UA13175U - Method for diagnosing changes in lungs at tuberculosis - Google Patents

Abstract

The proposed method for diagnosing changes in lungs at tuberculosis implies determining the content of apoptotic cells and determining the appotosis induction indicator as the ratio between the spontaneous apoptosis indicator and the induced apoptosis indicator. Infiltrative changes in the patient's lungs are diagnosed if the appotosis induction indicator is 0.400 ... 0.618. If the indicator is less than 0.400, pneumosclerosis is diagnosed. Destructive changes in the lungs are diagnosed if the indicator is more than 0.618.

Description

Description of the invention

A useful model relates to the field of medicine, namely to the field of phthisiology and immunology and can be used to diagnose pulmonary tuberculosis by detecting apoptotic cells.

The method of diagnosis by measuring apoptosis, which we have taken as the closest analogue, is known "Method of determination and/or quantitative detection and/or separation of apoptotic cells in a sample or part of a sample", patent for invention UMO 9527903 AT, dated 19.10.95, Изобретения стран мира (1996 ). - Vyipusk 084, Mo17. -

p. 48). The method consists in determining apoptosis to confirm the clinical diagnosis and control the effectiveness of the use of drugs and applied treatment. The method involves contact of the sample with a determining reagent that has a strong affinity for phosphatidylserine of the plasma membrane of apoptotic cells, qualitative and possible quantitative determination of cells, mainly intact cells that react with the reagent used, and separation of apoptotic cells from non-apoptotic cells that do not react with it.

The disadvantage of using absolute values of apoptosis for diagnosis is the dependence of the value of apoptosis on the time that has passed from the moment of taking cells to the beginning of the study, and their lability - dependence on the functional state of the organism. Also, the values of spontaneous, induced apoptosis and induction of apoptosis characterize each separate link of pathogenesis.

The task of the proposed method is to objectify the research method to eliminate the influence of factors of the external and internal environment.

The solution to the problem is achieved by the fact that in the well-known method - "Method of diagnosing changes in the lungs in tuberculosis" by determining apoptotic cells according to the proposed solution, the ratio of the apoptotic indices of spontaneous and induced apoptosis is determined, and with the index of induction of apoptosis from 0.400 to 0.618, the predominance of infiltrative changes in lungs, with values of the apoptosis induction index of less than 0.400, the predominance of pneumosclerosis is determined, with the apoptosis induction index of more than 0.618 29, the predominance of destructive changes in the lungs is diagnosed. -at

The claimed method is performed as follows. Mononuclear cells are isolated from the heparinized blood by centrifugation with an acceleration of 4009 for 25-35 minutes in a ficol-verografin density gradient (p-1.077-1.078), a ring of mononuclear cells is removed. The resulting cells are pipetted and centrifuged twice for 8-12 minutes with an acceleration of 2009 in a neutral buffer solution, for example, buffer solution with a pH of 7.2-7. Double cultures are prepared from the washed cells at a concentration of 5-x1O cells/ml, which are placed in a nutrient medium containing 1095 embryonic calf serum in a ratio of 1:1. A solution of an apoptosis inducer, for example, dexamethasone, is added to one of the double cultures in a final concentration of 1072-10-7M. Cell cultures are incubated for 12-72 hours at a temperature of 372C. After incubation, the cells are pipetted into a neutral buffer solution, such as buffer solution with a pH of 72-74, centrifuged for 3-7 minutes at 4009 acceleration. The percentage of live and dead cells is calculated using a standard post-mortem dye test, e.g. with trepan blue. Washed cells are incubated for 25-35 minutes. at a temperature of 372C with a dye solution, for example, Noespzi 33342 at a concentration of 0.05-0.15μg/ml in the ratio of cell suspension/dye solution - 1:1-2:1, centrifuged with the addition of a neutral buffer solution, for example with pH 7.2-7.4, centrifuged for 3-7 minutes 3 pt") with an acceleration of 4009. The cells are fixed with a fixing mixture, placed on a slide, covered with a coverslip and sealed. The preparations are viewed under the illumination of a fluorescent lamp at a magnification of 7x90. The apoptotic index is defined as the percentage ratio of the number of cells with signs of apoptosis and 2000 cells detected in the field of view of the microscope. The apoptotic index is determined both in cells that were incubated only in the nutrient medium - the index of spontaneous apoptosis, and in cells that were incubated - in the nutrient medium in the presence of dexamethasone - the index of induced apoptosis. The apoptosis induction index was determined as the ratio of spontaneous and induced apoptosis indices in this patient. With an index of apoptosis induction from 0.400 to 0.618, the predominance of infiltrative changes in the lungs is diagnosed, with values of the apoptosis induction index of less than 0.400, the predominance of pneumosclerosis is determined, with an index of apoptosis induction greater than 0.618, the predominance of destructive changes in the lungs is diagnosed. If there are several at the same time

Of these processes, the value of the apoptosis induction index characterizes the result of the influence of all the specified factors. Mr. Example 1

Patient K-nko O.T., medical history Mo1304. Diagnosis: disseminated pulmonary tuberculosis in the phase of infiltration and decay, MBT(s). X-ray picture on June 4, 2002, bilaterally more in the upper and middle two lung fields, various foci of infiltration with areas of lightening, on the right, in the upper lobe of CM 5x4 cm in

Zb CM with a diameter of 4 cm. Apoptosis induction index 11.06.2002 - 1.4285. with Example 2

Patient I-va K.V., case history Mo774/818. Diagnosis: infiltrative tuberculosis of the upper lobe of the left lung in the phase of decay, MBT(Zht). X-ray picture on April 1, 2002: the right lung is transparent, in the upper lobe of the left lung (8142) there is peribronchial infiltration, a small focus of infiltration of a heterogeneous structure, small foci of soft density. Apoptosis induction index 06/05/2002 - 0.6136.

Example C

Patient R-ko V.I., case history Mo827/1003. Diagnosis: tuberculoma 51 of the right lung in the phase of decay and seeding. X-ray picture on 04/19/2002 in the lower lobe of the left lung massive small focal 65 dissemination. A group of foci that merge into a conglomerate in 51 right lung. Apoptosis induction index 05/29/2002. - 0.5909.

Example 4

Patient S-lo O.V., medical history Mo2548. Diagnosis: focal tuberculosis of the upper lobe of the right lung in the infiltration phase, MBT/(-). X-ray picture on November 14, 2002: on the right, in the upper part, several polymorphic focal shadows are determined against the background of the enhanced pulmonary pattern. Apoptosis induction index on November 22, 2002 - 0.2715.

Example 5

Patient D-v Yu.M., medical history Mo1427. Diagnosis: infiltrative tuberculosis of the upper lobe of the right lung, phase of decay and seeding. Office(s). VDT. X-ray picture on June 18, 2001: in the upper lobe of the right lung, there are 70 many foci of various sizes, a massive infiltrate of the upper lobe, a cavity of Х4.5 cm with infiltration all around, peribronchial infiltration to the root of the lung, which is slightly pulled up. Apoptosis induction index 06/21/2001. - 0.8929.

Thus, the use of the method declared in the National Center for Disease Control and Prevention at the department of phthisiology of KMAPO named after them. P.L. Shupyk and in the work of the branches of the research institute of phthisiology and pulmonology named after F.G. Yanovsky allowed /5 to diagnose pathological changes in the lungs of patients with pulmonary tuberculosis. In addition, the claimed method made it possible to objectively evaluate the results of the apoptotic index study due to the cultivation of cells and the use of the apoptosis induction index for diagnosis.

Claims (1)

The formula of the invention is a method of diagnosing changes in the lungs in tuberculosis by determining apoptosis, which is characterized by the fact that the ratio of the apoptotic indices of spontaneous and induced apoptosis is determined, and when the apoptosis induction index is from 0.400 to 0.618, the prevalence of infiltrative changes in the lungs is diagnosed, with values of the apoptosis induction index less than 0.400 determines the prevalence of pneumosclerosis, with an apoptosis induction index greater than 0.618 diagnoses the prevalence of destructive changes in the lungs. - Official bulletin "Industrial Property". Book 1 "Inventions, useful models, topographies of integrated microcircuits", 2006, MZ, 15.03.2006. State Department of Intellectual Property of the Ministry of Education and Science of Ukraine. cha «- (ze) h- - with . and? - (95) - - 50 sl s 60 b5