RU2183326C2 - Method to predict hepatic fibrosis at chronic hepatitis in children - Google Patents

Abstract

FIELD: medicine, pediatrics. SUBSTANCE: method is performed by bloodless epidermal scarification of forearm skin. It is obtained the print of cellular exudate of cutaneous aseptic inflammatory response (AIR) which is fixed with ethanol, dyed by Romanovsky, subjected to microscopy to evaluate the degree of AIR area fibrosis. At the absence of collagenous fibers the absence of hepatic fibrosis is diagnosed. At accumulation of single gentle fibers in rare vision areas - slightly pronounced hepatic fibrosis is diagnosed, at accumulation of coarse single fibers in all vision areas - moderate hepatic fibrosis and considerable mass of fibers in all vision areas as bundles at the background of pronounced structural and functional degradation of macrophages and accumulation of fibroblasts the pronounced hepatic fibrosis or cirrhosis are diagnosed. EFFECT: increased safety for prediction. 4 dwg, 2 tbl

Description

 The method relates to medicine (pediatrics), the pathology of cellular immunity, to the diagnosis of chronic hepatitis in children by the histochemical method, which allows to establish the severity of the process and control its dynamics during treatment.

 A known method that allows you to obtain information about the state of the pathological process in chronic hepatitis using intravital puncture biopsy of the liver. For this, preliminary preparation is necessary to eliminate contraindications, during which the blood is examined for hemorrhagic syndrome with a determination of the level of platelets, the prothrombin index; ECG, X-ray examination of the chest. After contraindications are eliminated during a puncture biopsy of the liver, parenteral administration of muscle relaxants and the use of mask anesthesia are required, which disable the respiratory movements for apnea, at which point a puncture needle is made in the mid-axial line in the region of the 9 intercostal space with a puncture needle and a column of liver tissue is removed. In Lilly’s solution, he is delivered to the pathological department, where, after histological processing, a pathologist conducts a morphological study of punctate with a conclusion on the degree of activity of the pathological process in the liver and the stage of the process, i.e. the absence or a certain degree of severity of fibrosis using the histological index of activity (GIA) according to the Knodel system. In accordance with the results of GIA calculation, a morphological diagnosis is formulated taking into account the current classification of chronic hepatitis (Desmet V. et al, 1995), Table 1.

 The disadvantages of this method that can be eliminated by the claimed method include the need to perform a liver biopsy in a specialized surgical hospital with a well-equipped operating room and a highly qualified team of surgeons and anesthesiologists, which may be available only to individual central clinics of the country. In addition, taking into account the given anesthesia and surgical intervention, a liver biopsy is an unsafe method with which complications such as bleeding from the puncture site, infection of the pleura, and damage to neighboring organs are associated. This dictates the need for patients to comply with strict bed rest with control over its general condition, blood pressure and pulse. In addition, it is necessary to take into account those cases when it is not possible to obtain liver tissue - then the puncture is inconclusive, and in 2-15% of the examined patients a “dry” puncture is also possible.

 We can not ignore the significant duration in time from the moment of puncture to obtain a morphological conclusion (up to 10 days). A significant drawback is the inability to conduct repeated studies with the necessary frequency in the dynamics during treatment.

 In assessing the severity of liver tissue fibrosis, the results of the widely available method - ultrasound of the abdominal cavity organs - provide significant assistance. According to the new classification of chronic hepatitis (1995), the stage of chronic hepatitis is based on an assessment of the degree of fibrosis of the liver parenchyma: 1. no fibrosis; 2. fibrosis is mild; 3. moderate fibrosis; 4. severe (severe) fibrosis and 5. irreversible stage - cirrhosis of the liver (table 1).

25 years of experience in the hepatological clinic of the Department of Pediatric Infections of the Russian State Medical University for the study of the features of ultrasound results of the liver in children with chronic hepatitis and other liver pathologies (A. Pisarev, 1984; Table 2) confirms the coincidence of the latter with liver puncture biopsy data ( FSN) in 62% of cases of observation. These data formed the basis for the development of ultrasound criteria for stages of liver fibrosis in children with chronic hepatitis. So, when comparing with PBP data, when “there is no fibrosis”, the ultrasound signs are described accordingly: “the liver is not enlarged, the transplant is homogeneous (homogeneous) in all departments, it has a low zhogenicity, the vessels of the portal system are not dilated.”
The stages of “mild fibrosis” according to PBP are consistent with ultrasound signs: “the liver is slightly enlarged, the nose is homogeneous, evenly or only in the periportal zone, it is slightly densified due to small focal structures, and the vessels of the portal system are not dilated”.

 With these “moderate fibrosis” ultrasound tests, the results are described accordingly: “the liver is enlarged, the parenchyma is evenly or partially densified due to small focal different density structures (light gray and white signals prevail); the liver contour is smooth; the vessels of the portal system are usually not dilated "In the lower sections there is a weakening of ultrasound signals."

 With "pronounced fibrosis - cirrhosis", the ultrasound signs correspond to morphological data: "the liver is slightly enlarged due to the right lobe, its contour is often uneven, the parenchyma is dense due to small and / or medium focal structures of mainly high amplitude (white color predominates); bile ducts of medium caliber are poorly visualized; dense interlobular septa are expanded; a mosaic pattern due to areas of different densities is possible; the portal-splenic vein system is enlarged, convoluted; collaterals are visible, often multiple (angiomat naya transformation); in the lower sharply weakened the ultrasonic signal, the stage of hepatocellular insufficiency - ascites ".

 The disadvantage of this method is that in some cases, if there are morphologically characteristic signs of liver fibrosis, a mismatch of the described pattern of ultrasound of the liver is possible, where they are not captured because of pronounced parenchyma edema against the background of the cytolytic syndrome accompanying an exacerbation of the active (aggressive) hepatitis process. In these cases, parenchyma edema overlaps areas of its compaction, thereby hindering the objectivity of ultrasound evaluation of liver fibrosis.

Bibliography of analogue and prototype:
Mansurov H.Kh., Kutchak S.N. Liver biopsy, 1964, p. 19-27.

 Pisarev A.G. Ultrasonic ultrasound imaging. In the book: Diseases of the digestive system in children. Ed. A.V. Mazurina. M., 1984, p. 130-132.

 Czaja A.J. A new classification of chronic active hepatitis. Russian Journal of Gastroenterology, Hepatology, Coloproctologist, 1995, p. 46-51.

 Desmet V., Gerber M, Hoofnagle J.H. et al. Classification of chronic hepatitis: diagnosis, determination of the severity and stage of the course. - In the same place, with. 38-45.

 The main objective (goal) of the proposed method is to assess the degree of liver fibrosis in chronic hepatitis in children, as well as a dynamic assessment of the effectiveness of the therapy for this disease.

 An additional goal is the safety of the claimed method for the life of the patient (skin scarification test with minor trauma to the child).

 Currently, the fact of the development of chronic hepatitis of viral etiology in children as a primary chronic inflammatory process, as a result of which diffuse inflammatory liver fibrosis is formed, accompanied by depression of the mononuclear phagocyte system (SMF), is proven. Inhibition of the phagocytic activity of these cells is accompanied by a decrease in collagenolytic function in areas of excessive fibrogenesis (Mayansky D.Ya. et al., 1989). In this regard, the “resuscitation” of SMF during fibrogenesis allows one to substantially “postpone” the development of irreversible liver fibrosis and accelerate the involution of fibrous structures (D.N. Mayansky, 1992). Inflammation is considered as an emergency stereotypical version of the functioning of the "physiological system of connective tissue" and refers to the pathological process.

 One of the forms of chronic inflammation is mononuclear infiltrative inflammation (MIV) as an adaptive version of the functioning of connective tissue in an attempt to maintain homeostasis, but with costs for the organ parenchyma. MIW is pre-genetically programmed for a persistent version of the course, which is designated as primary chronic. In the focus of chronic MIV, the role of the “conductor” of fibrogenesis is taken by macrophages that produce collagenase, which breaks down preformed collagen, and the plasminogen activator, which inhibits the collagenolytic functions of MIB zone fibroblasts (Mayansky D.N., 1992).

 Using the model of aseptic inflammatory reaction (ABP), it became possible to "look" into the focus of inflammation, to examine the functional state of cells of its composition, to monitor its phases from acute to chronic, and from the latter to fibrosis (Mayansky D.N., 1992) .

 Classical clinical models include the “skin window” test according to Rebuck JW, Crawley J.A., (1955), with the help of which it is possible to assess the nature of the course of skin ABP, phase changes (from neutrophilic to macrophage), as well as the degree of fibrosis of the inflammatory zone . As an alternative to the histological method for assessing the degree of liver fibrosis, the claimed method proposed an immunological criterion for assessing the degree of liver fibrosis, which is based on the results of the course of ABP in the skin and comparing them with the data of ultrasound of the liver (prototype) in chronic hepatitis B, D and C in children.

 The solution to this problem is achieved by surface scarification of the skin on the front surface of the forearm in the upper third, pre-treated with ethyl alcohol.

A scarifier is removed from the epidermis, measuring 0.5 • 0.5 cm, without damaging the dermis and its capillaries. The wound is covered with sterile glass, which is fixed with a band-aid and exposed for 24 hours. The obtained imprint of ABP exudate on the glass is dried and fixed at 96 ^{° with} ethanol, then stained according to Romanovsky, dried and microscopic with immersion under a light microscope (vol. 90) with immersion. In addition to microscopy, in order to assess the process of fibrosis of the ABP zone by this method, it is possible, after special histochemical processing, to evaluate the activity of 5-nucleotidase in macrophage membranes, the activity of the lysosomal enzyme-esterase and the level of nuclear RNA in these cells.

 In the process of microscopy, the morphological integrity and maturation stage of mononuclear cells (macrophage monocytes), the accumulation of neutrophils, lymphocytes and fibroblasts were evaluated. The degree of fibrosis of the ABP zone was evaluated in points: "0" - the absence of fibrous structures in all fields of the ABP (Fig. 1); "1" - weak accumulation of collagenic tender fibers, i.e. in the form of single fibrous structures in rare fields of view (Fig. 2); "2" is a moderate accumulation of collagen in the form of single coarse scattered fibrous structures in all fields of view (Fig. 3), and "3" is a pronounced, significant accumulation of fibers with visible collagen production by fibroblasts in the form of massive bundles in all fields of view with significant accumulation exudate and macrophage elements with their rough degradation or their absence in the ABP zone (Fig. 4).

 The criteria for a scoring of liver fibrosis according to liver ultrasound were: "0" - the absence of changes in the liver parenchyma; "1" - the accumulation of small focal structures of increased echogenicity, mainly in the periportal zone; "2" - uniform accumulation in the parenchyma of small focal different density structures up to 1/2 maximum; “3” - dense parenchyma due to mid-focal structures of high amplitude (up to 1 / 2-2 / 3 maximum). If there are signs of portal hypertension - cirrhosis.

 175 children aged 6 months to 14 years were examined. 53 of them had CHB, 40 had CGD and 82 had CGS.

 Comparison of AVR and ultrasound data in scores testified to their correlation with chronic hepatitis B in 72% of patients, with chronic hepatitis C in 76% and with chronic hepatitis C in 67.5% of children.

 AVR data were recorded more reliably in relation to fibrosis than the results of liver ultrasound in 24.7% of children with chronic hepatitis B, in patients with CGD in 22.6% and in chronic hepatitis C in 25.7% of patients.

 Less reliable than ultrasound, AVR data were found in 43% of patients with chronic hepatitis B, in 1.1% of patients with CGD, and in 6.7% of children with chronic hepatitis C.

 Fibrosis of the ABP zone was accompanied by a pronounced depression of migration activity, the level of 5-nucleotidase ectoenzyme and macrophage element esterase activity, while macrophage apoptosis phenomena correlated with the degree of fibroblastic direction of the inflammatory reaction. The indicated violations of the functional state of macrophages testified to the loss of their ability to control fibrogenesis.

 The presented results of fibrosis of the ABP zone in the skin in children with chronic hepatitis of viral etiology are a stereotypical “window” into the pathological process in the liver and can be used as an immunological criterion for diagnosing the degree of liver fibrosis in this disease.

Sources of information
Aruin L.I. Apoptosis and liver pathology. Russian Journal of Gastroenterology, Hepatology, Coloproctology, 1998, 2, p. 6-10.

 Mayansky D.N., Kutina S.N., Schwartz J.S. et al. Kupffer cell responsiveness in liver fibrosis / - In: Cells of hepatic sinusoid., v. 2 - Eds. Wisse E., Knook D., Decker K. - Kupffer cell found, Rijswijk, 1989, p. 88-90.

 Mayansky D.N. The mechanisms of chronic hepatitis. Morphofunctional features of the stroma of the liver. In the book: Chronic inflammation., 1991, p. 205-239.

 Mayansky D.N., Wisse E., Dekker K. et al. The New Frontiers of Hepatology, 1992, p. 200-210.

 Rebuck J. W., Crowley J. A. - Leukocytic functions in vivo. - Ann. N.Y. Acad Sci, 1955, v. 59, p. 757-794.

Claims (1)

 A method for the diagnosis of liver fibrosis in chronic hepatitis in children, characterized in that they carry out superficial scarification of the skin on the front surface of the forearm in the upper third, using the contact method, imprint on a sterile glass of exudate from the zone of aseptic inflammatory reaction (ABP) in the skin, the resulting imprint is dried, fixed ethanol, stained according to Romanovsky and microscopic, and in the absence of collagen fibers, the absence of liver fibrosis is diagnosed, with the accumulation of single fibers in rare fields fibrosis - mild fibrosis, with the accumulation of single coarse fibers in all fields of vision - moderate fibrosis and with a significant accumulation of fibers in all fields of vision in the form of bundles against the background of pronounced structural degradation of macrophages and the accumulation of fibroblasts, severe fibrosis or cirrhosis of the liver is diagnosed.

Images