1Federal State Autonomous Educational Institution of Higher Professional Education "Belgorod State National Research University", Pobedy st., home 85, Belgorod, 308017
2Regional State Budgetary Institution of Health "Belgorod Regional Clinical Hospital of St. Joasaph", 308007, Belgorod, Nekrasov Street, 8/9
In order to determine the viability of the blood leukocytes of patients with ischemic heart disease in various conditions of their cultivation in vitro, a study was conducted of 33 blood samples of patients. When performing the analysis, 4 observation groups were identified: the 1st group was consisted of 30 patients who had all the necessary conditions for blood sampling, selection and transportation of leukocyte suspension, their cultivation and analysis; the 2nd was one patient, the test of which was "randomly" incubated without maintaining the appropriate gas composition in the thermostat, the 3rd was one patient who's leukocyte suspension was ?mistakenly? contaminated with microbial agents from nutrient medium; the 4th was one patient in whom the blood was collected correctly, but for technical reasons the analysis was performed the next day, i.e. yesterday's leukocytes, which stood under hypothermia (+ 4 C) for 24 hours, were analyzed. The 5th group consisted of 30 patients to whom a drug of metabolic series cytoflavin at a therapeutic concentration was introduced into a sample with leukocyte suspension in vitro.
Materials and research methods. Blood sampling was performed in the morning on an empty stomach into a vacuum tube with EDTA. Leukocytes of blood were collected under aseptic conditions, mixed with nutrient medium RPMI-1640 with glutamine (PanEco company, Russia), placed in wells for examination under a microscope. The preparations were stained with fluorescent dyes: MitoTracker - Red CMXRos - allows to evaluate the mitochondrial membrane potential; Calcein AM - stains only viable cells; Ethidium bromide - stains only dead cells. Samples were incubated for 3 hours in a CO2 incubator with 5% CO2 content at 37 C. The results were evaluated by light and fluorescence microscopy using an inverted fluorescence microscope and specialized software. The materials were processed statistically with the calculation of arithmetic averages and their errors, the calculation of the index of viability of leukocytes and the determination of the reliability of differences by the Student's t-test.
The results of the study. In all patients of the 1st observation group, cell viability remained high (IL = 0.41 - 0.05) with preservation of the energy supplying function of mitochondria (the fluorescence value of mitochondria was 120.20 - 4.18 rel. units). In patients of the 2nd and 3rd observation groups, all leukocytes were killed with destroyed mitochondria, which is why it is not possible to determine the magnitude of mitochondrial fluorescence and viability index. In the patient of the 4th observation group, we found the initial signs of leukocyte destruction with impaired energy-generating function of mitochondria (the fluorescence value was 113.15 ? 2.12 rel. units, p <0.05 compared with the 1st group of patients). In patients of 5th group, when cytoflavin was injected into a sample with leukocyte suspension, two variants of changes in cell viability were observed: in 80% of patients an increase in viability of leukocytes (IL increased from 0.02 - 0.01 to 0.62 - 0.02, p <0.001) and in 20% - a decrease in this indicator (IL decreased from -0.13 - 0.01 to -0.60 - 0.03, p = 0.09), while the fluorescence of mitochondria changed in both cases upwards, but without achieving significant differences; morphologically, the leukocytes did not change with the introduction of cytoflavin, they corresponded to the norm.
Conclusion. Studies of human blood leukocytes should be carried out under all the necessary conditions for blood sampling (in a vacuum tube in the morning on an empty stomach), selection of a leukocyte suspension (with the addition of a nutrient medium exclusively under aseptic conditions), and their cultivation (in a CO2 incubator with 5% carbon dioxide) and analysis (one day blood sampling). Failure to comply with any of these technical procedures is accompanied by impaired mitochondrial functions, followed by their destruction and leukocyte death. According to an in vitro study, the drug of the metabolic series cytoflavin in patients with coronary heart disease in 80% of cases helps to improve cell viability.
leukocytes, viability, coronary heart disease, patients, microscopy, cytoflavin, in vitro study
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