Diagnostic technique for severe clinical course in gastric ulcer

FIELD: medicine.

SUBSTANCE: with using indirect immunofluorescence and monoclonal antibodies, the patients suffering gastric ulcer (GU) are examined for an expression level of CD8, CDDR markers by peripheral blood lymphocytes with the CD8 level exceeding 24.5% and the CDDR level exceeding 15% enabling diagnosing a severe clinical course of GU.

EFFECT: use of the offered diagnostic technique provides high information value in diagnosing the severe clinical course of gastric ulcer, well-timed prediction of the severe clinical course of GU, instant diagnosing, provides well-timed and adequate complex of therapeutic measures.

1 tbl, 1 ex

 

The invention relates to medicine, in particular to therapy, gastroenterology, immunology. It can be used in the clinic for the diagnosis of severe peptic ulcer disease (PUD) in adults.

A known method for the diagnosis of severity YAB in the number of exacerbations of the disease during the year. The patients with three or more exacerbations per year can be considered a disease with a severe course. The disadvantages of this method are: the subjective evaluation of the test feeling sick, which can lead to errors in the diagnosis of the severity of the disease and choosing the wrong treatment. In addition, for the first time showed worsening YAB is not possible to estimate the future severity of disease, abdominal pain and dyspepsia associated with the error power can be perceived by patients as the aggravation, and, on the contrary, weak expression of complaints during the next exacerbation may not attract the attention of the patient. (Order of the Ministry of health of the USSR "on the procedure for conducting a General medical examination of the population" №770 from 30.05.1986 - P.62, (in edition of Order of Ministry of health of the Russian Federation No. 270 from 12.09.1997).

There is a way to diagnose the severity YAB according to the degree of contamination of the coolant Helicobacter pylori. The disadvantage is that the degree of contamination is estimated by the results of several biopsies of the coolant, but the degree of contamination can be servname is Noah, in addition, the invasive method is traumatic. (Arwin LI, Kapeller L.L., Isakov V.A. Morphological diagnosis of diseases of the stomach and intestines. - M.: Triada-X, 1998 - S. 483).

There is a method of determining the severity of the disease state fractions of water in the composition of biomacromolecule formed elements of blood. The heavier for patients, the observed greater loss of associated and increased free fraction of water. The disadvantages of this method is the ambiguity of interpretation of the results, the use of complex, non-traditional methods. (Patent for invention №2312584, 2007, C.1).

YAB is characterized by different etiology, complex pathogenesis of chronic relapsing course, morphological equivalent in the form of a defect in mucosal and submucosal layers with outcome in a connective tissue scar. In patients with severe during periods of remission short, not more than 3-4 months in a year, pain and dyspeptic syndromes are much. This disease is a cause of prolonged suffering of a large number of people, significantly impairing their quality of life. In addition, the disease is accompanied by a high level of destructive and neoplastic complications leading to trucapture, disability, death, which entails an increase in Econ the economic losses for society as a whole.

Due to the large number of syndromic clinical manifestations YAB for diagnosis necessary clinical and biochemical blood tests, fecal occult blood, histological and cytological examination of biopsy specimens, urease test, esophagogastroduodenoscopy with biopsy and brush Cytology, ultrasound examination of abdominal cavity organs and other methods. Additional studies are under suspicion of a malignant ulcer, the presence of complications and comorbidities.

These methods are combined with the data obtained in the study of the clinical picture and history, allow more reliability to establish the existence of YAB and the severity of its course. (Arwin LI, Kapeller L.L., Isakov V.A. Morphological diagnosis of diseases of the stomach and intestines. - M.: Triada-X, 1998 - 483 C. the Order of the Ministry of health of the USSR "on the procedure for conducting a General medical examination of the population" №770 from 30.05.1986 - P.62 (in edition of Order of Ministry of health of the Russian Federation No. 270 from 12.09.1997). Patent for invention No. 2312584, 2007, C.1). The disadvantages of the methods used diagnostic YAB is the long duration of time associated with the use of a large number of instrumental and special methods of research, the ambiguity of interpretation of the results. In addition to the CSO, most studies are of high cost, the involvement of consulting physicians of other specialties, the absence, in most cases, medical documentation that contains a full history.

The aim of the invention is to increase the efficiency differential diagnosis of severe YAB by determining the level of expression of the markers CD8, CDDR the peripheral blood lymphocytes, which leads to faster diagnosis, timely forecasting the severity of YAB, ensuring greater efficiency and more rational selection of therapeutic interventions.

The essence of the proposed method of diagnosis is that patients YAB determine the degree of expression of cell markers CD8, CDDR method of indirect immunofluorescence using monoclonal antibodies and the level of CD8 more than 24.5% and CDDR more than 15% of diagnosed severe YAB.

Determined the number of CD8 and CDDR markers. CD8 is a marker killer-cytotoxic lymphocyte subpopulations, characterizing the activity of the inflammatory process on the cell type, which occurs when YAB. CDDR-marker of the second stage of lymphocyte activation, describing the severity of their activation, which is also the case for YAB.

The dynamics of the immunological changes observed in patients with severe YAB p the relation to light and moderate flow, characterized by a pronounced immune inflammation cell type (possibly Thl response) involving killer-lexically subpopulations of lymphocytes (CD8) and a high degree of their activation (CDDR). In this regard, the determination of the degree of expression of cell markers CD8, CDDR can serve as a diagnostic criterion of severe YAB.

The method is as follows: from the sample of heparinized blood samples (4-5 ml), taken on an empty stomach in patients with PUD emit cells at the gradient ficoll-urografin.

Then 1-0,1 million cells in a volume of 50 μl contribute to the wells of 96-well round-bottom of the tablet. To the cells, add 5 µl of the tested monoclonal antibodies (CD8, CDDR) and incubated for 30-45 min at +4°C. Then add 150 μl of Hanks solution and centrifuged 5 min at 200 g. After removal of the supernatant contribute 50 μl of Hanks solution, the cells are suspended, add 150 μl of Hanks solution and centrifuged 5 min at 200 g.

To the precipitate washed cells add 50 μl of F(ab~)2 fragments of sheep antibody to mouse Ig labeled with FITZ and diluted 1:100 (for cultivation using the Hanks solution containing 0.5% gelatin and 0.1% sodium azide). Cells are suspended and incubated for 30 min at +4°C, the cells are then washed 2 times as described above.

Stained cells analyzed by fluorescence microscope. Cells are suspended, transferred to a subject article is Clough, cover cover glass and looking at drug under immersion with magnification (×3; lens ×90).

The number antigenpositive cells is defined as the percent of fluorescent cells while browsing 200 lymphocytes minus % fluorescent cells observed in the preparation of the negative control. As a negative control, using drugs, prepared similarly, but without the processing of monoclonal antibodies. On the expression of cell markers CD8, CDDR judged severe YAB.

EXAMPLE

Patient A., 46 years old, taxi driver. Went to the clinic with complaints of mild pain in the epigastric region, occurring in 1.5-2 hours after a meal, and a night of pain, ending after eating, heartburn, nausea. These complaints concerned within 6 days. Medical help is not sought, took but, silos and soda, which gave a temporary effect. Smokes 20 cigarettes a day.

Physical examination: General condition of moderate severity. The body temperature of 36.6°C. the Skin skin color. The vesicular breathing, 18 in 1 min heart Rhythm correct 68 minutes HELL 110/70 mm Hg Tongue coated white bloom. The abdomen is soft, painful epigastric and the area of the projection of the pylorus (2 cm to the right of the navel). Liver, spleen not enlarged. Symptom of Pasternack tricate the capacity. Physiological functions are normal.

History: 3 years ago for the first time diagnosed with peptic ulcer of the duodenum. Treatment at the time of inspection: omez 20 mg 2 R/d 10 days, Almagel 1 tablespoon an hour after meal 5 days. More care was not addressed. 3-4 times per year notes above symptoms. Took omez, ranitidine.

These additional methods.

Complete blood count: HB 140 g/l, er 4,1*1012/l L to 7.2*109/l, erythrocyte sedimentation rate of 7 mm/h.

Urinalysis: specific gravity 1020, the color is straw yellow, the reaction of the acid, L 1-2 p/SP er 0-1 in the p/SP.

ECG: sinus rhythm, heart rate of 72 per minute, the normal position of the EOS.

CCF: lung fields clear, heart without visible pathology.

Ultrasound of abdominal organs: no visible pathology.

The fecal occult blood test (reaction of yens Gregersen collection): negative. Fibrogastroduodenoscopy: ulcer bulb duodenal ulcer twelve 0,4*0,6 cm, chronic erosive gastritis, Bullit. Cicatricial deformity of the duodenal bulb 2-3 degrees.

Urease test: Helicobacter pylori dramatically positive

Microscopy of smears prints gram stained biopsy specimens from the antrum and body of stomach: Helicobacter pylori typical spiral shape in a very large number.

The level of expression of markers CD8 28,60%, CDDR 18.4% of lymphocytes per fericelli blood. That is, CD8 was more than 24.5% and CDDR more than 15%. That allowed us to diagnose Peptic localization of ulcers in the bulb 12 SC intestine, severe degree, 2 degree, acute stage, and assign timely treatment.

Concomitant chronic erosive gastritis.

Complication: cicatricial deformity of the duodenal bulb 2-3 degrees.

A method for the diagnosis of peptic ulcer disease in adults in the level of expression of markers CD8, CDDR by peripheral blood lymphocytes was tested in the comparison groups between 38 patients of mild severity YAB, 45 patients with an average severity of YAB and 10 patients with severe YAB.

All of the respondents had performed blood sampling and determination of the level of expression of markers CD8, CDDR the peripheral blood lymphocytes of the above method.

The results of the study are presented in the table.

The data obtained showed that under severe flow YAB level markers CD8, CDDR significantly increased compared with moderate and mild degree of severity YAB. Diagnosis in the studied groups was confirmed by the main diagnostic methods.

For processing the received data, and statistical analysis in the studied groups was used for the statistical processing of information "STATGRAPHICS PLUS". Vice the Lyali average and the average error (M±M). Comparison of study groups conducted in pairs, using the parametric methods because the study sample could be approximated by a normal distribution. The comparison was performed using t-test (t) (comparison means) and Fisher (comparison of mean square deviations). The confidence level was assessed value "p". Differences in the comparison was considered significant with a probability of more than 95% at P<0,05.

Thus, the obtained data showed that in severe YAB level markers CD8, CDDR increases significantly compared to the average and lightweight for YAB, therefore, the level of expression of CD8 molecules, CDDR the peripheral blood lymphocytes can be considered highly sensitive markers of severe flow YAB.

Using the proposed diagnostic method has the following advantages:

1) high information content for the differential diagnosis of severe peptic ulcer disease by determining the level of expression of CD8 molecules, CDDR the peripheral blood lymphocytes;

2) timely forecasting of severe YAB, because this will ensure efficiency and rational realization of a complex of therapeutic measures for patients with YAB;

3) rapid diagnosis, since it is only necessary to determine the level of expression of markers CD8 CDDR the peripheral blood lymphocytes;

4) due to rapid diagnosis ensures timely and efficient implementation of the complex of therapeutic measures.

CD8 (%) M±M (min, max)CDDR (%) M±M (min, max)
Easy
for(n=38)
22,03±0,92*
(19,0±24,0)
13,00±1,04
(10,0±15,0)
Average
for (n=45)
21,13±0,79*
(16,0±23,0)
a 12.05±0,78
(8,0±14,0)
Heavy
for (n=10)
25,6±1,16**
(24,5±30,0)
17,0±1,67*
(15,00±22,00)
Note: * - p<0,05 - significance of differences between groups when compared with severe YAB.
**p<0,05 - significance of differences between groups when compared with easy for YAB.

Method for the diagnosis of severe peptic ulcer disease, including the study of the peripheral blood, characterized in that the hold determining the extent of expression of cell markers CD8, CDDR method of indirect immunofluorescence using monoclonal antibodies and when the content in the peripheral blood markers CD8 more than 245%, CDDR more than 15% of diagnosed severe peptic ulcer disease.



 

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