Vol 49, No 6 (2021)

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Full Issue


Evaluation of diagnostic accuracy of the automatic system for the analysis of digital lung X-ray for detection of spherical masses

Gavrilov P.V., Smolnikova U.A.


Rationale: Most data on the effectiveness of systems for the analysis of digital X-ray images have been provided by their developers and require a  high-quality validation in databases prepared independently of the developer.

Aim: To analyze the information content of automatic identification of spherical lung masses with digital X-ray imaging using one of the widely available diagnostic algorithms on publicly unaccessible reference datasets.

Materials and methods: The study was based on the recognition and analysis of digital X-ray images from two publicly inaccessible reference datasets that have the state registration (Russian Federation) with one of the publicly available diagnostic algorithms (FutureMed Analyzer). The study was performed using two models of X-ray screening as examples: Model  1 consisted of 100  X-ray images of the lungs with a  normal: abnormal ratio of 94%: 6%; Model  2 consisted of 5150 chest X-ray images with a normal: abnormal ratio of 97%: 3%.

Results: According to the results of the analysis of the X-ray images with the diagnostic system, 98%  of the images were correctly interpreted with Model 1 and 95% of the images, with Model 2. 83% of the cases from Model  1 and 69% from Model  2% were interpreted as images with lung abnormalities. The percentage of correct answers for differentiation of the chest X-ray images into two categories (normal vs. abnormal) for Model 1 and Model  2 was 95% and 98%, respectively. The sensitivity for detection of abnormal masses ranged from 69% to 83%. The specificity was 99% for the Model 1 chest X-ray images and 96% for the Model  2 chest X-ray images. The underdiagnosis rate was quite low ranging for Model 1 – 17%, and for Model 2  – 31%. The area under the curve for Model 1 was 0.91 and for Model 2 0.85.

Conclusion: The diagnostic efficiency of the automatic image analysis based on the convolutional neuronal networks approaches that of the radiologists. This system of automatic identification of abnormalities was unable to solve the most complex problems of detecting low density spherical masses (like "ground glass" area on computed tomography) and that of shadow summation for abnormalities located in such difficult to interpret zones as lung apices, clavicles, ribs, etc. To select a  suitable system, medical institutions need to conduct preliminary testing in their own models equivalent to the studies performed in a  given institution (parameters for radiography, nature and frequency of abnormalities).

Almanac of Clinical Medicine. 2021;49(6):359-364
pages 359-364 views

Investigation of the formation of cardiac tissue on substrates of varying degrees of anisotropy and rigidity

Shcherbina S.A., Shutko A.V., Nizamieva A.A., Nikitina A.V., Slotvitsky M.M., Tsvelaya V.A., Agladze K.I.


In the last decade, in vitro experiments have shown that mechanical properties of the bases could markedly influence the efficacy of differentiation of the induced pluripotent and embryonic stem cells and their development into the mature phenotype. By changing of mechanical, elastic and structural characteristics of the base, it is possible to increase the percentage of stem cells that differentiate to cardiomyocytes. The study was aimed at evaluation of the effects induced by changing physical characteristics of the base on the formation of phenotypic characteristics of cardiac cells. This included the comparison of structural properties of the cultured layer of heart tissue obtained by changing of elasticity and structure of polymeric bases. The results showed significant differences in calcium activity and structural characteristics of cardiomyocytes depending on the base properties, as well as significant variation in the excitation conduction. As long as conduction abnormalities in the heart tissues can often lead to occurrence of life-threatening cardiac arrhythmias, the results can be used to determine patient groups at increased risk of death from heart failure.

Almanac of Clinical Medicine. 2021;49(6):365-374
pages 365-374 views

Finite element analysis in the modeling of the heart and aorta structures

Smirnov A.A., Ovsepyan A.L., Kvindt P.A., Paleev F.N., Borisova E.V., Yakovlev E.V.


Rationale: 3D modeling of various anatomical structures has recently become a separate area of topographical, anatomical, and biomechanical studies. Current in vivo visualization methods and quantitative analysis in silico allow to perform the precise modeling of these processes aimed at investigation into the pathophysiology of cardiovascular disorders, risk prediction, planning of surgical interventions and virtual refinement of their separate stages.

Aim: To develop tools for elaboration, analysis and validation of personalized models of various structures of the heart and aortal arch taking into account their morphological characteristics.

Materials and methods: We used the results of 14 computed tomography studies from randomized patients without any disease or anomaly of the heart, aortic valve and aortal bulb. The analysis and subsequent transformation of the images were done with Vidar DICOM Viewer, SolidWorks 2016, VMTKLab software. For the FSI modeling of the aortic arch based on the results of functional multiaxial computed (MAC) coronarography (a female patient of 55 years) we developed a personalized model of the ascending aorta and aortic arch at the beginning of the systole. Using HyperMesh software (Altair Engineering Inc., USA) we have built a network of finite element of the luminal area, adventitia, and aortic media. To model mechanical properties of the aortic structures we used an anisotropic hyperelastic material model by Holzapfel – Gasser – Ogden. Material modeling, choice of the limiting antecedents, and analysis of fluid-structure interaction were performed with Abaqus CAE 6.14 software (Simulia, Johnston, USA). Adaptive image meshing by Young was used to elaborate the finite element template of the left ventricle. The algorithm was realized within the IDE PyCharm software media in Python 3.7. The algorithm was realized based on the open-source libraries OpenCV, NumPy, Matplotlib, and SciPy.

Results: The first stage of the development of the aortic valve model included the design of its virtual 3D template. Thereafter, a cohesive geometric model was elaborated. Subsequent stage of the work included the transformation of the aortic valve geometric model into the parametric one. This was done through the use of the “Equations” tool within the SolidWorks. No problems with geometry of the model during its deformation were identified. Aortic segment modeling was based on the data obtained by functional MAC coronarography. Based on this and on Inobitec Dicom Viewer software, we generated a multiplane reconstruction of the zone of interest including anatomical structure of the heart and aortic valve. With the resulting set of contours, we created a 3D model, which then was converted into a polygonal stereolithographic model. We developed an algorithm for adaptive meshing to elaborate a polygonal template capable of deformation that can be used for registration both with the net methods (B-Spline) and based on the image characteristics (homologous pixels).

Conclusion: The resulting parametric 3D model of the aortic valve anatomical structures is capable of adequate transformation of its geometry under external factors. It can be used in simulators of endovascular cardiosurgical procedures.

Almanac of Clinical Medicine. 2021;49(6):375-384
pages 375-384 views

Efficiency of SpCas9 and AsCpf1 (Cas12a) programmable nucleases at genomic safe harbor loci in HEK293 cells

Pavlova S.V., Elisaphenko E.A., Shayakhmetova L.S., Medvedev S.P.


Rationale: The development of eukaryote genome engineering tools based on CRISPR-Cas programmable bacterial nucleases systems opens wide horizons for gene therapies, human disease cell modeling, as well as investigation into manifestation of disease phenotypes and visualization of cellular processes. The safety and approximation of experiments both at the cellular and organismal levels depend on the accuracy of introducing double-stranded breaks into the target DNA regions. The search for new variants of more accurate CRISPR-Cas nucleases and evaluation of their ability to hydrolyze nucleosome DNA in vivo is considered a critical task for the development of the genome engineering technologies.

Aim: To analyze the activity of the programmable nuclease AsCpf1 (Cas12a), with low level of off-target activity, in the human genome loci that are safe for the introduction of transgenic constructs (“safe harbor”) and to compare its efficiency with that of the widely used SpCas9 nuclease in HEK293 cells.

Materials and methods: We performed the bioinformatics analysis of the association between target regions with nucleosomes and other proteins in the safe harbor loci AAVS1 and GSH-Ch1 and the transcriptionally inactive gene MYBPC3 (cardiac myosin binding protein 3) based on ATAC-seq data for the HEK293FT cells obtained from the NCBI SRA database. Plasmids encoding SpCas9 and AsCpf1 nucleases and guide RNA to the target regions were constructed and transfected into the HEK293FT cells. Events in the target regions of the HEK293FT cell genome were studied in the sequenograms with the TIDE algorithm.

Results: The results of the ATAC-seq experiments for HEK293FT cells have shown that the AAVS1 locus can be referred as open chromatin with a low nucleosome density, while the GSH-Ch1 locus can be attributed to closed chromatin. In HEK293FT cells, the cardiac MYBPC3 gene has intermediate chromatin density. Assessment of the efficiency of introducing breaks into the studied HEK293FT cell chromatin loci by nucleases has shown that SpCas9 is able to cope with chromatin of any nucleosome density, while AsCpf1 can effectively introduce DNA breaks only at loci with open chromatin, such as AAVS1 and MYBPC3. Editing events occur at a very low rate at the GSH-Ch1 locus with a high nucleosome density.

Conclusion: We have found low efficiency of the AsCpf1 nuclease in the genomic safe harbor locus GSH-Ch1, which is characterized by a high nucleosome density. When planning an experiment on AsCpf1 nuclease genome editing, the epigenetic chromatin landscape and the nucleosome density should be considered, as well as chromatin opening substances should be used.

Almanac of Clinical Medicine. 2021;49(6):385-395
pages 385-395 views

Assessment of an antitumor effect of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone in A-549 tumor cell subcutaneous xenografts

Lukbanova E.A., Zaikina E.V., Sayapin Y.A., Gusakov E.A., Filippova S.Y., Zlatnik E.Y., Volkova A.V., Kurbanova L.Z., Khodakova D.V., Kaymakchi D.O., Lazutin J.N., Shevchenko A.N., Pandova O.V.


Rationale: Chemotherapy is one of the lung cancer treatment methods. The search for new substances with antitumor effect against malignant lung neoplasms is relevant because of low efficacy and side effects of cytotoxic agents. A promising substance class with various biological activities, including antitumor, includes alkaloids of the tropolone family, such as heptamerous non-benzoid aromatic compounds. 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)- 5,6,7-trichloro-1,3-tropolone has been synthesized in Institute of Physical and Organic Chemistry; it is a  new compound belonging to 2-quinoline-2-yl derivatives of 1,3-tropolone.

Aim: To assess the antitumor effect of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)- 5,6,7-trichloro-1,3-tropolone on subcutaneous xenografts of A-549 lung tumor cells in immunodeficient Balb/c Nude mice.

Materials and methods: The study included 50  immunodeficient Balb/c Nude mice divided into 4  experimental groups depending on the dosage of the study substance (0.0055, 0.055, 0.55, and 2.75  mg/g); group 5  was the control group. A-549  cells of lung cancer were used as a xenograft. The antitumor effect of tropolone was evaluated by the inhibition of tumor growth and the index of tumor growth. The experiment lasted for 36 days starting from the first administration of the substances.

Results: The mean tumor volumes on day 36  of the experiment in the control group and four experimental groups were 2729.5; 2150.8; 1746.4; 952.3  and 678.9  mm3 , respectively. The indices of tumor growth in groups  1, 2, 3 and 4 were significantly lower than in group 5 (control) starting from days 24, 21, 21 and 15, respectively, and till the end of the experiment. Maximal differences between groups 4 and 5 were observed at days 33 and 36 (by  3.7, p=0.01 and 4.1, p=0.003  times, respectively).

Discussion: The anti-tumor effect of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)- 5,6,7-trichloro-1,3-tropolone demonstrated in the study could be related to various mechanisms. For example, numerous studies have shown that its related compound hinokitiol exerts a cytotoxic effect associated with cessation of the cell cycle, apoptosis induction, DNA damage, and autophagic death of tumor cells.

Conclusion: The study demonstrated significant differences in xenograft volumes in all experimental groups, compared to the control group. In mice, 2.75  mg/g bodyweight was the most effective dosage of the studied compound leading to a slow decrease in tumor growth rates and a  decrease in the volumes of subcutaneous xenografts.

Almanac of Clinical Medicine. 2021;49(6):396-404
pages 396-404 views

Diagnostic criteria of lymphoproliferative diseases from the peripheral blood samples using a cell biochip

Fedyanina O.S., Chuksina Y.Y., Khmelevskaya A.N., Khvastunova A.N., Matveev Y.N., Kataeva E.V., Filatov A.V., Kuznetsova S.A.


Background: At present, the diagnosis of lymphoproliferative disorders is based on the combination of blood or bone marrow smear morphology and immunophenotyping by flow cytometry. Immunophenotypic testing by flow cytometry technique is available only in big medical centers, which is not always convenient for a  patient. Therefore, development of an available method for preliminary diagnosis of lymphoproliferative diseases not requiring special equipment seems relevant.

Materials and methods: Peripheral blood mononuclear cells from 17  patients admitted to the hospital with suspicion of a  lymphoproliferative disorder, and 17  healthy donors were studied on a cell biochip for determination of proportions of cells positive for various surface CD antigens. The diagnosis was verified by flow cytometry.

Results: Compared to healthy controls and patients with T-cell lymphoproliferative disorders (TCLPD), the patients with B-cell lymphoproliferative disorders (BCLPD) had significantly lower proportion of CD7+ cells (medians, 7% and 73% respectively, p=2×10-6 for comparison with healthy controls; median  7% and 93% for comparison with TCLPD, p=0.032). In addition, the patients with BCLPD had higher proportion of peripheral СD19+ mononuclear cells, compared to that in the patients with TCLPD and healthy donors (medians 84% and 13% for comparison between BCLPD and healthy control, p=2×10-5; 84% and 3% for comparison of BCLPD and TCLPD, p=0.033). The patients with B-cell chronic lymphocytic leukemia had significantly higher CD5+ cells in the cell biochip compared to the patients with other BCLPD (medians 72% and 9%, p=0.024). The patients with TCLPD had significantly lower proportion of CD19+ cells than the healthy controls (medians, 3% and 13%, respectively, р=0.042).

Conclusion: The study has demonstrated the potential to use the previously developed cell biochip for diagnosis of lymphoproliferative diseases. The biochip makes it possible to sort out white blood cells according to their surface differentiation antigen for their further morphological analysis. The cell biochip allows for the differential diagnosis between BCLPD and TCLPD and determination the lymphocyte clones based on the expression of immunoglobulin light chains.

Almanac of Clinical Medicine. 2021;49(6):405-411
pages 405-411 views

Comparison of modulation interference microscopy, DNA spectrometry, DNA cytometry, and flow cytofluorimetry in the assessment of phytohemagglutinin-induced activity of human blood lymphocytes

Sustretov A.S., Bogush V.V., Guseva O.S., Iliasov P.V., Limareva L.V.


Rationale: The study of the structural particulars and functional state of immune cells and primarily lymphocytes is of great importance for both fundamental and clinical medicine. It requires the development of simple and reliable analytic methods that would allow for fast and effective real-time assessment of cell activity.

Aim: To evaluate the effectiveness of the interference microscopy compared to DNA spectrometry, DNA cytometry, and flow cytometry with an internalized fluorescent label CFSE (carboxyfluorescein succinimidyl ester) in the assessment of PHA-induced proliferation of human blood lymphocytes.

Materials and methods: Phytohemagglutinin (PHA)-induced proliferative activity of blood lymphocytes from 10  healthy volunteers was studied with various methodological strategies. Blast transformation of lymphocytes was induced by their incubation for 5 days with PHA 5 μg/mL. The cell proliferative activity was assessed as follows: 1) by DNA spectrometry at 260/280 nm using Tecan Infinite 200  Pro with a  specialized NanoQuant Plate™; 2)  by cytophotometry followed by cell distribution analysis assessing deoxyribonucleic acid (DNA) content after staining with Felgen's dye with an imaging system based on an Olympus BX41 light microscope with a ProgRes CF camera; 3) by flow cytometry using an internalized fluorescent label CFSE; the analysis was performed with a BD FACS Calibur flow cytometer; 4) by measurement of the lymphocyte interference profile with a  modulation interference microscope MIM-340 (Schwabe, Russia). The functional activity of the nucleus (FAN) was determined and used as a criterion for assessment of the lymphocyte functional state.

Results: Incubation of lymphocytes with PHA led to an increase in the linear size by 22.2±2.8%, a  decrease in phase height by 46.3±4.7% (p=0.019), and an increase in FAN by 75.9±9.4%, vs control (p=0.046). As measured by isolated DNA spectroscopy, PHA stimulation of lymphocytes was associated with an increase in the amount of DNA by  55% vs baseline (409.8±22.3  ng/μL and 264.3±25.0  ng/μL, respectively, p=0.049). Felgen's reaction revealed that the proportion of nuclei containing more than 2n DNA was 2% in the control cells and 14.8% in the PHA-activated lymphocytes, with a  difference between the groups of 12.8%. CFSE staining with subsequent incubation and assessment by flow cytofluorimetry demonstrated an increase in the percentage of proliferating cells from 1.68±0.9% in the control to 55.56±5.6% (p=0.00068) in the mitogen-stimulated sample.

Conclusion: Modulation interference microscopy does not require the sample preparation and demonstrated comparable and even higher effectiveness compared to conventional methods for assessment of lymphocyte activity. At the same time, it allows for evaluation of the lymphocyte functional state in real time in the process of cultivation. This opens ample opportunities for evaluation immune cells for research and diagnostic purposes.


Almanac of Clinical Medicine. 2021;49(6):412-418
pages 412-418 views

Structural and functional abnormalities of cell membranes in the end-stage chronic kidney disease

Ievlev E.N., Kazakova I.A., Sukhenko E.P.


Background: Chronic kidney disease (CKD) is associated with abnormalities in all functions of the body systems including changes in intracellular processes. Assessment of erythrocyte electrophoretic mobility (EEM) in patients with CKD stage  5 on dialysis (5d) has becoming increasingly relevant, since this method characterizes the pathophysiological state of the patient and gives the possibility to modify treatment.

Aim: To identify EEM characteristics in patients on programmed hemodialysis and their association with clinical and laboratory parameters.

Materials and methods: We performed a  cross-sectional observational study in 220  patients with confirmed CKD 5d. The average age of the patients was 56.5±1.4  years (26 to 85 years) and the duration of dialysis therapy was 3.7±0.4 years. The Kt/V urea adequacy index was 1.54±0.08. The control group included 60 healthy blood donors, comparable for their age and sex. EEM was assessed with Cyto-Expert kit (Axion Holding, Izhevsk, 2010) and the WT-Cell program (LLC Westtrade LTD, 2019). Statistical analysis was performed with BioStat 2019 software.

Results: The patients on the programmed hemodialysis had lower values of oscillation amplitude (10.2±0.5  μm and 21.2±2.1  μm, p<0.001) and lower proportion of mobile red blood cells (69.5±1.8%, 89.7±9.9%, p<0.001), compared to the control group. Lower values of the oscillation amplitude were found in the age group of 25 to 44  years (9.0±1.0  μm, p<0.05). There was a  weak positive correlation between age and amplitude of erythrocyte oscillation (R=0.20, p<0.05). There were differences in the oscillation amplitude values in the patients with various dialysis experience: 1 to 2 years, 11.3±0.8 μm, 2 to 5 years, 9.9±0.7 μm, 6 to 10 years, 9.4±1.3 μm, and over 11 years, 7.4±0.9 μm (p<0.05). The duration of dialysis therapy demonstrated a weak negative correlation with the amplitude of erythrocyte oscillation (R=-0.24, p<0.01). The erythrocyte oscillation amplitude was associated with systolic blood pressure before hemodialysis procedure (R=0.34, p<0.05) and with pulse pressure before hemodialysis (R=0.37, p<0.05). The proportion of mobile erythrocytes correlated with parathyroid hormone level (R=0.32, p<0.05).

Conclusion: EEM in the patients receiving programmed hemodialysis have their specific characteristics related to a significant decrease in the oscillation amplitude proportional to the effective cell charge and lower proportions of mobile erythrocytes compared to those in the healthy control. The erythrocyte oscillation amplitude is negatively correlated with age and duration of dialysis therapy and is associated with blood pressure parameters and mineral bone indices.


Almanac of Clinical Medicine. 2021;49(6):419-426
pages 419-426 views

Atomic force microscopy in the assessment of erythrocyte membrane mechanical properties with exposure to various physicochemical agents

Sherstyukova E.A., Inozemtsev V.A., Kozlov A.P., Gudkova O.E., Sergunova V.A.


Background: Mechanical properties of cell membranes and their structural organization are considered among the most important biological parameters affecting the functional state of the cell. Under the influence of various pathogenic factors, erythrocyte membranes lose their elasticity. The resulting changes in their biomechanical characteristics is an important, but poorly studied topic. It is of interest to study the deformation of native erythrocytes to a depth compatible with their deformation in the bloodstream.

Aim: To investigate the patterns of deep deformation and the particulars of structural organization of native erythrocyte membranes before and after their exposure to physicochemical agents in vitro.

Materials and methods: Cell morphology, nanostructure characteristics, and membrane deformation of native erythrocytes in a  solution of hemoconservative CPD/SAGM were studied with atomic force microscope NTEGRA Prima. Hemin, zinc ions (Zn2+), and ultraviolet (UV) radiation were used as modifiers. To characterize the membrane stiffness, we measured the force curves F(h), hHz (the depth to which the probe immersion is described by interaction with a homogeneous medium), and the Young's modulus values of the erythrocyte membrane.

Results: Exposure to hemin, Zn2+ and UV radiation led to transformation of the cell shape, appearance of topological defects and changes in mechanical characteristics of erythrocyte membranes. Under exposure to hemin, Young's modulus increased from 10±4  kPa to 27.2±8.6  kPa (p<0.001), exposure to Zn2+, to 21.4±8.7  kPa (p=0.002), and UV, to 18.8±5.6  kPa (p=0.001). The hHz value was 815±210  nm for the control image and decreased under exposure to hemin to 420±80 nm (p<0.001), Zn2+, to 370±90 nm (p<0.001), and UV, to 614±120 nm (p=0.001).

Conclusion: The results obtained contribute to a  deeper understanding of interaction between membrane surfaces of native erythrocytes and small vessel walls. They can be useful in clinical medicine as additional characteristics for assessment of the quality of packed red blood cells, as well as serve as a basis for biophysical studies into the mechanisms of action of oxidative processes of various origins.

Almanac of Clinical Medicine. 2021;49(6):427-434
pages 427-434 views


Visual and acoustic feedback on the support reaction for upper and lower extremities: a case study of a female patient after a stroke

Isakova E.V., Egorova Y.V.


Stroke is the leading cause of adult disability worldwide. The consequences of a stroke can include various disorders, namely, motor, cognitive, emotional, and behavioral disorders, which, in their turn, lead to a decreased daily life activities and self-care ability, and affect quality of life of the patients. This article describes a  clinical case of the implementation of a  new comprehensive program for neurorehabilitation treatment in a female patient in the early recovery period from ischemic stroke with spastic hemiparesis, moderate cognitive impairment and affective disorders. The neurorehabilitation program is based on the use support reaction biofeedback through auditory and visual channels. Classical static and dynamic stabilometric trainings were sequentially used in the patient standing on the platform, as well as exercises with a force joystick for the upper limb in the sitting position. The multifaceted program also included classes with a speech therapist/neuropsychologist, as well as physical therapy with an instructor. The treatment resulted in an improvement in the patient's general condition, restoration of the upper limb movement function, normalization of cognitive functions, and regression of affective disorders. The treatment-related effects were persistent, and the assessment of the neurological status at 3 months' follow-up showed no deterioration of neurological symptoms. Thus, the multifaceted rehabilitation treatment based on the combination of increased physical activity and sensory stimulation, as well as cognitive tasks, may facilitate good recovery after a stroke.

Almanac of Clinical Medicine. 2021;49(6):435-442
pages 435-442 views

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