction induced by adrenaline and collagen platelet aggregation, in contrast to the increase (as with minimal fibrosis) indicators of spontaneous platelet aggregation, β-thromboglobulin and platelet factor 4. While the absolute value of the spontaneous aggregation of platelets, level β-thromboglobulin and activity of platelet factor 4 higher than the corresponding indices in patients with chronic viral hepatitis with minimal fibrosis. Indicators of functional activity of platelets following:

- adrenaline platelet aggregation 54-16%,

- collagen platelet aggregation 52-24%,

spontaneous platelet aggregation >2,6%,

- β-thromboglobulin >204 IU/ml,

- 4 platelet factor >7.8 seconds.

Index of fibrosis 4 points (the presence of cirrhosis at least class a Child-Pugh) (in addition to clinical and instrumental signs of portal hypertension) is characterized by maximum (relative to the s index of fibrosis 2-3) inhibition induced by adrenaline and collagen platelet aggregation, level β-thromboglobulin within normal limits or slightly increased, increased platelet factor 4 and increased spontaneous platelet aggregation. While the absolute values of these parameters are as follows:

- adrenaline platelet aggregation <16%,

- collagen platelet aggregation <24%,

spontaneous platelet aggregation 0,9-1,5%,

- β-thromboglobulin within normal limits or slightly increased,

- 4 platelet factor of 4.7-5.2 sec.

Table 2 shows the parameters of the functional activity of platelets to assess the severity of liver fibrosis.

Table 2
Sick
IndicatorsHealthyindex of fibrosis
0-1 points2-3 points4 points
Adrenaline
aggregation54-76%>76%54%-16%<16%
platelets
Collagen
aggregation52-68%>68%52%-24%<24%
platelets
Spontaneous
aggregation0,5-0,9%>0,9%>2,6%0,9-1,5%
platelets
170-182 IU/mlN
β-122-170>170>204or within theor
thromboglobulinIU/mlIU/mlIU/mlstandards
Platelet1,3-4,7>4,7>7.8 secondsa 4.7-5.2 sec
factor 4secsec
Note: ↑ - increase compared to the norm, ↓ - decrease in comparison with the norm N is within limits.

Developed non-invasive diagnostic criteria of severe fibrosis equally informative for any etiological forms of viral liver disease (b, C, b+C, B+D).

Clinical example

Patient A., aged 27. Case history No. 71. Was in the gastroenterological Department of the regional clinical hospital with 04.01.01 on 18.01.01, the Diagnosis of chronic viral hepatitis C (aHCV +, HCV RNA +) with moderate activity. Concomitant diagnosis: serological signs moved HBV infection.

Complaints about General weakness, decreased performance. Considers himself ill with 1999 the ode, when recovered from acute viral hepatitis b+C (HBs Ag +, aHCV +). In September 2000, he was troubled and General weakness. While the survey found a threefold increase in transaminases. From history: tonsillectomy in 7 years. Drug use between 1998 and 2000.

Objectively: the skin, sclera and the regular color. The lungs and heart without features. AD - 125/80 mm RT. Art., pulse 64 in 1 minute. The size of the liver by karlovu: 10×9×8 see Liver soft-elastic consistency, painless, smooth, the lower edge of the stands from under the costal arch to 1.5 cm Spleen not palpated.

Complete blood count: er. 4,2×l012/l; HB 142 g/l; blood clot. 210×109/l; lake. 4,2×109/l; E. 2%; p. 2%; S. 62%; limp. 28%; Mont. 4%; erythrocyte sedimentation rate of 6 mm/h

Urinalysis: Rel. density 1,019; without deviation from the norm; diastasis urine 128 ed,

Biochemical studies. Total protein 72 g/l; albumin 44%; globulins: α 20%; β 17%; γ 19%. Blood glucose 4.1 mmol/l Bilirubin: total 13,76 µmol/l AST 44 u/l; Alt 126 u/l; LDH 254 u/l; GGT 21 u/l; alkaline phosphatase 169 u/l Thymol test 7,7 u; CEC UE 180; Ceruloplasmin of 41.7 mg/DL; cholesterol 4.5 mmol/l Triglycerides: PETIT 87%; the clotting time 5 min 43 sec; duration of bleeding 40 C.

Markers of hepatitis: HBs Ag (-); aHBs (+); HBe Ag (-); aHBe (+); aHBc Ig M (-); aHBc amounts. (+); aHCV (+); HCV-PHK (+).

Ultrasonography. Liver: right lobe 108 mm, the left share of 79 mm, echo is normal, tissue is homogeneous. Intrahepatic ducts are not expanded. Portal vein 10 mm, splenic 5 mm, choledoch 5 mm Gallbladder 71×13 mm, wall thickness 3 mm Pancreas: head 22 mm, the body is 15 mm, the tail is 23 mm, echo is increased. Spleen 127×52 mm

Liver biopsy. Lobular structure saved. Portal tracts unevenly expanded expressed lymphomacrophagal infiltration with formation of lymphoid follicles. Marked proliferation of the bile ducts, significant focal proliferation kupperbusch cells. Lobular component in the form of chains, and on-site 3-4 hepatocytes. Fibrosis of the stroma of the walls of the Central veins and stroma slices in 3 area. Hydropic degeneration of hepatocytes. Conclusion: chronic hepatitis with moderate activity. Index Knodes 7 points. Index Desmet 1 point.

The studied parameters: induced adrenaline and collagen platelet aggregation 78,6%, 70.0% respectively, spontaneous platelet aggregation 1,49%, the content of β-thromboglobulin 172,2 IU/ml, activity 4 platelet factor 4,88 C. As can be seen from this example, the patient with a minimum degree of fibrosis determined by the elevated levels of spontaneous and induced platelet aggregation and indicators thrombocytopathies peptides.

The use of the proposed method of evaluation grade is liver fibrosis lets not resort to biopsy of the liver. The study is safe for the patient, there is no need for invasive procedures, is available for practical health care institutions, not time-consuming.

Sources of information:

1. Sherlock W., J. Dooley. Diseases of the liver and biliary tract. - M., 1999. - 864 S.

2. Coverdale S.A., Samarasinghe D.A., Lin, R., Kench, J., Byth K., Khan M.H., Crewe E., Liddle C., George J., Farrell G.C. Changes in antipyrine clearance and platelet count, but not conventional liver tests, correlate with fibrotic change in chronic hepatitis C: value for predicting fibrotic progression // Am. J. Gastroenterol., 2003. - Vol.98 (6).- P.1384-1390.

3. Mattiello K., A. Favaro, Bemardinello E., Cavaletto L., Mezzocolli I., Luise S., De Carlo, S., D. Sacerdoti, Gatta A., Chemello L. Non-invasive diagnosis of liver fibrosis progression by clinical and usdoppler predictive model during chronic HCV infection // J. Hepatol. - 2003. - Vol. 38, Suppl. 2. - P.155.

4. Penz m, Oesterreicher C, Wrba F., C. Datz, Gschwandtler M., Hofer H., Novacek G., Gangl.A., Ferenci P. Routine serum laboratory parameters for the staging of fibrosis in patients with chronic hepatitis C // J. Hepatol. - 2003. - Vol.38, Suppl. 2. - P.163.

5. G. Borroni, Ceriani R, Tommasini M.A., Maltempo C., Fellini C., Contu L., Cazzaniga M. Platelet count: a simple marker of liver cirrhosis in chronic hepatitis C (CHC) infection // J. Hepatol. - 2002. - Vol.36, Suppl. 1. - P.47.

6. Wright m, Goldin R, Thursz M. Construction of a predictive model to identify rapid fibrosis progression in patients with chronic hepatitis C virus infection // J. Hepatol. - 2003. - Vol.38, Suppl. 2. - P.17.

7. Ceriani R., G. Borroni, Tommasini M.A. et al. Platelet count and AST/ALT ratio can predict liver cirrhosis in chronic HCV infection // Hepatology Rapid Literature Rewiew. - March 1999. - P/C02/17.

8. Fasoli A., Romagnoli P., Botta F. et al. Platelets/spleen area ratio reflects progressive liver disease // Hepatology Rapid Literature Rewiew. - March, 1999. - P/C 11/13.

9. Renou, S., Muller P., Halfon Ph. et al. Relevance of moderate isolated thrombocyopenia as a strong predictive marker of cirrhosis in patient with chronic hepatitis With vims // Hepatology Rapid Literature Rewiew. - March 1999. - P/C02/17.

10. Poynard T., Bedossa P. Age and platelet count: a simple index for predicting the presence of histological lesions in patients with antibodies to hepatitis With virus. METAVIR and CLINIVIR Cooperative Study Groups // J. Viral Hepat, 1997. - Vol. 4. - P.199-208.

11. Nakamura N., M. Shibazaki, Y. Nitta, Y. Endo Translocation of platelet into Disse spaces and their entry into hepatocytes in response to lipolysaccharides, interleukin-1 and tumor necrosis factor: the role ofKupffer cells // J. Hepatol. - 1998. - Vol.(6). - P.991-999.

12. Takemura, S., Minamiyama y, Kawada N. et al. Increasing interest among nucleotides modulate the portal circulation with generation of nitric oxide // Hepatology Research. - 1998. - Vol. 13 (1). - P.29-36.

13. Papatheodoridis g, Papakonstantinou E., Andtioti E., E. Cholongitas, K. Petraki, Kontopoulou I., Hadziyannis S. Thrombotic risk factors and extent of liver fibrosis in patients with chronic viral hepatitis (CH) // J. Hepatol. - 2002. - Vol.36, Suppl. 1. - P.175.

14. Shiraishi T., Morimoto, S., Koh, E., et al. Increased release of platelet-derived growth factor from platelets in chronic liver disease // Eur. J. Clin. Chem. Clin. Biochem. - 1994. - Vol. 32 (1). - P.5-9.

15. Gressner A.M. Hepatic fibrogenesis: the puzzle of interacting cells, fibrogenic cytokines, regulatory loops and increasing interest among matrix molecules // Z. Gastroenterol., 1992. Vol. 30, Suppi 1. - P.5-16.

16. Gressner A.M. Cytokines and cellular crosstalk involved in the activation of fat-storing cells J. Hepatol. - 1995. - Vol.22 (2 Suppl). - P.28-36. 17.Gressner A.M., Chunfang G. A cascade-mechanism of fat storing cell activation forms the basis of the fibrogenic reaction of the liver // Verh. Dtsch. Ges. Pathol. - 1995. - Vol. 79. - P.1-14.

18. Mallat, A., Gallois, C., Tao J.C. et al. Platelet-derived growth factor-BB and thrombin generate positive and negative signals for human hepatic stellate cell proliferation. Role of a prostaglandin/cyclic AMF pathway and cross-talk with endothelin receptors // J. Biol. Chem. - 1998. - Vol. 273 (42). - P.27300-27305.

19. Pinzani m, Milani s, Herbst H. et al. Expression of platelet-derived growth factor and its receptors in normal human liver and during active hepatic fibrogenesis // Am. J. Pathol. - 1996. - Vol.148 (3). -P.785-800.

The way to diagnose the severity of liver fibrosis in patients with chronic viral hepatitis, including the identification of liver fibrosis, characterized in that identify indicators of spontaneous and induced adrenaline and collagen platelet aggregation, activity β-thromboglobulin and platelet factor 4 in plasma and at the parameter values: adrenaline platelet aggregation >76%, collagen platelet aggregation >68%, spontaneous platelet aggregation >0.9 per cent, activity β-thromboglobulin >170 IU/ml, 4 platelet factor >with a 4.7 - define chronic viral hepatitis with minimal fibrosis on Desmet (0-1 point), when the values of the parameters: adrenaline platelet aggregation 54-16%, collagen platelet aggregation 52-24%, spontaneous platelet aggregation >2,6%, the activity of p-thromboglobulin >204 IU/ml, 4 platelet factor >7,8 - define chronic viral hepatitis with moderate to severe fibrosis by Desmet (2-3 points), with parameter values: adrenaline platelet aggregation <16%, collagen platelet aggregation <24%, spontaneous platelet aggregation 0,9-1,5%, activity β-thromboglobulin within normal limits 122-170 IU/ml or slightly increased, 4 platelet factor 4,7-5,2 to determine fibrosis by Desmet 4 points (the presence of cirrhosis at least CL the SSA And Child-Pugh).



 

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1 dwg, 2 ex, 1 tbl

FIELD: veterinary medicine, biochemistry.

SUBSTANCE: the present innovation deals with boiling an extract, cooling, centrifuging, dissolving a residue, cooling, centrifuging, dissolving a residue, adding sulfuric acid into a tube and 1%-condensate's solution followed by heating, cooling, photometry against the control at wave length being 315 nm, as a condensate one should apply resorcinol.

EFFECT: higher accuracy and economy of detection.

2 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: method involves fixing material and setting histochemical reaction on detecting cholinergic nerve structures. The fixed material under study is incubated during 60-90 min in thermostat at 37°C in mixture containing acetylthiocholine iodide as substrate, washed, treated with alcohols in growing concentrations, cleared and enclosed into balsam. The material is fixed with 2% glyoxylic acid prepared on 0.1 M phosphate buffer solution having pH of 7,0. Histochemical reaction is set concurrently with material fixation for detecting adrenergic nerve structures containing neuromediators forming luminescent reaction products with glyoxylic acid, in which the material is incubated during 15-20 min at temperature of 20-25°C. Then it is dried and heated during 5-10 min in thermostat at 80°C, studied with luminescent microscope, subjected to morphometric study and photographed. Then, histochemical reaction is set on the same material for detecting cholinergic nerve structures.

EFFECT: high comparison accuracy on the same portion of tissue being used.

4 dwg

FIELD: medicine.

SUBSTANCE: method involves determining proportion of cyclic adenosine monophosphate to cyclic guanosine monophosphate, serotonin and histamine level and serotonin/histamine ratio coefficient, content of prostaglandin E2 and prostaglandin F2α and adenylatecyclase index. Cyclic adenosine monophosphate to cyclic guanosine monophosphate ratio being equal to 2.7-3.3, serotonin level of 4.5-6.6, content of prostaglandin E2 being equal to 670-1340 mg/g of protein and prostaglandin F2α to 320-960 mg/g of protein, label index being equal to 10.1-35,0% and adenylatecyclase index being equal to 19.2-30.0 rmole/g of protein/min, stomach and duodenal ulcer healing is predicted.

EFFECT: high accuracy of prognosis.

FIELD: medicine, oncology.

SUBSTANCE: before carrying out hyperbaric oxygenation (HBO) one should detect the content of lipid peroxidation products in the condensate of expired air. If the content of TBC-active products is below 14 conventional units it is necessary to perform about 3-4 HBO seances at 1.3 absolute atmosphere for 40 min both in pre- and postoperative periods. If the content of TBC-active products varies 14-24 conventional units one should conduct about 3-4 HBO seances at 1.3 absolute atmosphere for 40 min in preoperational period only. If the content of TBC-active products is above 24 conventional units HBO séances should not be carried out . The method is considered to be noninvasive, of high information value and enables to increase efficiency to apply HBO in patients with pulmonary cancer planned for operative treatment.

EFFECT: higher efficiency.

3 ex

FIELD: medicine, obstetrics.

SUBSTANCE: one should study placental tissue to prepare 10% homogenate and detect NADPH-oxidase activity according to reduction of 2.6-dichlorophenylindophenol at the presence of NADPH. Results of reaction should be specified spectrophotometrically at wave length being 600 nm. Method is atraumatic and enables to predict the development of encephalopathy in neonatals at high accuracy.

EFFECT: higher efficiency of prediction.

2 ex

FIELD: medicine.

SUBSTANCE: method involves determining lactate content in peripheral venous blood sample taken from cadaver. The value being less than 16 mmole/l, hypoglycemic coma is diagnosed.

EFFECT: high reliability of diagnosis independently on death outcome time.

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