Method for determining severity in patients with bronchopulmonary diseases

FIELD: medicine.

SUBSTANCE: invention involves patient's induced sputum sampling, cell substance recovery and preparation for the examination. A cell alteration value is determined by apoptosis evaluation, while the cell substance is additionally examined for a cell necrosis alteration value. The apoptosis and necrosis values are determined in two or more examinations every 1-3 days. The derived values are expressed in the form of a necrosis index (NI) and an apoptosis index (AI) to calculate a cell alteration index (CAI) by formula: CAI=NI/AI, wherein NI is the necrosis index; AI is the apoptosis index. A severe clinical course of the bronchopulmonary disease is stated by the CAI values within 0.5 to 1.0. A moderate degree of severity is shown by the CAI values falling within the range 1.0 to 1.3. And a mild degree is indicated by the CAI values in the range 1.3 to 1.7. The clinical effectiveness of the bronchopulmonary disease can be determined by comparing the initial CAI values and the CAI value during treatment, and if observing increasing the CAI values, the improvement is diagnosed.

EFFECT: using the method enables higher accuracy of determination with providing more simple and fast method.

2 ex, 2 tbl

 

The invention relates to medicine, namely to diagnosis, and can be used to determine the severity of bronchopulmonary diseases.

The development of inflammation in bronchopulmonary diseases depends on the duration of the recruitment of cells from the bloodstream into the lining of the bronchi and activation of these cells. A Central role in the inflammation site is given to the mechanisms of regulation of cell viability. There are two forms of cell death - necrosis and apoptosis. The first is cell death caused by exposure to harmful factors leading to the violation of the integrity of cell membranes, resulting in cell literoitca and its contents poured into the intercellular space. Cell necrosis is usually accompanied by inflammation. Apoptosis is a special type of programmed cell death by dividing them into parts ("apoptotic Taurus"), which are then phagocytized by neighboring cells of different types that does not lead to the development of inflammation. These forms of damage or alteration of the cells are of great importance in homeostasis and pathological conditions of the body. It is considered that if the cells die due to necrosis, the possibility of pharmacological intervention may not give a positive result, even if it is only the initial stage is uridine. However, according to Professor V.S. Novikov (Programmed cell death / Usenbekov. - St. Petersburg: Nauka, 1996), therapeutic effectiveness in the inhibition of realization of cellular collapse (apoptosis) can be achieved with the early and comprehensive application of pharmacological agents.

Known way to determine the severity of bronchopulmonary diseases - bronchial asthma (Bespalova I.D., Crnogorac GA, V.T. Volkov EN 2277710 C1 from 2006.06.10), consisting in the determination of inflammatory markers in biological fluids, using the definition of the reaction of a skin window. Received strokes, after 12 and 24 hours, fixed with methanol and stained by Bernhard. Determine the number of neutrophils and macrophages, counting from 100 to 500 cells in each smear and the percentage of neutrophils 57,78±4,40 and 32,35±3,61 after 12 and 24 hours respectively define first degree of inflammatory activity, while the percentage of neutrophils 62,56±5,50 12 hours, and 40,88±6,22 24 hours determine the second degree of activity of inflammation.

The disadvantage of this method is that it is traumatic, because as inflammation markers in biological fluids using clarificatory the skin of patients, time-consuming for the researcher and long.

There is a method of assessment effectivelyintegratedinto therapy Chlamydophila pneumoniae in patients with bronchial asthma (Fedorov G., Grigoriev, V.N., Petushkova so-CALLED. EN 2230319 C1 from 2004.06.10). At the same time as inflammation markers in biological fluids using peripheral blood of patients, in which method an indirect immunofluorescence assay to determine subpopulation structure of lymphocytes. When CD4+lymphocytes of less than 36,67% and D95 lymphocytes of less than 57,95% 3-4 weeks after treatment antibiotic therapy aimed at the eradication of the pathogen, it is considered effective.

The disadvantages of this method are:

the lack of data about the state of local immunity of patients;

the subjectivity of the received data, as the results will largely depend on the skill of the person fulfilling it;

- high costs in connection with the use, to identify subpopulations of cells monoclonal antibodies.

The closest to our proposed method is the evaluation of apoptosis of neutrophils in patients with sepsis, accompanied by acute respiratory distress syndrome (Fialkow L., Filho, L.F., Bozzetti M.C., Milani A.R., Filho E.M.R., Ladniuk R., Pierozan P., de Moura R., Prollal J.C., E. Vachon, G.P. Downey Neutrophil apoptosis: a marker of disease severity in sepsis and sepsis-induced acute respiratory distress syndrome Critical Care. - 2006, 10(6):R155). With this method neutrophils isolated from the peripheral blood of patients by adding dextran and use specific gradient in density is of astora, after sedimentation of the cells washed twice from these solutions, the concentration is brought to 1×106cells in 1 ml RPMI medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 mg/ml penicillin, 100 mg/ml streptomycin, after incubation for 24 h, colouring neutrophils by Giemsa and assesses the status of the cells for the identification of changes to the kernel 500 cells (chromatin condensation and reduction of nuclear structure), which is expressed in percentages. Thus the dependence of the degree of apoptosis of neutrophils of patients with sepsis from the severity of the disease.

The disadvantage of this method is that this method captures only system disorders in patients, and in the appointment of therapy in patients with bronchopulmonary diseases, it is necessary to investigate the condition of the cells not only systemic blood flow, but also directly target organ (lung). It is known that in the target organ cells reach the last stage of functional maturation, which, ultimately, leads to the initiation of the process of their death. In addition to the disadvantages of this method is the long period of incubation of the cells for which use additional reagents and determination of apoptotic activity only neutrophils, without regard to other cells.

adaca of the proposed technical solution is to obtain high precision data, simplification and acceleration of the method.

The problem is solved in that in the method for the diagnosis of disease severity in patients with bronchopulmonary diseases, including selection of subject matter, the allocation of a cellular substance, preparation to study, determine the magnitude of alteration of cells by assessing their apoptosis according to the invention as the study material used induced sputum (IM) patient, in the cellular substance additionally determine the amount of alteration of cells by necrosis, indicators of apoptosis and necrosis is determined at two or more surveys with an interval of 1-3 days, the resulting performance is expressed as the apoptotic index (AI) and index of necrosis (JN), calculate the index of alteration of the cell (FCC) by the formula:

IAC=IN/AI, where

IN the index of necrosis;

AI - apoptotic index;

the degree of severity of the disease in patients determined on the basis of the values of IAC and diagnose severe currents bronchopulmonary disease with indicators IAK from 05 to 1.0, the average from more than 1.0 to 1.3 and a mild degree of flow at rates ranging from more than 1.3 to 1.7

The essence of the method is illustrated by the following sequence of operations:

As the material for research using induced sputum (MI) patients with BD who holehouse diseases. THEY receive after inhalation 3-5% sterile hypertonic saline using compression nebulizer "Omron CX-3 (Japan). Then IM using sterile disposable syringe (5 or 10 ml) is transferred into a test tube. For dispersion and homogenization of sputum using 0.1% solution of dithiothreitol, which is added to the sputum in a 1:1 ratio. The mixture is then shaken for 10 minutes and filtered through nylon gauze. Filtered THEM centrifuged at 1200 rpm for 10 minutes, then the supernatant is drained, and a suspension of the cells washed 3 times in Hanks solution without phenol red by centrifugation at 1200 rpm for 10 minutes.

Sputum examination shall be held not later than 3 hours after receipt of material throughout this time the samples are stored at a temperature of +4°C.

To determine necrosis cell suspension added at the rate of 1:2 solution of 0.1% Triton X-100, 0.08 N HCl, M NaCl, gently mix and leave for 15 seconds, then bring the rate of 1:2 cold solution of dye acridine orange (5 mg/ml, on the basis of citrate buffer (pH 6.0)containing 1 mM EDTA-Na, M NaCl, 0.2M NaPO4, 0.1M. After coloring within 5-10 minutes preparing medicines and immediately analyzed under fluorescent microscope.

To determine apoptosis, after mixing the cell suspension with a solution of Triton (composition of the solution specified above), cells fixed with 4% paraformaldehyde on the basis of distilled water containing 1% of cacodylate sodium and 1% sodium chloride, and then stained with a specific dye Hoechst 33342 (5 mM) for detection of apoptosis for 15 min and analyzed under fluorescent microscope.

Damage (alteration) cells induced sputum appreciate by viewing drugs under fluorescent microscope with exciting filters FS, SS, ES and locking filter type LGL=18+LGL=19 using a water immersion lens. The results are obtained on the basis of the evaluation of color, intensity and morphology of the fluorescent cells. In the case of alteration of cells by necrosis count 100 cells, of which define % yellow cells, including dye is acridine orange, and expressed as an index of necrosis (JN). Apoptotic cell activity is expressed as the apoptotic index (AI) - % of green cells, including dye Hoechst 33342, and with the presence of morphological signs of apoptosis - modified nuclei, consisting of 3 or more items, and reduced cytoplasm.

Calculate the index of alteration of the cell (FCC) by the formula: IAC=IN/AI, where JN is an indicator of necrosis; AI - apoptotic index.

Compare the index of alteration of the cells of patients in exacerbation with indicator IAK healthy individuals (counter is eh). Reduction IAK phagocytes THEM in a patient, compared with values in healthy donors, indicates a violation of local protection in the target organ. Diagnosed with severe currents bronchopulmonary disease with indicators IAK from 05 to 1.0, the average from more than 1.0 to 1.3 and a mild degree of flow at rates ranging from more than 1.3 to 1.7

The basis for determining the effectiveness of the proposed method of evaluation of bronchopulmonary diseases was based on the results of the study of induced sputum of patients with community-acquired pneumonia (EP), bronchial asthma (BA) mild, moderate and severe in the process of inpatient treatment in the pulmonary Department of the General hospital of the Pacific fleet, Vladivostok) and therapeutic Department DOMC (Vladivostok). Clinical diagnosis of bronchopulmonary disease was established on the basis of common criteria. Assessment of the severity of patients at the time of admission was carried out according to the criteria proposed Lignorance in 1996 (A. Okorokov. Diagnosis of respiratory diseases / Anecortave. - M: Honey. lit., 2000. - 448 C.). The study included patients with mild illness - 60 (38.7 per cent), with medium-heavy - 48 (37.4 per cent) and heavy 22 (23.9 percent). The control group consisted of 20 healthy persons with no history of chronic bronchopulmonary pathology is, not suffering from an acute illness within 6 months. This IAK in healthy individuals is equal to 2.0.

A study of indicators of alteration of cellular factors of local immunity (MI) patients with bronchopulmonary disease has made it possible to identify a distinct change their values compared with those for healthy persons. the lowest value IAK was noted in the period of exacerbation in patients with severe EAP (2.7 times) and BA (2.6 times). This marked a clear difference values IAK depending on the severity of the disease, the more severe is the disease, the lower the IAK. The lowest values IAK was noted in patients EAP that pointed to the failure of local factors protect the lungs in this disease.

Examples of specific performance.

Example 1

Patients with a diagnosis of community-acquired pneumonia (EP) remained conservative inpatient treatment pulmonary Department of the General hospital of the Pacific fleet. The EAP was diagnosed with a combination of newly arising infiltrative changes on the radiograph of the lungs and the following clinical signs (Torres, 1997): a) cough b) purulent or Muco-purulent sputum, rales on auscultation or signs of seal lung tissue; d) dyspnea or tachypnea; d) peripheral blood leukocytes >10000 or <4500 m the or > 10% band neutrophils. Considering the fact that the patients included in the study were of the same sex, age and had no comorbidities, the severity of the EAP was estimated on the basis of clinical manifestations according to the criteria developed by Butler LI (1996). Patients the study MI by the proposed method. The data presented in table 1.

Table 1
Indicators of alteration of cells induced sputum (PAC) in patients with RR
PAKEAP first severity n=26VP second severity n=18EAP third degree n=10Control group, n=20
IN %96±6,859±3,849±3,545,2±2,4
AI, %70,01±6,854±4,666,7±5,222,5±1,6
JAMES1,41,10,7The values of IAC, is presented in the table, in the period of exacerbation, patients EAP can be divided according to the severity of the disease. Patients of the first (easy) degree EAP is IAK amounted to 1.4, the second (middle) of 1.1, whereas a third of patients (severe) disease 0,7.

Example # 2

Patients with a diagnosis of bronchial asthma (BA) of various degrees of severity of the disease was on the conservative inpatient treatment therapeutic Department DOMZ. For a diagnosis of ad, determine severity were used recommendations adapted to the Russian conditions of consensus (GINA 2002), assessment of the clinical course BA was performed according to the change of day and night symptoms, measured in points on a scale from 0 to 5, frequency and duration of exacerbations of the disease, indicators of external respiration functions. Patients the study MI by the proposed method. The data presented in table 2.

Table 2
Indicators of alteration of cells induced sputum (PAC) in patients with BA
PAKBA, mild disease severity n=16BA, the average degree t is tin disease n=14 BA, severe degree of severity of the disease (n=8Control group, n=20
IN %42,3±2,444,6±3,439,3±1,645,2±2,4
AI, %28,8±1,837,3±1,843,7±3,422,5±1,6
JAMES1,51,20,92,0

Thus, the value of IAC in the period of exacerbation with bronchial asthma can be classified according to the severity of the disease. Thus, in patients with mild disease BA is IAK amounted to 1.5, the average is 1.2, and the heavy - 0,9.

The way to diagnose the severity of the disease patients with bronchopulmonary diseases, including selection of subject matter, the allocation of a cellular substance, preparation to study, determine the magnitude of alteration of cells by assessing their apoptosis, characterized in that as the study material used induced sputum of a sick person, in the cellular substance additionally determine the amount of alteration of cells by necrosis, however, is ately apoptosis and necrosis is determined at two or more surveys with an interval of 1-3 days, the indicators expressed as an index of necrosis (JN) and apoptotic index (AI) and calculate the index of alteration of the cell (FCC) by the formula
IAC=IN/AI,
the degree of severity of the disease in patients determined based on the value of IAC and diagnose severe currents bronchopulmonary disease with indicators IAK from 0.5 to 1.0, the average from more than 1.0 to 1.3 and a mild degree of flow at rates ranging from more than 1.3 to 1.7.



 

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