SU1343358A1 - Method of diagnostics of celiac disease - Google Patents

Abstract

The invention relates to immunology. The purpose of the invention is to improve the accuracy of the method. A suspension of lymphocytes is mixed with an equal volume of gliadin solution. After incubation, the lymphocytes are precipitated. . The supernatant is drained, the precipitate is resuspended, washed and centrifuged. A solution of a negatively charged chromophore is added to the tubes and the fluorescence is recorded. Intense luminescence of lymphocytes is characteristic of celiac disease. The method allows the diagnosis of celiac disease at an early date. U) 00 tn eo

Description

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The invention relates to medicine, in particular to immunological diagnostic methods, and can be used for the differential diagnosis of chronic diseases of the small intestine and specific treatment approaches.

The purpose of the invention is to improve the accuracy of the differential diagnosis of celiac disease and chronic enteritis.

The method is carried out as follows.

In patients with an empty stomach, 4 ml of blood are taken from the cubital vein into heparin tubes (25 units per 1 ml). An equal volume of saline is added to the tubes. To isolate lymphocytes, 4 ml of diluted blood is applied to 2 ml of ficoll-verografine solution (density 1.08) and centrifuged for 30 minutes at 3 thousand rpm. The obtained white ring (lymphocytes and monocytes) is sucked off with a pipette into a dry tube and washed twice with a 199 section (5 ml) with repeated centrifugations for 5 minutes at 1500 rpm. The obtained suspension was incubated on plastic cups Peter in an incubator at 37 ° C for 30 minutes to remove adherent cells. Then the lymphocytes are counted in the Gorev chamber. The concentration of lymphocytes is adjusted to 10 in 1 ml. A suspension of lymphocytes is introduced into test tubes (at least 5 parallel samples) of 0.1 ml and mixed with an equal volume of a solution of antigen-gliadin prepared in physiological solution pH 7.2-7.4, and in the test tube - 0.1 ml saline instead of antigen. The tubes are incubated for 1 hour in a thermosta7.

those at 37 ° C. The doses of gliadin taken are optimal, since doses below 3.5 mg / ml do not give stability to luminescence, and doses greater than 5.5 mg / ml are toxic to the α-lymphocyte membrane. After incubation, the lymphocytes are precipitated by centrifugation for 5 minutes at 1500 rpm, the supernatant is carefully drained, the precipitate is resuspended for 1 minute and washed twice in 5 ml of physiological solution for 5 minutes, followed by centrifugation.

To determine the fluorescence, 2.5 ml of physiological solution and 0.05 ml of 5i10 are added to the tubes.

a solution of 1-anilino-B-naphthalene sulfonate (ANS), a negatively charged chromophore, the fluorescence is recorded on a spectrofluorimeter. The quantum yield is excited at 470 and recorded at 470 nm. Quantitative indications on the instrument scale indicate the intensity of the luminescence of the lymphocyte membrane, and express them in relative units.

A study of peripheral blood lymphocytes in patients with celiac disease and chronic enteritis of the second degree of severity under the action of gliadin revealed significant differences in the intensity of fluorescence.

The data obtained show that only in patients with celiac disease, an intense fluorescence of lymphocytes is revealed after treatment with gliadin. The luminescence intensity of lymphocytes in patients with celiac disease under the action of gliadin was quite different from the fluorescence indicators in patients with chromic enteritis of the II degree of severity (p 0.001). Consequently, the reaction revealed a specific lymphocyte response in patients with celiac disease after pretreatment with gliadin.

Example 1. Ill T., 39 years. Complaints: frequent loose stools, pain throughout the abdomen, reduced appetite,

5 periodic muscle cramps in the limbs, weight loss. Anamnesis: ill since the age of 18, when abdominal pain and diarrhea periodically appeared. From the age of 30, annual 2-3 times of exacerbation, began to lose weight. It was treated on an outpatient basis, but without effect.

Inspection: Sharply reduced nutrition (height 170 cm, weight 49 kg). The skin and visible mucous membranes are pronouncedly pale. Increased brittle nails and hair loss. The tongue is coated with white bloom with imprints of the teeth along the edges. The symptom of the tiny roller is positive. The abdomen is swollen, painful along the colon.

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A splash noise in the cecum is determined. The liver and spleen are not palpable.

Blood test: e 3.8, mute. 11.0%, CPU 0.89, l 4.0 p. 1, p 51, u 2, lim 36, m 10, ESR 4 mm / h. Total protein 7.54 g7., Alia. 4.29g% glob. 3.25g% Absorbability tests: with D-xylose 2 h - 0.8 g, 5 h - 1.2 g (moderate decrease); total fatty acids: 7.6 g / h, free fatty acids 4.3 g / h - moderate increase. Serum immunoglobulins: A 110 mg% (55-251), M 125 mg7o (68-350 G 260 mg% (97-235). X-ray examination of the small intestine: intestinal loops are evenly distributed. The tone is somewhat reduced. The passage is accelerated. The relief is uniformly feathery without gross visible changes. Preliminary diagnosis: chronic enteritis II degree of severity.Additional research of peripheral blood lymphocytes after stimulation with gliadin allowed a final diagnosis — chronic enteritis of II degree of severity — as well as fluorescence intensity of 1.1, what b lo significantly lower than in patients with celiac disease, the arithmetic mean of the intensity of which is equal to familiarize 5.79 units.

The confirmation of the correctness of the diagnosis using the method of fluorescent probes using gliadin lymphocyte treatment showed recovery from the treatment carried out.

As another example, a case history is presented, where it was also difficult to diagnose celiac disease. Since there was an atypical clinical course of the disease, the presence of symptoms that were rarely encountered in this pathology. I

Example 2: Sick S., 29 years old. Complaints: frequent loose stools, abdominal distension, pain all over the abdomen, weakness, dizziness, blood flow of the gums, appearance of bluish skin, cramps in the hands, weight loss .

Anamnesis: ill since the age of 24, when, after childbirth, stubborn constipation of shifts to diarrhea, abdominal distension, later gestational bleeding from 26 years, cramps of the limb muscles appeared.

Inspection: reduced nutrition (height 169 cm, weight. 58 kg), measured dry skin, thinness and brittle nails. Laxity and bleeding gums. Tongue wet, nipples smoothed. The symptom of the roller roller is positive, the stomach is moderately swollen in the new sections, a painless, soft noise of splashing in the blind

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KHuiKO is not defined. Ground and tails are not palpable.

Blood test: er. 3.9, gsm. , 12.6g7 „, l. 4.6, p. 2, p. 60, e-2. 61, lim. 24, m. 11, ESR - 5 mm / h. Total protein 6.86g, alb. 3.43, gamma glob. 15.2%. Samples for absorption with D-xylose: 2 h, 0.38 g, 10 5 h, 0.65 g (reduced), total rpm, | e acid 38.1 g / h, free fatty acids 22.3 g / h ( increased). Serum immunoglobulins: A 80 mg%, (55-251), K 110 mg% (68-350), G 15 210 mg% (97-235).

X-ray examination of the small intestine: marked changes in the duodenal and upper loops of the jejunum

2Q guts - mucus, fluid, hypotonia and hypokinesis. The relief is preserved, but flattened and rebuilt in some places,

Preliminary diagnosis: chronic enteritis, grade II, Celiac disease.

Using the fluorescent probe method, the luminescence intensity of lympho-3Q cells stimulated with in-vitro gliadin is 7.12. The resulting sharply positive lymphocyte response to gliadin made it possible to diagnose celiac disease. Final diagnosis: Celiac.

Wound diagnosis of celiac disease in patients with chronic diseases of the small intestine ensures that treatment is prescribed in accordance with a diet that excludes gliadin from the diet, and, as a result, the ability to work is maintained. A total of 52 4ejv (celiac, chronic enteritis II grade, donors) were examined.

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Thus, the proposed method allows differentiation of celiac and chronic enteritis of the II degree of severity according to changes)) of the intensity of fluorescence of peripheral blood lymphocytes under the action of gliadins. In comparison with the norm, intense luminescence of gliadin-stimulated lymphocytes is characteristic of celiac disease. For patients with chronic enteritis, low fluorescence values of lymphocytes treated with gliadin are characteristic. This makes it possible to increase the accuracy in the early stages of diagnosis of celiac disease.

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Claims (1)

The invention claims cues and chronic enteritis, assessment performed with a fluorescent probe Method for the diagnosis of celiac disease pu-on the intensity of the glow of lymphocytes, In this case, the addition of gliadin to lymphocyte, while gliadin is added to concentrates of peripheral blood of patients with a walkie-talkie of 3.5–5.5 mg / ml per ly10 lymphocyte, followed by an assessment of the reaction, about t and when the luminescence intensity differs from from tsez, 3 to 9.7 units. they are diagnosed with goals of increasing the accuracy of the method by way of a cue; differential diagnosis of targets - 0.4-2.8 units. - chronic enteritis.