RU2090887C1 - Method for diagnosing chronic hepatitis in children - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves determining functional state of skin macrophages by applying histochemical examination method. The cells are incubated with adenosine-5-monophosphate, stained with ammonium or sodium sulfide for detecting 5-nucleotidase. Its contents in cellular cytoplasmic membrane being equal to 11.3 ± 1.7, chronic persistent hepatitis is diagnosed. Its contents in cellular cytoplasmic membrane being equal to 5.3 + 0.4, chronic active hepatitis is diagnosed. EFFECT: enhanced accuracy of diagnosis. 4 dwg

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 you to establish the severity of the process and control its dynamics during treatment.

 The known method / 1 analogue /, which allows using the described complex of pathological symptoms or clinic / Malakhovsky Yu.E. in the book. under. ed. N.I. Nisevich, Len. "Medicine", 1981. "Diseases of the liver and biliary tract in children," p. 249,271; Golzand I.V. in the book. "Successes of hepatology", Riga, 1977 6, p. 245,256; ch. "The course of chronic hepatitis in children" / follow the dynamics of the disease.

 Of the subjective symptoms, the following are taken into account: 1 / dyspeptic complaints: loss of appetite, belching, poor fat tolerance, pain in the right hypochondrium and epigastrium, flatulence, unstable stools; 2 / complaints of an astheno-vegetative nature: general weakness, fatigue, irritability, sweating, headaches. However, in young children / up to 10 years / subjective signs are rarely detected.

 Of the objective symptoms of chronic hepatitis, hepatomegaly is usually detected. The liver is characterized by a dense, smooth, sensitive, often enlarged due to the left lobe.

 The spleen in 50 60% of cases in patients protrudes from the costal arch by 1 3 cm. But more often pronounced splenomegaly is determined in children under 5 years of age in cases of aggressive hepatitis.

 Jaundice of varying intensity and duration is found in patients / in 25–40% / with chronically active hepatitis and, as a rule, is absent in inactive forms.

 A number of patients have "hepatic palms", single telangiectasias, hemorrhages / in the form of rare nosebleeds, petechiae or ecchymoses /.

 It should be noted that the clinical manifestations of chronic hepatitis are very variable, individual, and the disadvantage of this method, excluded by the claimed method, is the factor of subjectivity in the doctor’s assessment of clinical symptoms.

The known method / 2 analogue / biochemical functional tests, widely used in the examination of patients with chronic hepatitis / see Todorov I. "Clinical laboratory research in pediatrics" Sofia, 1966 Blyuger A. F. "Viral hepatitis and its outcomes", Riga, 1975 / It uses the following methods: 1 / modernized Iendrassik-Kleghorn method for quantitative determination of the content bound bilirubin to the total in Normally, the bilirubin content does not exceed 1.05 mg% / Predtechensky V.K. "Guidelines for clinical and laboratory research", Medgiz, 1960 /; 2 / a method for determining the activity of aminotransferases in blood serum / Umbreitefal, 1957 / is based on the transamination reaction between alanine / for the determination of AlAT / and α ketoglutaric acid. Determined by dinitrophenylhydrazine, pyruvic acid, which is calorimetrically formed as a result of these reactions, is determined; normal AlAT activity is 5 units. AsAT up to 40 units. 3 / widespread biuret and refractometric methods for determining the total protein, which are based on the ability of proteins to form a complex compound of purple color / cm with copper sulfite in an alkaline medium. Predtechensky V.E. 1960 /; normally, the total protein content is 5.7 ± 0.7%. To determine the content of protein fractions in blood serum, there is a method of electrophoresis on paper according to Grassman. Hamming as modified by A. G. Gurevich, 1955. Normally, in the blood serum, using this method, usually 5 fractions are usually isolated: albumin 59.7 ± 4.5% a ₁ globulins 3.8 ± 1.8% α ₂ globulins 6 , 9 ± 2.3% β globulins 9.7 ± 3% and g globulins 19.7 ± 4% The content of total cholesterol in blood serum is determined by the known method of Engelhard and Smirnova. The principle is based on the dissolution of cholesterol in chloroform, which then gives a green color with acetic anhydride and acetic acid. Its normal content is 180-250 mg%. Determination of the prothrombin complex factors / Quick method / is based on determining the time of formation of a blood plasma clot when an excess of thromboplastin and the optimal amount of calcium are added to the plasma / Todorov I. 1966 /. Normally, his indicators range from 70-110%
The disadvantages of this method, excluded by the claimed method, include: the need for blood sampling from a vein in large quantities, multi-stage techniques, the need for sophisticated equipment with a large number of reagents, special training of personnel. In addition, the known method gives an unlikely analysis and prognosis of the disease. The listed methods are schematic and only approximately give an idea of the pathological process in the liver, in which the main reaction is pathogenetically the immune system reaction.

 The most accurate information about the state of the pathological process in chronic hepatitis can be obtained using intravital morphological examination of liver tissue, mainly by puncture biopsy of the liver [1] A.F. Blyuger "Viral hepatitis and its outcomes", Riga, 1970 p.272-279.

 After a thorough examination, to avoid contraindications, the patient determines the number of platelets, blood coagulation, bleeding time, prothrombin complex. The operation of a puncture biopsy in children is as follows / I.I. Bogomazov "Puncture biopsy of the liver in children", diss. Cand. 1965 /. The child is placed on the operating table in a supine position; he is given mask anesthesia and muscle relaxants are administered parenterally, which disables respiratory movements at the time of liver puncture. During the apnea period, a child is punctured in the 9th intercostal space on the right in the mid-axillary line and a puncture needle is inserted into the resulting hole and a column of liver tissue is removed. Immediately taken a piece of the liver should be placed in a fixative solution / 10% formalin or Lilly fixative / and delivered immediately to the pathological department, where further processing of the biopsy material is carried out. To do this, the resulting piece of liver is carried out through alcohols, poured into paraffin. Then, tissue sections are prepared from the paraffin block, dewaxed and sequentially stained with standard dyes / hematoxylin-eosin according to Van Gieson, etc. /. At the last stage, a specialist pathologist reviews the preparations and gives an opinion on the state of the pathological process in the liver.

 The disadvantages of this method, which is 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 specially trained highly qualified personnel: a surgeon and an anesthetist. This may be available only to individual central clinics of the country. In addition, the puncture of the liver is an operative measure and, given the given anesthesia, is regarded as an unsafe manipulation. Known complications are associated with liver biopsy, which in rare cases can occur: bleeding from the puncture site, infection of the pleura and abdominal cavity, damage to neighboring organs. Therefore, after a puncture, the patient is in bed for 24 hours with mandatory monitoring of the general condition, blood pressure and pulse. It should be remembered that during puncture, it is not always possible to obtain liver tissue, and there may be inconclusive puncture, and in 2-15% "dry" puncture is also possible (A. F. Bluger "Viral hepatitis and its outcomes", Riga, 1970 p. 272 279). Finally, you should consider the significant length of time from the moment of puncture to obtain the morphological conclusion of a specialist pathologist / it ranges from 3 to 10 days /. An important disadvantage is the impossibility of conducting this study with the necessary frequency in the dynamics during treatment.

 The main objective / purpose / of the claimed invention is a dynamic assessment of the severity of the pathological process in chronic hepatitis in children during therapy.

 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 /.

The solution to this problem is achieved (in contrast to the prototype) by the fact that the proposed method excludes anesthesia, punctate collection, histological processing and punctate assessment, and surface skin scarification is performed on the front surface of the forearm in the upper third pre-treated with ethyl alcohol. A 0.5 x 0.5 cm section of the epidermis is removed with a scarifier 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 cell exudate on glass is dried and fixed in formalin vapor, then placed in an incubation medium for 30 minutes at a temperature of 37 ^o C, consisting of adenosine-5-monophosphate / 30 mg /, 0.1 M acetate buffer pH 5.6; 0.1 M solution of lead nitrate / 1 ml /. After incubation and washing, the drug is placed for 2 min in a 5% solution of ammonium sulfide or sodium. After washing and drying, it is microscopically immersed. In the fields of vision, macrophage cells with a brown or black color of the cytoplasmic membrane are fixed, indicating the fixation of the activity of the 5-nucleotidase ectoenzyme, which reflects the activation state of macrophages normally functioning in the inflammation zone. Normally, the average number of macrophages saturated with this ectoenzyme is 23 ± 5.4 positively stained cells.

 The claimed method examined 89 children aged 1 year to 14 years suffering from chronic hepatitis. 2 groups of patients with significantly different contents of macrophages saturated with 5-nucleotidase were allocated. Each group corresponded to a specific variant of the inflammatory process in the liver according to puncture biopsy of the liver.

 In 1 group of patients / 20 people /, mainly with chronic persistent hepatitis / CPG / and persistent release of HBsAg, or anti-HCV / markers of hepatitis B or C viruses /, with low symptomness of the clinical picture, the content of macrophages in the area of aseptic inflammatory reaction in skin / ABP / with activity in their cytoplasmic membrane 5-nucleotidase averaged 11.3 ± 1.7 / positively stained cells /.

 In punctate of the liver, moderate infiltrative and dystrophic manifestations without significant destructive changes in the liver parenchyma were characteristic of this group of patients. There was moderate fibrosis in the region of the portal tracts, combined with moderate lymphocytic and macrophage infiltration.

 Thus, in patients with sluggish chronic hepatitis a moderate decrease in 5-nucleotidase activity was detected, which, in turn, testified to the functional inferiority of these cells in the mechanisms of elimination of viral antigens and the transfer of antigenic information to other immunocompetent cells.

 In the second group of patients (69 children) with chronic active hepatitis, in which the clinical manifestations of the disease were more manifest, which was expressed by the presence of hepatolienal syndrome in all patients, and astheno-vegetative and hemorrhagic syndromes were noted in 40-50%. In addition, there were extrahepatic manifestations in the form of palmar erythema, telangiectasias, and in biochemical tests hyperbilirubinemia, hyperfermentemia and dysproteinemia.

 In this group of patients, a deeper inhibition of the functional activity of macrophages was recorded, which was expressed by a decrease in their number to 5.3 ± 0.4 with positive staining for 5-nucleotidase. A distinctive morphological variant of the liver punctures of this group of children was that they were characterized by destructive phenomena with destruction of the border plate and the accumulation of significant periportal infiltrates from lymphohistiocytic and macrophage infiltrates with the presence of plasma cells and active fibroblasts; pronounced was the growth of connective tissue in the portal tracts with its introduction with cellular infiltrates into the lobules. In some children with high activity of the process, stepwise necrosis was revealed in a large number.

 Thus, this group was characterized by parallelism between the depth of the pathological process in the liver with the degree of inhibition of functional activity and degradation of macrophage cells.

 Example: Patient Gudkov A, 8 years old; medical history N 656; from 1 normally proceeding pregnancy and childbirth, weighing 3 kg; from 6 months He received multiple parenteral manipulations with the introduction of blood products in connection with pneumonia, and from 2.5 years in connection with a nephrectomy and several chemotherapy courses for nephroblastoma. From the age of 7 there were complaints of pain in the epigastric region and the right hypochondrium, which tended to intensify for six months. When examined in the diagnostic center of the RCCH diagnosed with gastroduodenitis, pancreatitis, dysbiosis; in addition, against the background of hepatomegaly, hyperfermentemia was detected and in the ELISA method, hepatitis B and C / HBsAg, HBeAg, anti-HCV / markers were detected. To clarify the nature of liver damage and resolve the issue of treatment tactics, he entered our clinic / department of pediatric infections, Russian State Medical University /. The condition at receipt of moderate severity, manifestations of astheno-vegetative syndrome, extrahepatic signs in the form of palmar erythema and ecchymosis are expressed. Soreness in the epigastric region, the liver is enlarged to 2x2xv / 3 below the costal margin and tightened, the spleen was palpated 1 cm below the costal margin. According to abdominal ultrasound, the liver is diverse and significantly densified in the periportal zone. Gall bladder with constriction and S-shaped deformation. Enlarged spleen. In blood biochemistry from 29.5.92 / at admission /: protein total. 71 g / l, AlAT 155 units. AsAT 83 units. / with a norm of up to 40 units /, cholesterol 4.17 mmol / l / norm of 3.7 6.5 /.

In immunological status: from 2 / 6-93 g. OCT ₃ -38% OCT ₄ - 19% OCT ₈ 18% / normal /; in the AVR zone of macrophages 0% / chemotaxis /, therefore, the determination of 5-nucleotidase was impossible.

 In blood tests, urine tests for other organs without pathology.

 Puncture biopsy of the liver N445 from 25 / 6-92, chronic active hepatitis of moderate activity.

 Given the activity of the process in the liver, as well as the reproductive phase of HB-viral and HC-viral infections, a course of reaferon in the form of suppositories was prescribed for 3 months. which has, in addition to the antiviral effect, an immunomodulatory effect. 3 months later during the follow-up examination: no complaints, the liver at the costal margin became less dense; the spleen is not enlarged. In a biochemical analysis of blood / from 2 / 11-92 g / AlAT and AsAT 90 units. / normal to 40 units /, i.e., data from a clinical examination and functional liver tests indicated the onset of incomplete remission of the process. This period was characterized by an improvement in the functional state of macrophages, characterized by an improvement in the migratory activity of these cells (chemotaxis was 29.6% at a rate of 75%), and 6 cells saturated with 5-nucleotidase were determined in the fields of view. Subsequently, after 6 months. after treatment, HBeAg seroconversion to anti-HBe occurred, indicating an improvement in the phagocytic activity of these cells and the inclusion of mechanisms for the elimination of viral antigens. This example demonstrates the value of the proposed method both in assessing and predicting the depth of the pathological process in the liver in chronic hepatitis, and in assessing the effect of antiviral and immunomodulating therapy (reaferon).

 A positive effect of the application of the claimed method for a patient is that monitoring the dynamics of the severity of the pathological process in the liver allows you to set certain periods of therapy, thereby reducing the rehabilitation time, eliminating the side effects of therapy and stopping the progression of the pathological process in the liver in a timely manner, ahead of the results of repeated liver biopsy (prototype), which for a patient is traumatic and life-threatening than the claimed method.

 The disadvantages of the clinical assessment of the current pathological process (1 analogue), excluded by the claimed method, are low-symptom in a number of cases (both with chronic hepatitis C and with chronic hepatitis C), as well as the subjectivity of the doctor in assessing the symptoms.

 Biochemical methods for assessing the current pathological process / 2 analogue / do not give, in comparison with the claimed method, a sufficient duration of the forecast / t.k. only mild cytolysis syndrome is recorded during a period of high activity of the pathological process directly in the liver. In addition, indicators of biochemical tests may be normal even with an actively ongoing process in the liver in patients with chronic hepatitis.

 Basic immunological method for assessing the functional state of the liver / T. V. Cherednichenko, Bull. N 44 UDC 616.36.002.1.059.31 / 088.8 obl. November 30, 1995 / where the depth of the pathological process in the liver is judged by the level of reduction of macrophage chemotaxis in the area of aseptic inflammation in the skin, in some cases (even with cirrhosis) this indicator remains normal. At the same time, in the claimed method, against the background of normal indicators of macrophage chemotaxis, a sharp inhibition of 5-nucleotidase activity in the cytoplasmic membrane of these cells is detected, which correlates with the depth of the pathological process in the liver. Thus, a significant difference between the claimed method from the basic immunological is its higher accuracy.

Claims (1)

 A method for diagnosing chronic hepatitis in children by examining cells, characterized in that the preparation of skin macrophage cells is obtained by contact, incubated with adenosine-5-monophosphate, stained with ammonium or sodium sulfide on 5-nucleotidase, by the presence of which the functional the state of the macrophage immunity link and its content in 11.3 ± 1.7 cells is diagnosed as chronic persistent hepatitis, and in 5.3 ± 0.4 cells as chronic active hepatitis.