RU2249433C2 - Method for diagnosing chronic pancreatitis cases developed on the background of duodenal peptic ulcer disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves determining clinical symptoms and pancreatic enzymes blood level. Enzyme level being in norm in blood serum, chronic pancreatitis is diagnosed by additionally determining deviations from reference level after intramuscularly introducing Immunophane 5 times and by detecting specific changes in patient immunity status.

EFFECT: high accuracy of diagnosis.

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Description

The present invention relates to medicine, in particular to gastroenterology and immunology, and may find application in the diagnosis of chronic pancreatitis (CP).

Statistical analysis shows that about 10% of the world's population suffers from peptic ulcer of the duodenum (ulcer of duodenal ulcer). The long, often recurring course of ulcerous duodenal ulcer in 63-100% of cases leads to irreversible changes in the structure and function of the pancreas, contributing to the development of secondary CP (Sukhova N.A., 1992, Kokueva O.V., 1998). Damage to the pancreas occurs with the participation of immunocompetent cells as a result of depletion of the reserves of the immune response and disruption of immunological tolerance in patients with ulcerous duodenum. The chronic pancreatitis that developed against the background of ulcer disease is forming a qualitatively new symptom complex that changes and aggravates the course of the underlying disease. It is possible to prevent the development of CP in patients with pulmonary ulcer disease when diagnosing it at a very early stage, long before the manifestation of functional disorders.

In the diagnosis of CP, an important role is played by the detection of hyperfermentemia, which is investigated by quantifying the content of amylase, lipase, and trypsin in the blood serum (Reference Medical Laboratory Technologies, St. Petersburg, 1999, v. 2, pp. 19-20, 39- 41, 20-21. Biochemical research methods in the clinic. M., 1969, pp. 186-191, 206-208. Boger MM, Methods for the study of the pancreas, Novosibirsk: Nauka, 1982, pp. 80-94) .

In clinical practice, the activity of amylase is most often investigated, since there is a specific substrate for this enzyme and its determination methods are simple. There are three main groups of methods for studying amylase in biological fluids:

1) reductometric, based on the determination of sugars formed from starch;

2) amyloclastic, based on the determination of the amount of unsplit starch by its reaction with iodine;

3) chromolytic, based on the use of substrate-dye complexes, which decompose under the action of amylase to form a water-soluble dye.

Lipase activity is determined in most methods based on titrometric determination of the amount of fatty acids released by the enzyme. These methods differ in the substrate used: olive oil, tween, terbutyrin. The photometric method for determining lipase involves the introduction of special reagents into the reaction mixture. A turbidimetric method is used as a unified method, in which olive oil is used as a substrate.

Trypsin is determined in blood serum by examining its activity according to Erlanger et al. in the modification of V.A. Shornikova. The method is based on trypsin cleavage of a colorless synthetic substrate-benzoylarginine-p-nitroanilide with the formation of colored p-nitroaniline, the amount of which is determined calimetrically.

However, it is often difficult to judge the presence and severity of CP by the activity of pancreatic enzymes in the blood, since the disease in combination with YAB DPK is often not accompanied by an increase in the activity of enzymes. In these cases, the methods used are not significant and CP may not be diagnosed in time, which will lead to the progression of the inflammatory and degenerative process in the pancreas with a violation of its functional activity, the manifestation of the disease and the development of complications due to the lack of timely pathogenetic treatment.

As an auxiliary method for the detection of CP, an immunological study of peripheral blood is used. The applied immunological methods are divided into 3 groups (Velbri S.K. Immunological diagnosis of pancreatic diseases, M., 1985):

1) the definition of normal and altered tissue antigens and individual constituent components of the pancreas in the blood and biological fluids as a sign of damage or impaired pancreatic function;

2) the determination of antibodies or sensitized lymphocytes against pancreatic antigens or its individual components, antigen-antibody complexes as indicators of the immune response. These indicators may be diagnostic criteria for the course of the disease;

3) determination of immunological parameters that do not directly indicate damage to the pancreas, but characterize the state of the immune system as a whole, the degree of its reactivity as the body's ability to withstand the development of the main pathological process, and its resistance to complications.

It is important that in CP, the level of pancreatic enzymes in the blood and urine usually rises later and normalizes before the pancreas antigen disappears from the blood. Therefore, the detection of the latter in blood serum is a highly specific method for the diagnosis of CP. But with a combination of CP with YAB DPK, the effectiveness of the method may be unsatisfactory, pancreatic antigen in this category of patients is detected only in 1 of 7 cases. The presence of antipancreatic antibodies is not specific for CP, because they can circulate in the blood with cancer, cystic fibrosis of the pancreas and be absent in severe CP and reduced patient reactivity, which, of course, reduces their diagnostic value.

In the work, we used a comprehensive study of the immune status of level II with an assessment of the state of the cellular, humoral, phagocytic parts of the immune system.

Test material: peripheral venous blood. Blood sampling is carried out under aseptic conditions from the vein of the elbow bend into sterile silicone tubes. In one of them, a heparin solution is preliminarily added at the rate of 10 PIECES / ml of blood, then 5 ml of venous blood is added. In another (dry) tube make 3 ml of blood.

Assessment of the state of the cell link is carried out using monoclonal antibodies to CD3, CD4, CD8, CD16, CD19 lymphocyte receptors.

The course of the study.

Heparinized blood is mixed in a volume ratio of 2: 1 with a 3% gelatin solution on medium 199, mixed and placed for 20-25 minutes in a thermostat at a temperature of + 37 ° C. After delamination, the top layer is carefully transferred with a Pasteur pipette to centrifuge tubes and centrifuged for 10 minutes at 1000 rpm in the cold. To remove red blood cells, the precipitate is resuspended in 1 ml in 1 ml of hemolytic buffer or in 0.5 ml of distilled water (exposure time 20-30 s), followed by dilution with a 20-fold volume of phosphate-buffered saline (PBS). Cells are pelleted by centrifugation at 400-430 rpm for 20 minutes in the cold and collected in an interphase containing lymphocytes using a Pasteur pipette. The resulting suspension of lymphocytes is washed three times in PBS.

The isolated lymphocytes are resuspended in the required volume of PBS, bringing their concentration to 1-2 million / ml, and 100 ml of cell suspensions are introduced into the U-shaped wells of a 96-well plate. The plates are centrifuged in the cold for 2 minutes at 1000 rpm, the supernatant is removed, and the pellet is resuspended in 100 μl of the required dilution monoclonal antibodies in phosphate buffered isotonic solution with 0.5% bovine serum albumin and 0.02% sodium azide. Cells are incubated with antibodies at + 4 ° C for 30 minutes with occasional gentle shaking. After the end of the incubation period, the cells are centrifuged at + 4 ° C for 2 minutes, the pellet is resuspended in 150-200 μl PBS or medium 199, precipitated, and subjected to repeated washing.

The obtained fixed samples are analyzed under a microscope in ultraviolet light. To evaluate the results of the reaction with a sufficient degree of reliability, it is necessary to count at least 200-300 cells in each sample.

Physiological standards for the relative content of lymphocytes accepted in the laboratory:

- CD3- 54-77%;

- CD4- 35-50%;

- CD8 - 18-25%;

- CD16-5-15%;

- CD19-3-13%.

The titer of immunoglobulins of classes A, M, G is calculated mathematically based on the data of radial immunodiffusion in agar gel according to the Mancini method.

The course of the study.

At a temperature of 55-56 ° C, agar is mixed with the corresponding antiserum in a ratio of 3: 1. Quickly pour the mixture into a glass Petri dish lying on a horizontal table. After the agar has solidified in it, 33 holes are knocked out using a punch, 5 ml of the test serum are added to each well with a Pasteur pipette, test serum with a known content of immunoglobulins is added in 4-fold dilutions. After this, the plates are placed in a moist environment in a strictly horizontal position for 24-48 hours at 4 ° C (for JgA and JgG - 24 hours, for JgM - 48 hours). After 24-48 hours, the reaction is taken into account. Under oblique illumination, the precipitation ring becomes visible and is measured with a ruler. According to the diameter of the ring around the wells with test serum, a calibration curve is built on semilogarithmic paper. When constructing it, the fact that in a certain range of concentration of immunoglobulins the diameter of the ring is directly proportional to the logarithm of the concentration of immunoglobulins is taken into account.

After measuring the diameter of the ring in the test samples, the concentration of immunoglobulin of the corresponding class in the test samples is estimated from the calibration curve.

The concentration is expressed in ME or in mg protein per 100 ml (mg%) or g / l (SI system).

Physiologically normal content of immunoglobulins:

- JgA - 0.7-3.0 g / l;

- JgG - 8.0-16.0 g / l;

- JgM - 0.8-2.5 g / l.

The serum level of the total fraction of circulating immune complexes (CEC) is estimated by the precipitation method with a 3.5% solution of polyethylene glycol (PEG).

Necessary materials and equipment:

- isotonic sodium chloride solution;

- 7% PEG solution;

- 0.1 n sodium hydroxide solution;

- centrifuge;

- spectrophotometer;

- centrifuge tubes;

- pipettes with a capacity of 1 ml and 5 ml.

Reagents

7% PEG solution: 7 g of PEG are dissolved in 100 ml of borate buffer (pH 8.4). Borate buffer is prepared by mixing 55 ml of a 1.24% solution of boric acid and 45 ml of a 1.9% solution of borax, adjusted to 200 ml with distilled water. 3.5% PEG solution: 20 ml of a 7% PEG solution and 20 ml of borate buffer. 0.1 n sodium hydroxide solution: 4 g of NaOH are diluted in 1 ml of distilled water.

Statement of the method.

Blood serum is diluted with isotonic sodium chloride in a ratio of 1:25 (0.1 ml + 2.5 ml). Diluted serum is mixed with 7% PEG in a ratio of 1: 1 (0.5 ml + 0.5 ml). For the precipitation reaction, the tubes are placed in a refrigerator at 4 ° C for 18 hours. The tubes are centrifuged for 15 minutes at 1500 rpm and the supernatant is removed. Next, the precipitate is dissolved in 2.5 ml of 0.1 N. NaOH solution, mix very carefully and leave at room temperature for at least 30 minutes. Samples are measured on a spectrophotometer, wavelength 280 nm against 0.1 N. NaOH solution. CEC levels are expressed in units of optical density. CEC levels determined by precipitation with a 3.5% PEG solution should normally not exceed 0.110 optical density units.

The absorption activity of neutrophilic granulocytes and the degree of completion of phagocytosis are investigated by reaction with a live culture of Staphylococcus aureus (strain 209).

The course of the study.

A drop of venous blood in an amount of 0.1 ml is placed on a clean fat-free coverslip. It is kept in a humid chamber at 37 ° C for 45 minutes, after which the resulting clot is carefully removed with tweezers. The coverslip with attached neutrophils is washed in a Hanks solution and the calculated dose of the daily Staf culture is applied. aureus and again placed in a humid chamber at 37 ° C for 30 minutes. After incubation, the glass is washed in a Hanks solution to remove unabsorbed particles and dried. The dried preparation is fixed and stained according to Romanovsky-Giemsa.

Accounting for phagocytic activity is carried out by determining the percentage of neutrophils with phagocytic material - phagocytic number - and the number of particles absorbed per one neutrophil - phagocytic index. In each preparation, at least 200 cells are counted. Physiological standards in the laboratory:

- absorption activity - 50-70%;

- phagocytic number - 2.2-4.3;

- phagocytic index - 1.4-2.5;

- digesting activity - 70-94%;

- digestion index - 1.3-2.0.

In order to diagnose the level of enzyme, we used:

I. Spectrophotometric determination of the change in turbidity of a suspension of olive oil under the action of lipase (Reference “Medical laboratory technology”, v.2, St. Petersburg, 1999, S. 39-41).

The most specific substrate for lipase is triomine (olive oil).

Determination of lipase activity according to Titz.

Under the influence of lipase, olive oil breaks down to form fatty acids, which are titrated with alkali. The amount of alkali spent on titration is used to judge lipase activity.

Reagents:

1. Olive oil emulsion: 93 ml of distilled water; 0.2 g of sodium benzoate and 7 ml of gum arabic are mixed well. To this mixture was added 93 mg of olive oil (slow). Then the mixture is emulsified for 10-15 minutes, first at low and then at high speeds.

2. 0.2 M Tris buffer with pH 8.0: 50 ml of a 0.4 M Tris-hydroxymethylaminomethane solution (48.4 g / L) was mixed with 26.8 ml of 0.4 N hydrochloric acid and bring the volume to 100 ml with distilled water.

3. Indicator: 1 g of thymolphthalein is dissolved in 100 ml of ethanol.

4. 0.05 n. a carbonate-free sodium hydroxide solution is titrated until a blue color appears. The difference in volume is 0.05 n. sodium hydroxide, spent on experience and control, is the activity of lipase in units of Titz.

Table 1 study progress experience the control buffer solution 1,0 1,0 olive oil emulsion 0.5 0.5 distilled water 2,5 2,5 blood serum 1,0 - mix, then incubate in a thermostat for 2 hours at 37 ° C transfer the contents of all tubes to Erlenmeyer flasks 50 ml ethanol 3.0 3.0 blood serum - 1,0 1% thymolphthalein solution 4 drops 4 drops titration of 0.05 N. sodium hydroxide

II. Determination of trypsin levels by photometric method. The method is based on the ability of trypsin to hydrolyze the chromogenic substrate N-α-tosyl-L-arginine-4-nitroanilide. The resulting 4-nitroanilide is determined photometrically.

Determination of trypsin activity according to Haverbeck-Erlanger.

Equipment:

- spectrophotometer, thermostat. Base reagents:

- tris;

- calcium chloride;

- dimethylformamide;

- 0.001 n. hydrochloric acid solution;

- nitroaniline 0.1 μM: 13.8 g of nitroanaline are diluted in 1 l of distilled water;

- potassium chloride 0.14 n .;

- benzoyl-arginine-para-nitroanaline (DAPNA);

- acetic acid, 30% solution.

Working solutions:

1. Tris buffer 0.2 M solution with a pH of 7.8: 24.23 g of Tris and 3.67 g of calcium chloride are diluted in 800 ml of distilled water, the pH is adjusted to 7.8 with 2 N. hydrochloric acid solution. Then the volume was adjusted to 1000 ml with distilled water.

2. Substrate solution (20 mg%): 2 mg of BAPNA are dissolved by gentle heating in 0.1 ml of dimethylformamide, then Tris buffer up to 10 ml is added.

3. 30% acetic acid solution.

Blood serum is diluted 1: 2 0.14 N. sodium chloride solution. The optical density of the liquid is determined on a spectrophotometer at a wavelength of 410 nm.

To calculate the concentration of para-nitroaniline, a calibration curve should be constructed. For a unit of activity of trypsin, one takes such activity in which 1 μM substrate per minute is cleaved. Trypsin activity is determined by the difference in the concentration of paranitroaniline in the experimental and control tubes. Given the time of incubation and dilution, the activity of trypsin in serum is calculated by the formula:

Eo is the extinction of experience;

Ek - extinction control;

And - the optical density of the factor obtained from the separation of the concentration of blood in the tubes of the calibration curve by their optical density.

table 2 study progress experience the control diluted blood serum 0.5 0.5 substrate 4.0 4.0 acetic acid solution - 1 thermostating for 30 minutes at 37 ° C acetic acid solution 1 - photometry

In healthy people, the trypsin activity in the blood is in the range of 0-4 honey / ml.

III. To study the activity of amylase as a prototype, we used a unified amyloclastic method with persistent starch substrate (Karavei method) (Reference Medical Laboratory Technologies, St. Petersburg, 1999, v.2, p.20-21). The principle of the method is that amylase hydrolyzes the degradation of starch with the formation of end products that do not give a color reaction with iodine. Amylase activity is judged by a decrease in color intensity.

Equipment:

- photoelectric colorimeter, water bath.

Reagents:

1. 2% starch solution: to 1 ml of cold distilled water add 200 mg of starch, stir it, then add 6-7 ml of hot distilled water and put in a boiling water bath until completely dissolved. After that, the volume of distilled water is adjusted to 10 ml. The substrate must be transparent.

2. Phosphate buffer solution with a pH of 7.1. To prepare it, 7.62 g of potassium hydrogen phosphate (KP ₂ PO ₄ ) and 20.45 g of sodium disodium phosphate (Na ₂ HPO ₄ ) are mixed, the volume is made up to 1000 ml with distilled water.

3. 3% solution of sodium chloride.

4. 0.1 n. iodine solution: 3 g of potassium iodide are added to 2.5 ml of distilled water, 1.2 g of crystalline iodine is added and the volume is adjusted with distilled water to 10 ml.

5.1 n. hydrochloric acid solution.

Table 3 study progress experience the control starch solution 5 5 buffer solution 3 3 salt solution 1 1 mix and heat for 10 minutes at 37 ° C blood serum 1 1 1 n hydrochloric acid solution - 2 mix and thermostat for 30 minutes at 37 ° C 1 n hydrochloric acid solution 2 - 0.2 ml from each experiment and control are added to the flask with iodine solution, mixed photometry

To prepare a working solution of iodine in a flask of 50 ml make 40 ml of distilled water, 0.5 ml of 1 N. hydrochloric acid solution and 0.1 N. iodine solution. The number of flasks should correspond to the number of tubes with experience and control. Photometry is performed in a 1 ml cuvette with a red light filter (wavelength 630-69 nmk) against water.

Ek - extinction control;

Eo is the extinction of experience;

And - the optical density of the factor obtained from the separation of the concentration of blood in the tubes of the calibration curve by their optical density. This factor reflects the concentration of para-nitroaniline corresponding to the unit of optical density.

A unit of amylase activity corresponds to 10 mg of split starch, subject to incubation for 30 minutes at 37 ° C. Normally, serum amylase activity is 80-120 units.

The main disadvantage of the methods used to study fermentemia lies in their low information content in patients with ulcerative duodenal ulcer complicated by CP, when the level of enzyme content corresponds to normal values.

The objective of the invention is to improve the accuracy and timeliness of the diagnosis of CP, which developed against the background of YAB duodenum.

The essence of the invention lies in the fact that with a normal content of pancreatic enzymes in the peripheral blood, CP is diagnosed by further determining the deviation of their level from physiological values and by the characteristic changes in the patient’s immunogram after five times intramuscular injection of 0.005% immunotropic drug 1 ml every other day into the patient.

The method is as follows. In a patient suffering from ulcerous duodenal ulcer, characteristic complaints of pain in the left hypochondrium, epigastrium, radiating to the back, weight loss, unstable stool, and nausea are revealed. An objective study reveals pain on palpation in the pancreatoduodenal zone, positive symptoms of Mayo-Robson and Kach. An instrumental study is performed, confirming the presence of ulcerative duodenum and signs of CP: esophagogastroduodenoscopy, ultrasound. Blood is taken from the patient's vein and the level of amylase, lipase and trypsin is determined, a comprehensive study of the immune status is performed. In the study of peripheral blood, data for hyperfermentemia were not obtained, indicators of the immune status correspond to physiological values, that is, there were no diagnostic signs of exacerbation of CP. Immunofan of 0.005% 1 ml intramuscularly is administered to the patient every other day No. 5. The drug has an immunostimulating effect, is able to increase antibody production in the first 2-3 days after administration and, due to the increased opsonizing ability of blood serum, stimulate the phagocytic function of neutrophils. In patients with autoimmune diseases, regardless of the activity of the process, the administration of the drug causes a short-term aggravation of immune inflammation. After 3-4 days, the patient is repeatedly taken blood from a peripheral vein to study the level of enzyme content and a comprehensive assessment of the immune status in dynamics. When hyperfermentemia is detected with respect to the normal and hyposuppressive type of the immune system (imbalance of T-lymphocyte subpopulations with prevailing helper cell content, deficiency of CD3 and CD8-lymphocytes and an increase in the immunoregulatory index; hyperimmunoglobulinemia of all major or individual classes of immunoglobulins and an increase in the level of circulation of immune systems functional activity of phagocytes and incomplete phagocytosis) are diagnosed with CP, which developed against the background of ulcerative duodenum.

Example 1

Patient S., medical history No. 4731.

She was admitted to the gastroenterological therapeutic department of the Krasnodar Regional Clinical Hospital with complaints of epigastric pain, nausea after eating, weakness.

From the anamnesis: I have ulcerative duodenal ulcer for about 20 years.

Objective upon admission: the abdomen is painful on palpation in the epigastrium, both hypochondria, along the projection of the pancreas, positive symptoms of Mayo-Robson and Kacha.

During instrumental and laboratory studies, it was revealed: on esophagogastroduodenoscopy, scar-ulcerative deformity of the duodenal bulb, catarrhal gastritis, bulbitis; indirect signs of the pathology of the pancreatic-biliary zone (the mucosa of the post-bulbar sections with teleimphoangioectasias).

Ultrasound examination: the size of the pancreas is within normal limits, its contours are uneven, ECHO genesis is increased, the structure is diffusely heterogeneous. Conclusion: ultrasound signs of CP.

In the study of the enzymatic activity of the pancreas, the indicators did not go beyond the upper limit of the norm.

In the immunogram, moderate T-cell deficiency due to CD4 lymphocytes, depression of the absorption activity of neutrophils. The content of immunoglobulins and CEC is within normal limits.

Five times the patient was administered intramuscularly 1 ml of a 0.005% solution of immunofan every other day. 3 days after the last injection, the levels of amylase, lipase, trypsin and the immunogram were examined again. The indicators are expressed in the following figures compared to the source data.

Table 4 test value baseline level after administration of immunofan Amylase 25.0 g / l 35.9 g l / h Trypsin 175.4 mmol / l 290 mmol / l Lipase 1.2 units 1.8 units CD3 lymphocytes 52% 49% CD4 lymphocytes 28% 35% SD8 lymphocytes 20% 14% immunoglobulin A 0.6 mg / ml 0.9 mg / ml immunoglobulin M 1.2 mg / ml 2.8 mg / ml immunoglobulin G 9.3 mg / ml 20 mg / ml CEC 0.06 units 0.201 units % phagocytosis 43% 54% % digestion 69% 51%

The data obtained made it possible to make a clinical diagnosis: CP, pain, recurrent course in the acute stage.

Cicatricial-ulcerative deformity of the duodenal bulb, gastritis, bulbitis.

The study confirmed the fact of the attachment of CP to ulcer of duodenum and made it possible to conduct adequate pathogenetic therapy in a timely manner.

Example 2

Patient M., medical history No. 1625.

I entered the gastroenterological therapeutic department of the Krasnodar Regional Clinical Hospital with complaints of pain in the epigastrium, left hypochondrium, radiating to the back, having a paroxysmal nature, heartburn, weight loss of 6 kg for 2 months.

From the anamnesis: YP duodenal ulcer suffers for 10 years. Repeatedly treated in a hospital. She was hospitalized in the clinic with a diagnosis of YAB duodenal ulcer in the stage of decaying exacerbation.

Objective data on admission: the abdomen is painful in the epigastrium, pancreatic-duodenal zone during palpation, positive symptoms of Mayo-Robson and Kacha.

The examination revealed the following pathological changes: on esophagogastroduodenoscopy, scar-ulcerative deformity of the duodenal bulb, chronic papillitis.

According to ultrasound, the heterogeneous, hyperechoic structure of the pancreas. Conclusion: ultrasound signs of CP.

The content of pancreatic enzymes is within normal limits.

In the immunogram, an imbalance of subpopulations of T-lymphocytes with a predominance of suppressors, a decrease in the suppressor index, hyperimmunoglobulinemia A, a moderate increase in the absorption activity of neutrophils and the content of the CEC.

The patient was injected intramuscularly five times with a 0.005% immunofan solution of 1 ml every other day. After 4 days, a repeated study of peripheral blood was performed.

The data obtained are shown in table 5.

Table 5 test value baseline level after administration of immunofan amylase 18.0 g / l / h 33 g l / h trypsin 205 mmol / l 245 mmol / l lipase 1.3 units 1.7 units CD3 lymphocytes 55% 52% CD4 lymphocytes 32% 33% SD8 lymphocytes 29% 18% immunoglobulin A 2.1 mg / ml 1.1 mg / ml immunoglobulin M 2.0 mg / ml 3.2 mg / ml immunoglobulin G 10.4 mg / ml 18 mg / ml CEC 0.160 units 0.290 units % phagocytosis 65% 60% % digestion 74% 53%

Diagnosed with CP, pain, recurrent course in the acute stage. YAB duodenum, cicatricial-ulcerative deformity of the duodenal bulb.

Thus, the proposed method simplifies, facilitates, and makes timely the diagnosis of CP that has developed against the background of ulcerous duodenal ulcer, which will allow timely adequate pathogenetic treatment, reduce the frequency of complications and exacerbation of both ulcerous duodenal ulcer and chronic kidney disease, reduce the frequency of repeated hospitalizations and the length of stay of patients in in a hospital.

Claims (1)

A method for the diagnosis of chronic pancreatitis that developed against the background of duodenal ulcer, including the determination of clinical signs and the level of pancreatic enzymes in the blood, characterized in that with normal serum enzymes, chronic pancreatitis is diagnosed by additionally determining deviations from the normal content of their level after 5 -fold intramuscular injection of immunofan to the patient and according to characteristic changes in the indicators of the patient's immune status.