RU2456598C2 - Method for determining severity in patients with bronchopulmonary diseases - Google Patents

Abstract

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

Description

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 to the bronchial mucosa and the activation of these cells. In this case, the central role at the site of inflammation 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 disruption of the integrity of the cell membrane, as a result of which the cell is lysed and its contents are poured into the intercellular space. Cell necrosis is usually accompanied by the development of inflammation. Apoptosis is a special type of programmed cell death by dividing them into parts ("apoptotic bodies"), which are subsequently phagocytosed by neighboring cells of different types, which does not lead to the development of inflammation. These forms of damage or alteration of cells are of great importance both in homeostasis and in pathological conditions of the body. It is generally accepted that if a cell dies due to necrosis, then the possibility of pharmacological intervention cannot give a positive result, even if only the initial stage of damage occurs. However, according to Professor B.C. Novikov (Programmed cell death / B.C. Novikov. - St. Petersburg: Nauka, 1996), the greatest therapeutic efficacy in inhibiting the implementation of cell collapse (apoptosis) can be achieved with early and complex use of pharmacological agents.

A known method for determining the severity of bronchopulmonary disease is bronchial asthma (Bespalova I.D., Chernogoryuk G.E., Volkov V.T. RU 2277710 C1 from 2006.06.10), which consists in determining markers of inflammation in biological fluids of the body, by determining reactions of the “skin window”. The obtained smears, after 12 and 24 hours, are fixed with methanol and stained according to Noht. The number of neutrophils and macrophages is determined, counting from 100 to 500 cells in each smear and with a percentage of neutrophils of 57.78 ± 4.40 and 32.35 ± 3.61 after 12 and 24 hours, respectively, the first degree of activity of the inflammatory process is determined, and when the percentage of neutrophils 62.56 ± 5.50 after 12 hours, and 40.88 ± 6.22 after 24 hours determine the second degree of inflammation activity.

The disadvantage of this method is that it is traumatic, since a scarified area of the skin of patients is used as markers of inflammation in biological fluids of the body, it is laborious for the researcher and is long.

There is a method of evaluating the effectiveness of antibacterial therapy of Chlamydophila pneumoniae in patients with bronchial asthma (Fedorov G.N., Grigoryeva V.N., Petushkova T.N. RU 2230319 C1 from 2004.06.10). At the same time, peripheral blood of patients is used as markers of inflammation in body fluids, in which the subpopulation composition of lymphocytes is determined by indirect immunofluorescence. When the content of CD4 ⁺ lymphocytes is less than 36.67% and CD95 lymphocytes less than 57.95% 3-4 weeks after the end of treatment, antibiotic therapy aimed at eradicating the pathogen is considered effective.

The disadvantages of this method are:

- lack of data on the state of local immunity of patients;

- the subjectivity of the data, because the results largely depend on the qualifications of the specialist performing it;

- high cost in connection with the use of monoclonal antibodies to detect subpopulations of cells.

Closest to our proposed method is the assessment of neutrophil apoptosis in patients with sepsis accompanied by acute respiratory distress syndrome (Fialkow L., Filho LF, Bozzetti MC, Milani AR, Filho EMR, Ladniuk RM, Pierozan P., de Moura RM, Prollal JC , Vachon E., Downey GP Neutrophil apoptosis: a marker of disease severity in sepsis and sepsis-induced acute respiratory distress syndrome Critical Care. - 2006, 10 (6): R155). In this method, neutrophils are isolated from the peripheral blood of patients by adding dextran and using a specific density-gradient solution, after precipitation the cells are washed twice from these solutions, their concentration is adjusted to 1 × 10 ⁶ cells per 1 ml of RPMI medium containing 10% calf fetal serum , 2 mM L-glutamine, 100 mg / ml penicillin, 100 mg / ml streptomycin, after incubation for 24 hours, Giemsa stain is stained and the state of the cells is assessed by identifying a core change of 500 k etkah (chromatin condensation and reduction in nuclear structure), which is expressed as a percentage. In this case, the dependence of the degree of apoptosis of neutrophils in patients with sepsis on the severity of the disease was established.

The disadvantage of this method is that this method only fixes systemic disorders in patients, and when prescribing therapy to patients with bronchopulmonary diseases, it is necessary to study the condition of cells not only of the systemic blood flow, but also directly of the target organ (lungs). 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, the disadvantages of this method include a long period of incubation of cells, for which additional reagents are used, as well as the determination of the apoptotic activity of only neutrophils, excluding other cells.

The objective of the proposed technical solution is to obtain high accuracy of data, simplification and acceleration of the method.

The problem is solved in that in a method for diagnosing the severity of a disease in patients with bronchopulmonary diseases, including selecting the test material, isolating the cellular substance from it, preparing it for the study, determining the cell alteration by evaluating their apoptosis according to the invention, induced sputum is used as the test material ( MI) of the patient, in the cell substance, the value of cell alteration by necrosis is additionally determined, while the indicators of apoptosis and necrosis are determined yayut when two or more inspections at intervals of 1-3 days, the figures obtained are expressed as apoptotic index (AI) and necrotic index (TI) was calculated index alteration cells (FAC) of the formula:

IAK = IN / AI, where

IN - necrosis index;

AI - apoptotic index;

wherein the severity of the disease in patients is determined on the basis of the value of the IAA indicator and diagnose a severe degree of bronchopulmonary disease with indicators of IAA from 05 to 1.0, an average of more than 1.0 to 1.3 and a mild course with indicators of more than 1, 3 to 1.7

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

Inducted sputum (MI) of patients with bronchopulmonary diseases is used as a material for research. MI is obtained after inhalation of a 3-5% sterile hypertonic saline solution using an Omron CX-3 compression nebulizer (Japan). Then, they are transferred into test tubes using a sterile disposable syringe (5 or 10 ml). For dispersion and homogenization of sputum, a 0.1% dithiothreitol solution is used, which is added to the sputum in a ratio of 1: 1. The mixture is then shaken for 10 minutes and filtered through nylon gauze. The filtered MI is centrifuged at 1200 rpm for 10 minutes, then the supernatant is drained, and the cell suspension is washed 3 times in Hanks solution without phenol red by centrifugation at 1200 rpm for 10 minutes.

Sputum examination is carried out no later than 3 hours after receiving the material, throughout this time the samples are stored at a temperature of + 4 ° C.

To determine necrosis, a 0.1: 1 solution of Triton X-100, 0.08 N HCl, 0.15 M NaCl is added to the cell suspension at the rate of 1: 2, carefully mixed and left for 15 seconds, then a 5: 1 solution of cold acridine orange dye is added at a rate of 1: 2 / ml, based on citrate buffer (pH 6.0), including 1 mM EDTA-Na, 0.15 M NaCl, 0.2 M NaPO ₄ , 0.1 M. After staining, preparations are prepared for 5-10 minutes and immediately analyzed under a luminescent microscope.

To determine apoptosis, after mixing the cell suspension with a triton solution (the composition of the solution is indicated above), the cells are fixed with 4% paraformaldehyde based on distilled water, including 1% sodium cacodylate and 1% sodium chloride, then stained with specific Hoechst 33342 dye (5 mM) for detect apoptosis for 15 min and are analyzed under a luminescent microscope.

Damage (alteration) of induced sputum cells is assessed by viewing the preparations under a fluorescent microscope with exciting filters FS, SS, ES and a blocking filter type ЖS = 18 + ЖС = 19 using an immersion water lens. The results are obtained based on the evaluation of the color, intensity and morphology of fluorescent cells. In the case of evaluating cell alteration by necrosis, 100 cells are counted, of which% of yellow cells, including the dye - acridine orange, are determined and expressed as necrosis index (IN). Apoptotic cell activity is expressed as the apoptotic index (AI) -% of green cells containing Hoechst 33342 dye, and with the presence of morphological signs of apoptosis - altered nuclei consisting of 3 or more elements, and a reduced cytoplasm.

The cell alteration index (IAA) is calculated by the formula: IAA = IN / AI, where IN is an indicator of necrosis; AI - apoptotic index.

Compare the index of alteration of the cells of patients in the period of exacerbation with the IAK indicator of healthy individuals (control). A decrease in the IAA of myocardial phagocytes in the patient, compared with the values of healthy donors, indicates a violation of local protection in the target organ. Diagnose a severe degree of bronchopulmonary disease with an IAC from 05 to 1.0, an average from more than 1.0 to 1.3 and a mild course with indicators from more than 1.3 to 1.7

The basis for determining the effectiveness of the proposed method for evaluating bronchopulmonary diseases was the results of a study of induced sputum in patients with community-acquired pneumonia (CAP), bronchial asthma (BA) of mild, moderate and severe degree during inpatient treatment in the pulmonology department of the Main Hospital of the Pacific Fleet (Vladivostok) and therapeutic Branches of the Center for Industrial and Commercial Use (Vladivostok). The clinical diagnosis of bronchopulmonary diseases was established on the basis of generally accepted criteria. Assessment of the severity of the condition of patients at the time of admission was carried out according to the criteria proposed by L. I. Dvoretsky in 1996 (A. Okorokov. Diagnosis of respiratory diseases / A. N. Okorokov. - M .: Med. Lit., 2000. - 448 from.). The study included patients with a mild course of the disease - 60 (38.7%), with moderate-severe - 48 (37.4%) and severe 22 (23.9%) people. The control group consisted of 20 healthy individuals who did not have a history of chronic bronchopulmonary pathology, who had not had acute illnesses for 6 months. In this case, the IAC in healthy individuals is 2.0.

The study of indicators of alteration of cellular factors of local immunity (MI) in patients with bronchopulmonary diseases revealed clear changes in their values compared with those for healthy individuals. The lowest IAA value was observed during the exacerbation period in patients with severe course of CAP (2.7 times) and AD (2.6 times). At the same time, there was a clear difference in the values of IAA depending on the severity of the disease, the more severe the disease progresses, the lower the indicator of IAA. The lowest IAA values were observed in patients with CAP, which indicated the failure of local factors of lung protection in this disease.

Examples of specific performance.

Example No. 1

Patients diagnosed with community-acquired pneumonia (CAP) were treated with conservative inpatient treatment at the pulmonology department of the Main Hospital of the Pacific Fleet. CAP was diagnosed with a combination of newly occurring infiltrative changes in the chest radiograph and the following clinical signs (Torres, 1997): a) cough; b) purulent or mucopurulent nature of sputum; c) wheezing during auscultation or signs of compaction of the lung tissue; d) shortness of breath or tachypnea; e) peripheral blood leukocytes> 10,000 or <4,500 per ml or> 10% of stab neutrophils. Given that the patients included in the study were of the same sex, age, and had no concomitant diseases, the severity of the course of the EP was evaluated on the basis of the severity of clinical manifestations according to the criteria developed by Dvoretsky L.I. (1996). Patients underwent a MI study using the proposed technique. The data are presented in table 1.

Table 1 Indices of induced sputum cell alteration (PAA) in patients with CAP PACK VP of the first severity n = 26 VP of the second severity n = 18 VP of the third degree of severity n = 10 The control group, n = 20 IN,% 96 ± 6.8 59 ± 3.8 49 ± 3,5 45.2 ± 2.4 AI,% 70.01 ± 6.8 54 ± 4.6 66.7 ± 5.2 22.5 ± 1.6 Iak 1.4 1,1 0.7 2.0

According to the IAA values presented in the table, during the period of exacerbation, patients with CAP can be distributed according to the severity of the disease. In patients with the first (mild) degree of CAP, the value of IAA was 1.4, and in the second (medium) 1.1, while in patients of the third (severe) degree of the disease, 0.7.

Example No. 2

Patients with a diagnosis of bronchial asthma (AD) of varying severity of the disease were treated with conservative inpatient treatment at the therapeutic department of the ICEC. To diagnose AD and determine its severity, recommendations of a consensus adapted to Russian conditions were used (GINA, 2002), the clinical course of AD was assessed by changing the day and night symptoms, measured in points on a special scale from 0 to 5, frequency and the duration of exacerbations of the disease, indicators of the functions of external respiration. Patients underwent a MI study using the proposed technique. The data are presented in table 2.

table 2 Indices of induced sputum cell alteration (PAA) in AD patients PACK AD, mild disease severity n = 16 AD, moderate disease severity n = 14 AD, severe disease severity n = 8 The control group, n = 20 IN,% 42.3 ± 2.4 44.6 ± 3.4 39.3 ± 1.6 45.2 ± 2.4 AI,% 28.8 ± 1.8 37.3 ± 1.8 43.7 ± 3.4 22.5 ± 1.6 Iak 1,5 1,2 0.9 2.0

Thus, according to the values of IAA in the period of exacerbation of patients with AD can be distributed according to the severity of the disease. So, in patients with mild asthma disease, the value of IAA was 1.5, moderate - 1.2, and severe - 0.9.

Claims (1)

A method for diagnosing the severity of a disease in patients with bronchopulmonary diseases, including selecting the test material, isolating the cellular substance from it, preparing it for the study, determining the cell alteration by evaluating their apoptosis, characterized in that the induced sputum of the sick person is used as the test material in the cellular substance additionally determine the magnitude of cell alteration by necrosis, while apoptosis and necrosis are determined in two or more examinations with an interval of 1-3 days, the obtained indicators are expressed as the index of necrosis (IN) and apoptotic index (AI) and calculate the cell alteration index (IAC) according to the formula
IAK = IN / AI,
the severity of the disease in patients is determined on the basis of the value of IAA and diagnosed with a severe degree of bronchopulmonary disease with an IAA of 0.5 to 1.0, an average of more than 1.0 to 1.3 and a mild course with indicators of more than 1 , 3 to 1.7.