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INVENTION
Russian Federation Patent RU2249433

A method for diagnosing chronic pancreatitis, except
THE BACKGROUND duodenal ulcer

Name of the inventor: Kokueva OV (RU); Romanov JL (RU); Mihayloshina EV (RU); Cymbalyuk YM
The name of the patentee: Rossysky Center for Functional Surgical Gastroenterology (RU); Kokueva Olga (RU); Jeanne L. Romanova (RU); Mihayloshina Elena (RU); Cymbalyuk Yuri Mikhailovich
Address for correspondence: 350063, Krasnodar, ul. Sedin, 4, RTSFHG, Head. Patent Department TA Doroninoj
Starting date of the patent: 2002.04.15

The invention relates to medicine, in particular to gastroenterology. The method improves the accuracy of diagnosis of chronic pancreatitis that developed on the background of duodenal ulcer, and moreover, it is easy to perform. Carry out the clinical signs and levels of pancreatic enzymes in the blood, while with normal serum enzymes chronic pancreatitis is diagnosed by further determining the deviation from the normal content of their levels after 5-fold intramuscular patient imunofana and characteristic changes of immune status of the patient.

DESCRIPTION OF THE INVENTION

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

Statistical analysis shows that duodenal ulcer (duodenal ulcer) suffer about 10% of the population in the world. Long, often relapsing course of duodenal ulcer in 63-100% of cases leads to irreversible changes in pancreatic structure and function, contributing to the development of the secondary CP (Sukhov NA, 1992, Kokueva OV, 1998). The defeat of the pancreas is mediated by immune cells as a result of the depletion of the reserves of the immune response and breakdown of immunological tolerance in patients with duodenal ulcer. Develop on the background of chronic pancreatitis duodenal ulcer produces a new symptom, and changing the weighting for the underlying disease. To prevent the development of CP in patients with duodenal ulcer may at diagnosing it at an early stage, long before the manifestation of functional disorders.

In the diagnosis of CP plays an important role revealing hyperenzymemia, which was investigated by the quantitative determination of serum amylase blood lipase, trypsin (Reference "Medical Laboratory Technology", St. Petersburg, 1999, Volume 2, str.19-20, 39- ... 41, 20-21, Biochemical methods of investigation in the clinic Moscow, 1969, str.186-191, Boger 206-208 MM, research methods pancreas, Novosibirsk: Nauka, 1982, str.80-94) .

In clinical practice often examined amylase activity, since there is a specific substrate, and determining its uncomplicated methods for this enzyme. There are three main groups of methods of amylase in biological fluids study:

1) reduktometricheskie based on the determination of the starch produced from sugars;

2) amiloklasticheskie based on the determination of the amount of non-cleavage of starch by reaction with iodine;

3) hromoliticheskie based on the use of the dye-substrate complexes that decompose under the action of amylase to form water-soluble dye.

The lipase activity was determined in most titrimetric methods based on determining the amount of the liberated fatty acid by the action of the enzyme. These methods differ in the substrate used: olive oil, Tween, terbutirin. The photometric method for determining lipase associated with the introduction of special reaction reagent. As used unified turbidimetric method in which used as a substrate olive oil.

Trypsin was determined in serum by investigating its activity Erlanger et al. modification VA Shornikova. The method is based on cleavage with trypsin colorless synthetic substrate benzoilarginin-p-nitroanilide forming colored p-nitroaniline, whose number is determined kalibrimetricheski.

However, the activity of pancreatic enzymes in the blood is often difficult to judge the presence and severity of CP, because the disease combined with duodenal ulcer is often not accompanied by an increase in the activity of enzymes. In these cases, the methods used have no significant value and HP may be not diagnosed in time, which will cause the progression of inflammatory and degenerative processes in the pancreas with infringement of its functional activity, the manifestation of the disease and complications due to lack of timely pathogenetic treatment.

As a subsidiary of the method used to identify the HP immunological study of peripheral blood. Applied immunological methods are divided into 3 groups (Velbri SK Immunological diagnosis of pancreatic diseases, M., 1985):

1) Determination of normal tissues and modified antigens of individual components and blood components in the pancreas and body fluids as an indication of damage or disorder of the pancreas;

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

3) determination of immunological parameters, not pointing directly to the defeat of the pancreas, but characterizing the state of the immune system as a whole, the degree of reactivity as the body's ability to resist the development of the main pathological process and its resistance to complications.

Important is the fact that when KP level of pancreatic enzymes in the blood and urine usually rises after normalization and before pancreas antigen disappears from the blood. Therefore, the identification of the latter in the blood serum is a highly specific method for diagnosis of CP. But when combined with HP UDD method performance may be unsatisfactory because pancreatic antigen in these patients detected in only 1 of 7 cases. Availability antipankreaticheskih antibody is not specific to HP, because they can circulate in the blood for cancer, cystic fibrosis of the pancreas and absent in severe CP and reduced reactivity of the patient, which naturally reduces their diagnostic value.

In this paper we have used a comprehensive study of the immune status of a Level II assessment of the state of cellular, humoral, phagocytic parts of the immune system.

Test material: peripheral venous blood. Taking blood is produced in aseptic conditions from a vein in the elbow sterile siliconized tubes. In one of them pre-made by calculation of the heparin solution 10 U / ml of blood, and then 5 ml of venous blood. In another (dry) make 3 ml vial of blood.

Assessment of cell component produced with the use of monoclonal antibodies against CD3-, CD4-, CD8-, CD16-, CD19-lymphocyte receptors.

Progress in research

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

The isolated lymphocytes were resuspended in the required volume PBS, bringing them up to a concentration of 1-2 million / ml and introduced 100 ml of cell suspensions in U-shaped 96-well plate. Plates were centrifuged in the cold for 2 minutes at 1000 rev / min, the supernatant removed and the pellet was resuspended in 100 ul of dilutions of monoclonal antibodies to the desired isotonic phosphate buffered solution with 0.5% bovine serum albumin and 0.02% sodium azide. Cells were incubated with antibodies at 4 ° C for 30 minutes with occasional gentle shaking. After expiration of the incubation cells were centrifuged at 4 ° C for 2 minutes, the pellet was resuspended in 150-200 ul of PBS or media 199 is deposited, washing of reextracted.

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

Physiological norm of the relative content of lymphocytes taken in the laboratory:

- CD3- 54-77%;

- CD4- 35-50%;

- CD8- 18-25%;

- CD16- 5-15%;

- CD19- 3-13%.

Titer immunoglobulin classes A, M, G calculated mathematically based on the data in the radial immunodiffusion according to Mancini agar gel method.

Progress in research

At a temperature of 55-56 ° C is mixed with agar appropriate antiserum in a ratio of 3: 1. Quickly the mixture was poured into a glass Petri dish, lying on a horizontal table. After solidification of agar stenciled therein emboss punch 33 wells, each well Pasteur pipetted 5 ml of the test serum in medium was added to four wells of a 4-fold dilutions of test serum with known immunoglobulins. The plates placed in a moist environment in a strictly horizontal position for 24-48 hours at 4 ° C (and for JgA JgG - 24 hours for JgM - 48 hours). After 24-48 hours the reaction into account. With oblique illumination precipitation ring becomes visible and measured by a ruler. Diameter rings around the wells with the test serum calibration curve on semilogarithmic paper. When its construction take into account the fact that in a certain range of concentrations of immunoglobulins diameter of the ring is directly proportional to the logarithm of the concentration of immunoglobulins.

After measuring the diameter of the ring in the test samples from the calibration curve estimate the concentration of the relevant class of immunoglobulin in the test samples.

Concentration expressed as ME or mg protein per 100ml (mg%) or g / L (the SI system).

Physiologically normal immunoglobulin content:

- JgA - 0,7-3,0 g / l;

- JgG - 8,0-16,0 g / l;

- JgM - 0,8-2,5 g / l.

The level of serum total fraction of circulating immune complexes (CIC) was assessed by precipitation with a solution of 3.5% polyethylene glycol (PEG).

Materials and Equipment:

- Isotonic sodium chloride solution;

- 7% PEG solution;

- 0.1N. sodium hydroxide solution;

- Centrifuge;

- Spectrophotometer;

- Centrifuge tubes;

- Pipette 1 ml and 5 ml.

Reagents.

7% solution PEG 7 g PEG dissolved 100 ml of borate buffer (pH 8.4). The borate buffer was prepared by mixing 55 ml of 1.24% boric acid solution and 1.9 ml of 45% borax solution was adjusted to 200 ml with distilled water. 3.5% PEG solution: 20 ml of 7% PEG solution and 20 mL of borate buffer. 0.1N. Sodium hydroxide solution: 4 g NaOH diluted in 1 ml of distilled water.

Production method

The serum was diluted with isotonic sodium chloride solution at a ratio of 1:25 (0.1 mL + 2.5 mL). The diluted serum was mixed with 7% PEG solution in a ratio of 1: 1 (0.5 ml + 0.5 ml). For carrying out the precipitation reaction tubes placed in a refrigerator at 4 ° C for 18 hours. The tubes were centrifuged for 15 minutes at 1500 rev / min, and the supernatant removed. Next, the precipitate was dissolved in 2.5 mL of 0.1N. NaOH solution, stirred thoroughly and left at room temperature for at least 30 minutes. The samples is measured with a spectrophotometer, a wavelength of 280 nm against 0.1N. NaOH solution. CEC levels expressed in optical density units. CEC level determined by precipitation of 3.5% solution of PEG, normally not to exceed 0,110 units of optical density.

Absorbing activity of neutrophils and the completeness of phagocytosis was examined by reaction with the vibrant culture of Staphylococcus aureus (strain 209).

Progress in research

venous blood drop in the amount of 0.1 ml is placed on a clean skim the cover glass. Its kept in a moist chamber at 37 ° C for 45 minutes, after which the clot was carefully removed with forceps. The cover glass with attached neutrophils were washed in Hanks' solution, causing the calculated daily dose Staf culture. aureus and again placed in a moist chamber at 37 ° C for 30 minutes. After incubation, the slide is washed in Hanks solution to remove non-absorbed particles and dried. The dried drug fix and paint Romanovsky-Giemsa.

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

- Absorbing activity - 50-70%;

- Phagocytic number - 2,2-4,3;

- Phagocytic index - 1.4-2.5;

- Digestive activity - 70-94%;

- Digestion index - 1.3-2.0.

With a view to the diagnosis of enzyme levels we used:

I. The spectrophotometric determination of the change in turbidity of olive oil suspension under the action of lipase (Reference "Medical Laboratory Technology", Volume 2, St. Petersburg, 1999, s.39-41).

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

Determination of lipase activity Tietze.

Under the influence of lipase olive oil is split into fatty acids, which are titrated with alkali. According to the number who went to the titration of alkali judged on lipase activity.

Reagents:

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

2. 0.2 M Tris pH 8.0 buffer: 50 mL 0.4 M Tris gidroksimetilaminometana solution (48.4 g / l) was mixed with 26.8 ml of 0.4 n. hydrochloric acid and the volume was adjusted to 100 ml with distilled water.

3. Indicator: 1 g of Thymolphthalein was dissolved in 100 ml of ethanol.

4. 0.05 n. sodium hydroxide solution containing no carbonates, titrated until the blue color. The difference in the amount of 0.05N. sodium hydroxide consumed on the experience and control of the lipase activity in the Tietz units.

II. Determining the level of trypsin photometric method. The method is based on the ability of trypsin to hydrolyze the chromogenic substrate N- a -tozil-L-arginine-4-nitroanilide. The resulting 4-nitroanilide is determined photometrically.

Determination of trypsin activity Heverbeku-Erlanger.

Equipment:

- Spectrophotometer, thermostat. Basic reagents:

- Tris;

- calcium chloride;

- Dimethylformamide;

- 0,001 BC. hydrochloric acid;

- 0.1 uM nitroaniline: 13.8 g nitroaniline diluted in 1 liter of distilled water;

- Potassium chloride 0.14 N .;

- Benzoyl-arginine-p-nitroaniline (BAPNA);

- Acetic acid, 30% solution.

Working solutions:

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

2. The substrate solution (20 mg%) 2 mg BAPNA dissolved with gentle warming in 0.1 ml of dimethylformamide, then added to Tris buffer 10 ml.

3. 30% acetic acid solution.

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

To calculate the concentration of p-nitroaniline follows construct a calibration curve. One unit of trypsin activity is defined such activity in which 1 mM of substrate cleaved per minute. Trypsin activity is determined by the difference in concentration of para-nitroaniline test and control tubes. Given the incubation time and dilution of trypsin activity in blood serum is calculated by the formula:

Eo - the extinction of experience;

Ek - extinction control;

A - Optical density factor obtained by dividing the concentration of blood in the tubes of the calibration curve for absorbance.

In healthy people, the trypsin activity in the blood is in the range 0-4 Med. / Ml.

III. To investigate the activity of amylase as a prototype we used a standardized method amiloklastichesky with resistant starch substrate (method Karaveya) (Reference "Medical Laboratory Technology", St. Petersburg, 1999, Volume 2, s.20-21). The principle of the method is that the splitting amylase hydrolyzes starch to form the final product does not give a color reaction with iodine. The activity of amylase judge to reduce color intensity.

Equipment:

- Fotoelektrokolorimert water bath.

Reagents:

1. 2% starch solution: to 1 ml of cold distilled water were added 200 mg of starch, stir it, and then topped up with 7.6 ml of hot distilled water and put in a boiling water bath until dissolved. Thereafter the volume was adjusted with distilled water to 10 ml. The substrate must be transparent.

2. The phosphate buffered saline pH 7.1. For its preparation was mixed 7.62 g of potassium dihydrogen phosphate (KP ₂ PO ₄₎ and 20.45 g of disodium hydrogen phosphate (Na ₂ HPO _4), the volume was adjusted with distilled water to 1000 ml.

3. 3% sodium chloride solution.

4. 0.1N. Iodine solution: 3 g of potassium iodide were introduced into 2.5 ml of distilled water, 1.2 g of crystalline iodine and volume adjusted with distilled water to 10 ml.

5. 1N. hydrochloric acid solution.

To prepare the working solution in the iodine flask is made 50 ml with 40 ml of distilled water, 0.5 ml of 1N. hydrochloric acid solution and 0.1N. iodine solution. Number of flasks must match the number of tubes with experience and control. Photometry is performed in cuvette 1 ml with a red filter (wavelength 630-69 NMC) against water.

Ek - extinction control;

Eo - the extinction of experience;

A - Optical density factor obtained by dividing the concentration of blood in the tubes of the calibration curve for absorbance. This factor reflects the concentration of para-nitroaniline corresponding one of the optical density.

Unit amylase activity corresponding to 10 mg of starch digested provided incubation for 30 minutes at 37 ° C. Normally amylase activity in blood serum is 80-120 units.

The main drawback of the methods used in the study of enzyme concluded their low information content in patients with duodenal ulcer complicated by HP, when the level of enzyme corresponds to the normal range.

Object of the invention is to improve the accuracy and timeliness of diagnosis of CP that developed on the background of duodenal ulcer.

The essence of the invention is that with normal pancreatic enzyme in peripheral blood HP diagnosed by further determining the deviation of the level from physiological values ​​and parameters characteristic changes of the patient after fivefold immunogram intramuscular patient immunotropic preparation imunofana 0.005% to 1 mL of a day.

The process is as follows. A patient suffering from duodenal ulcer, identify specific complaints of pain in the left upper quadrant, epigastric pain, radiating to the back, weight loss, unstable stool, nausea. Objective examination reveals tenderness in pancreatoduodenal zone, positive symptoms Mayo-Robson and Kacha. Produce instrumental study, confirming the presence of duodenal ulcer and features HP: esophagogastroduodenoscopy, ultrasonic examination. exercise blood samples from diseased veins and determine the level of amylase, lipase and trypsin, perform a comprehensive study of immune status. When peripheral blood analysis data for hyperfermentemia received, indicators of immune status correspond to physiological values, ie diagnostic HP acute symptoms have been identified. The patient is administered imunofan 0.005% to 1 mL in a day intramuscularly № 5. The drug has immunostimulating effect capable in the first 2-3 days after injection and to increase the production of antibodies by elevated serum opsonizing ability to stimulate phagocytic neutrophils. Patients with autoimmune diseases, regardless of the activity process of administration of the drug causes a transient exacerbation of immune inflammation. After 3-4 days, the patient re-take blood from a peripheral vein to study the levels of enzymes and comprehensive assessment of the immune status dynamics. In identifying hyperenzymemia respect to the norm and giposupressornogo type of the immune system (the imbalance subpopulations of T-lymphocytes with a prevalence of content-helper cells, shortage of CD3 and CD8 lymphocytes and increased immunoregulatory index; hyperimmune all major or individual classes of immunoglobulins and increased circulating immune complexes, depletion functional activity of phagocytes and incomplete phagocytosis) are diagnosed with CP, which developed against the background of duodenal ulcer.

example 1

Patient S., medical history number 4731.

He received gastroenterological medicine department of the Krasnodar Regional Hospital with complaints of epigastric pain, nausea after eating, weakness.

From history: UDD sick for about 20 years.

Objectively at admission: stomach pain on palpation in the epigastric, both hypochondria, in the course of the projection of the pancreas, the positive symptoms of the Mayo-Robson and Kacha.

When the instrumental and laboratory studies were identified: on esophagogastroduodenoscopy scar-peptic duodenal bulb deformation esogastritis, bulbit; indirect signs of pathology panktreato-biliary zone (mucous postbulbarnyh departments with telelimfoangioektaziyami).

Ultrasound examination: pancreas sizes within the normal range, the contours of her rough, ECHO geneity enhanced diffuse non-uniform structure. Conclusion: The ultrasound signs of CP.

In the study of the enzymatic activity of the pancreas indicators do not go beyond the upper limit of normal.

In the immunological moderate T-cell deficiency due to CD4 lymphocytes, depression absorptive activity of neutrophils. The content of immunoglobulins and CEC - in the normal range.

Five-time patient produced intramuscular administration of 1 ml of 0.005% solution imunofana a day. 3 days after the last administration of amylase, lipase and trypsin were examined again immunogram. Indicators expressed in the following figures, compared with the original data.

The data allowed to put a clinical diagnosis: HP, painful form, relapsing course in the acute stage.

Rubtsovo-peptic duodenal bulb deformation, gastritis, bulbit.

The study confirmed that joining HP in duodenal ulcer, and has made it possible to conduct the adequate pathogenetic therapy.

example 2

Patient M., medical history number 1625.

Received gastroenterological medicine department of the Krasnodar Regional Hospital with complaints of epigastric pain, left upper quadrant, radiating to the back, wearing paroxysmal in nature, heartburn, weight loss of 6 kg over 2 months.

From history: suffering from duodenal ulcer for 10 years. Repeatedly treated in a hospital. Hospitalized in a clinic with a diagnosis of duodenal ulcer in the process of fading exacerbation.

Objective data on admission: painful stomach epigastric pancreatic-duodenal zone palpation, positive symptoms Mayo-Robson and Kacha.

The examination revealed the following abnormalities: on esophagogastroduodenoscopy scar-peptic duodenal bulb deformity, chronic papillitis.

On ultrasound data heterogeneous, hyperechoic pancreatic structure. Conclusion: The ultrasound signs of CP.

The content of pancreatic enzymes - in the normal range.

In the immunological imbalance subpopulations of T-lymphocytes with a predominance of suppressor, suppressor reduction index hyperimmune A moderate increase in the absorptive activity of neutrophils and content of the CEC.

The patient was administered intramuscularly five times 0.005% solution imunofana 1 ml a day. After 4 days repeated research produced peripheral blood.

The data obtained are shown in Table 5.

CLAIM

A method for diagnosing chronic pancreatitis that developed against the background of duodenal ulcer, comprising determining the clinical signs and levels of pancreatic enzymes in the blood, characterized in that with normal serum enzymes chronic pancreatitis is diagnosed by further determining the deviation from the normal content of their levels after 5 -fold imunofana intramuscular injection to the patient and the characteristic changes of immune status of the patient.

print version
Publication date 29.03.2007gg

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