Criteria and Method for Detection of Circulating Tumor Cells
The purpose of the work is to form a complex of criteria for tumor malignancy evaluation and to improve the method of detecting circulating tumor cells in human blood. Мета роботи — формування комплексу критеріїв оцінювання злоякісності пухлини та удосконалення методу виявлення циркулюючих пухлинних...
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Chernyshova, T.A. 2019-12-19T16:28:24Z 2019-12-19T16:28:24Z 2019 Criteria and Method for Detection of Circulating Tumor Cells / T.A. Chernyshova // Cybernetics and computer engineering. — 2019. — № 1 (195). — С. 82-97. — Бібліогр.: 35 назв. — англ. 2663-2578 DOI: https:// 10.15407/kvt195.01.082 https://nasplib.isofts.kiev.ua/handle/123456789/161696 602.9:611.018 The purpose of the work is to form a complex of criteria for tumor malignancy evaluation and to improve the method of detecting circulating tumor cells in human blood. Мета роботи — формування комплексу критеріїв оцінювання злоякісності пухлини та удосконалення методу виявлення циркулюючих пухлинних клітин в крові людини. Предложенный метод определения циркулирующих опухолевых клеток, который является усовершенствованием технологии ISET, объединяет два этапа. На первом этапе осуществляется выделение ЦОК с совершенствованием за счет установки двух дополнительных поликарбонатных фильтров с порами диаметром 5 и 3 мкм и обеспечении режима 100% герметизации камеры гемолизации и постоянного давления в течение всего процесса фильтрации. На втором этапе при использовании сформированного комплекса критериев осуществляется определение степени злокачественности выделенных клеток. Использование разработанного метода в автоматизированной системе анализа цифровых микроскопических изображений циркулирующих опухолевых клеток обеспечивает определение и расчет характерных признаков для отнесения объекта к определенному классу злокачественности и формирования базы данных сканированных изображений с занесением имеющихся клеток или их образований, а также окончательную проверку результатов оценки злокачественности опухоли по шаблонаммаскам ЦОК и доброкачественной опухолевой клетки. en Міжнародний науково-навчальний центр інформаційних технологій і систем НАН України та МОН України Кибернетика и вычислительная техника Medical and Biological Cybernetics Criteria and Method for Detection of Circulating Tumor Cells Критерії та метод виявлення циркулюючих пухлинних клітин Критерии и метод выявления циркулирующих опухолевых клеток Article published earlier |
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| title |
Criteria and Method for Detection of Circulating Tumor Cells |
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Criteria and Method for Detection of Circulating Tumor Cells Chernyshova, T.A. Medical and Biological Cybernetics |
| title_short |
Criteria and Method for Detection of Circulating Tumor Cells |
| title_full |
Criteria and Method for Detection of Circulating Tumor Cells |
| title_fullStr |
Criteria and Method for Detection of Circulating Tumor Cells |
| title_full_unstemmed |
Criteria and Method for Detection of Circulating Tumor Cells |
| title_sort |
criteria and method for detection of circulating tumor cells |
| author |
Chernyshova, T.A. |
| author_facet |
Chernyshova, T.A. |
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Medical and Biological Cybernetics |
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Medical and Biological Cybernetics |
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2019 |
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English |
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Кибернетика и вычислительная техника |
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Міжнародний науково-навчальний центр інформаційних технологій і систем НАН України та МОН України |
| format |
Article |
| title_alt |
Критерії та метод виявлення циркулюючих пухлинних клітин Критерии и метод выявления циркулирующих опухолевых клеток |
| description |
The purpose of the work is to form a complex of criteria for tumor malignancy evaluation and to improve the method of detecting circulating tumor cells in human blood.
Мета роботи — формування комплексу критеріїв оцінювання злоякісності пухлини та удосконалення методу виявлення циркулюючих пухлинних клітин в крові людини.
Предложенный метод определения циркулирующих опухолевых клеток, который является усовершенствованием технологии ISET, объединяет два этапа. На первом этапе осуществляется выделение ЦОК с совершенствованием за счет установки двух дополнительных поликарбонатных фильтров с порами диаметром 5 и 3 мкм и обеспечении режима 100% герметизации камеры гемолизации и постоянного давления в течение всего процесса фильтрации. На втором этапе при использовании сформированного комплекса критериев осуществляется определение степени злокачественности выделенных клеток. Использование разработанного метода в автоматизированной системе анализа цифровых микроскопических изображений циркулирующих опухолевых клеток обеспечивает определение и расчет характерных признаков для отнесения объекта к определенному классу злокачественности и формирования базы данных сканированных изображений с занесением имеющихся клеток или их образований, а также окончательную проверку результатов оценки злокачественности опухоли по шаблонаммаскам ЦОК и доброкачественной опухолевой клетки.
|
| issn |
2663-2578 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/161696 |
| citation_txt |
Criteria and Method for Detection of Circulating Tumor Cells / T.A. Chernyshova // Cybernetics and computer engineering. — 2019. — № 1 (195). — С. 82-97. — Бібліогр.: 35 назв. — англ. |
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2025-11-25T22:47:33Z |
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| fulltext |
ISSN 2663-2586 (Online), ISSN 2663-2578 (Print). Cyb. and comp. eng. 2019. № 1 (195)
DOI: https:// 10.15407/kvt195.01.082
UDC 602.9:611.018
CHERNYSHOVA T.A., physician,
e-mail: tetyana.che@gmail.com
Aviation Medical Center of the
National Aviation University,
Komarova av., 1, Kyiv, 03058, Ukraine
CRITERIA AND METHOD FOR DETECTION OF CIRCULATING TUMOR CELLS
Introduction. Modern advances in science and technology have substantially expanded the possibili-
ties for detecting malignant neoplasms. A great number of methods for the detection and allocation of
circulating tumor cells clearly indicates the interest of researchers to this problem.
The purpose of the work is to form a complex of criteria for tumor malignancy evaluation
and to improve the method of detecting circulating tumor cells in human blood.
Results. The proposed method for determining circulating tumor cells, which is an im-
provement of ISET technology, combines two stages. At the first stage the improvement is in
adding two additional polycarbonate filters with 5 and 3 micron diameter pores, and provid-
ing a mode of 100% sealing of the chamber with hemolysis, and constant pressure through-
out the filtration process. At the second stage, we carried out the determination of malig-
nancy degree of the isolated cells using the developed set of criteria. The use of the developed
method in the automated system for the analysis of digital microscopic images of circulating
tumor cells provides the detection and calculation of characteristic features for assigning an
object to a certain class of malignancy and the creation of scanned images database with
recorded existing cells or their entities, as well as the final verification of the results of tumor
malignancy evaluation for template masks of circulating tumour cells and benign tumor cells.
Conclusions. The application of the proposed method for the detection of circulating tu-
mor cells allows detecting smaller cells than in case of using traditional methods, ensures their
integrity and intactness.
Keywords: circulating tumor cells, criteria for evaluation of tumormalignancy, method of de-
termining circulating tumor cells in human blood.
INTRODUCTION
Modern achievements in science and technology have substantially expanded the
possibilities for detecting malignant neoplasms. However, despite the wide arsenal of
auxiliary research methods, the physician often has significant difficulties in making
the accurate diagnosis, which is ultimately reflected in the treatment. The problem of
detecting and recognizing the circulating tumor cells (hereinafter — CTС) has become
relevant in the last few decades because it is important for the treatment of a patient for
© CHERNYSHOVA T.A., 2019
82
Criteria and Method for Detection of Circulating Tumor Cells
ISSN 2663-2586 (Online), ISSN 2663-2578 (Print). Cyb. and comp. eng. 2019. № 1 (195) 83
a number of reasons [1–2]. At the beginning of the primary tumor development, the
detecting of CTC allows making an early diagnosis. The amount of CTC is reduced in
response to treatment, that is why monitoring the CTC amount let us analyze the effec-
tiveness of a particular treatment regimen [3]. At the same time, the change in the CTC
amount can be used as a means for tracking relapse in patients with no signs of disease
in case of adjuvant chemotherapy [4].
There are a great number of methods for CTC detecting and allocating which
clearly point to researchers interest to this problem. Despite the diversity of meth-
ods, the findings of patient screening and determining the clinical prognosis of
existing CTC is always associated with a negative prognosis for survival.
THE PROBLEM STATEMENT
Now, the tremendous efforts of doctors and scientists are focused on optimizing
the methodological, informational and technical aspects of the CTC detection and
evaluation for various types of cancer and, above all, breast cancer, prostate can-
cer, skin cancer (melanoma), colorectal cancer and micellar lung cancer.
These efforts are mainly aimed at reducing the qualitative and quantitative er-
rors in the CTC calculation, as well as minimizing the CTC loss caused by exces-
sive blood transfusion during treatment. Most of the available approaches for cap-
turing CTC are based on the differences in these cells from normal cellular blood
components by physical (size, density, potential difference) and molecular (e.g.,
surface expression) characteristics.
Optical method for estimating the CTC amount in a blood sample is based on
automatic digital microscopy. To reduce the time of scanning large areas of cyto-
logical preparations, the technology of massive optical fiber scanning is proposed,
which can scan 500 times bigger area than automatic digital microscopy technology
without degrading the sensitivity of this process for a shorter period of time [5, 6].
There are a number of technologies and systems based on the methods of im-
munofluorescence, immunomagnetic division, flow cytometry and other immu-
nomagnetic methods that allow the tumor cells isolation by means of magnetic field,
using the ability of these cells to interact with antibodies against CTC markers with
conjugated magnetic particles [7–14]. But there is no analytical consideration of
using such technologies for the purpose of early non-invasive detection of cancer.
The CTC selection was also performed morphologically. One of the direc-
tions of such methods development is the detection of the studied CTC by size
using microfilters [15, 16]. Although these methods allow a significant increase in
resolution, cells are often deformed and destroyed during detection [16, 17], which
stimulates to pay special attention to prevent such destruction or to increase the
integrity of the detected cellular fractions [15, 17].
One of the most informative markers for the tumor process progression is the
CTC in blood, the increased number of which can lead to a more thorough examina-
tion of patients in the "asymptomatic period" [18, 19]. According to the results of
randomized trials conducted in several clinical and research centers, authors deter-
mined the role of the CTC level increasing in the development of tumor progression
in breast cancer, prostate and colorectal cancer [20]. In the studied patients, elevated
levels of CTC (from 3 to 400 cells per specimen) were detected in 75% of cases.
Chernyshova T.A.
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Table 1. Dependence of the amount of CTC in peripheral blood on the degree of the tumor
differentiation [20]
Degree of the tumor differentiation
Number of patients,
absolute numbers (%)
with CTC > 3n = 30
Number of patients,
absolute numbers (%)
with CTC < 3n = 10
Well differentiated adenocarcinoma 5 (12,5 %) 3 (7,5 %)
Moderately differentiated adenocar-
cinoma 9 (25 %) 5 (10 %)
Poorly differentiated adenocarci-
noma 16 (42,5 %)* 2 (2,5 %)*
Note: * - a statistically significant difference in the detection of patients with CTC > 3 and < 3 with
poorly differentiated adenocarcinoma, p = 0.0235.
According to the standardized requirements for the CTC study, the sample is consid-
ered to be positive for any number of CTC which exceeds 3 cells. The degree of tumor
differentiation affects the amount of CTC in peripheral blood (Table 1).
Retrospective analysis showed that early relapses and metastases (up to 24 months)
developed more frequently in patients with the CTC level of more than 3 cells.
Due to the analysis of numerous studies in this area, the presence of circulating tu-
mor cells in the blood indicates the acquisition of fundamentally new qualities of tumor
such as invasiveness and the ability to metastasize. Undoubtedly, CTCs are not the only
key to understanding the biology of metastasis, but also a prognostic and predictive
newcomer oncomarker that reflects the effectiveness of antitumor treatment. One of the
most important issues for improving the methods of determining the CTC is to ensure
the separation of integral and intact CTCs with further evaluation and determination of
effective criteria for assigning selected cells to malignant neoplasms.
The purpose of the work is to form a complex of criteria for tumor malig-
nancy evaluation and to improve the method of detecting circulating tumor cells
in human blood.
COMPLEX OF CRITERIA FOR DETERMINING
THE MALIGNANCY OF CIRCULATING TUMOR CELLS
The principle of forming a set of criteria is a combination of the sequential defini-
tion of two groups of criteria and the expansion of the criterion space to 5 micron
and 3 micron cells.
The 1st group of criteria. By the ISET method [21–24], the definition of tumor
malignancy is carried out according to five criterion characteristics: the variation of
the nuclei size of isolated cells, anisonucleosis — C1; exceeding the size of the cell
nucleus for a triple calibrated pore size — C2; presence of abnormalities of the nu-
cleus (shape, contours, area) — C3; the value of nuclear-cytoplasmic ratio (NCR),
that is, the ratio of the area of the nucleus (Sn) to the area of the cytoplasm of the cell
(Sc) — C4; the presence of cellular complexes-C5 (Table 2).
Criteria and Method for Detection of Circulating Tumor Cells
ISSN 2663-2586 (Online), ISSN 2663-2578 (Print). Cyb. and comp. eng. 2019. № 1 (195) 85
Table 2. Criteria for tumor malignancy according to ISET in our version
Criterion characteristics Criterion
1 Anioniculosis is the ratio of different sizes of cell nuclei C1 > 0,5
- for 8 µm pore size C2 > 24 µm
- for 5 µm pore size C2 > 15 µm 2 Nuscleus
size
- for 3 µm pore size C2 >7,5
3 Abnormality of the nucleus (shape, contours, squares) present
4 Nuclear-cytoplasmic ratio high
5 Cellular complexes present
Table 3. Criteria for the degree of the tumor malignancy according to P. Scarft,
H. Bloom, W. Richardson [25]
Index Criterion Points
R1 >75% 1
75% > R1 >10% 2 Formation of tubular and flow
structures
R1 <10% 3
R2 <10% 1
20% > R2 >10% 2 The number of mitoses
R2>20% 3
cells of the same size and shape, shallow,
with dispersed distribution of chromatin,
without nucleoli
1
small polymorphism of the nuclei and some
aggregation of cells 2 Cellular polymorphism
The nuclei are large, of different shapes,
with one or more nucleoli and coarse chro-
matin 3
A detected CTC is considered to be malignant if it meets 4 of the above 5 criteria.
The 2nd group of criteria. To improve the results reliability in the case of some
forms of cancer and, when determining the degree of tumor malignancy becomes a
complex and time-consuming process, we additionally use the modified scheme of
P. Scarft, H. Bloom, W. Richardson [25]. It reveals 3 groups of malignancy ac-
cording to such characteristics as formation of tubular and flow-like structures
(R1), number of mitoses (R2), cellular polymorphism (R3) (Table 3).
The degree of tumour malignancy (hereinafter — DTM) is determined by the
sum of points according to three characteristics:
DTM = R1 + R2 + R2
Classification of the degree of malignancy:
I (low) — 3–5 points;
II (moderate) — 6–7 points;
III (high) — 8–9 points.
Thus, the developed set of criteria for the DTM is based on a combination of
sequential determination on two criteria scales: 1) ISET with the proposed expan-
sion of the criterion space on the cell range (5 microns and 3 microns), and 2) the
modified scheme of P. Scarft, H. Bloom, W. Richardson [25].
Chernyshova T.A.
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DETECTION AND EVALUATION OF MALIGNANCY OF CIRCULATING TUMOR CELLS
Using the Isolation by Size of Tumor Cells Isolation Method. The basic method
of our study is the Isolation by Size of Tumor Cells (ISET®) method [21, 22, 26].
This innovative, proprietary method allows for the loss of isolation from CTCs (as
well as circulating tumor microembolism or clusters from several CTCs), provided
that their integrity is maintained and analyzed by various methods, including blood
cytopathology [27, 28]. In addition, ISET® Cytopathology for blood is the most
sensitive method for CTC diagnosis, which is an early sign of cancer invasion.
Long before the metastases, the CTCs flow in the bloodstream and can be diag-
nosed using this technology. Thus, oncologists receive additional information
about the invasive potential of cancer which helps to stop cancer invasion before
the formation of metastases [28].
The ISET system is based on the classic cytopatological criteria: tumor cells,
originating from various solid tumors (more than 16 microns in diameter) are lar-
ger than blood cells. The ISET technology allows quick processing of a blood
sample on a filter without using antibodies and isolated unharmed fixed tumor
cells up to 8.5 μm in diameter using a standard membrane with a nominal pore
size of 8 μm [29].
Improvement of the method. The question of the hypothetical loss of
"small" CTC through pores of 8 microns is really important. We emphasize that to
this day the minimum size of the CTC diagnosed with different methods was more
than 8 microns.
Improvement of the basic method is carried out due to:
- installation of two additional polycarbonate filters with 5 and 3 microns
diameter pores,
- providing a mode of 100% sealing of the camera with hemolysis and con-
stant pressure throughout the filtration process,
- use of the developed set of criteria,
- application of the proposed method for processing microscopic CTC im-
ages, which enables to detect CTC of any size, integral and intact.
The research was carried out on an experimental installation, which was de-
veloped by an author team of scientists from Vinnitsa and Mariupol [30]. Differ-
ences in the methods, approaches, information provision and hardware and soft-
ware used provided practically 100% sealing of the cylinder and the uniform dis-
tribution of pressure on CTC during their filtration, as well as the exclusion of the
influence of the subjective factor in the process of creating pressure on the sample.
The proposed method involves two stages which are two blocks of research.
The first stage is the allocation of integral itact CTC. The principle of this stage
involves filtering of patients' blood through a polycarbonate membrane with specified
pore sizes using the ISET system [22] with the inclusion of two additional polycar-
bonate filters with pores diameter of 5 and 3 microns. Thus, the filtration module con-
sists of 10 membranes; each of them allows to simultaneously filter 1 ml of blood (10
ml of blood) through 10 membranes with 8 μm diameter pores, then 10 membranes
with 5 μm diameter pores and 10 membranes with 3 μm diameter pores.
At the first, preparatory step, a blood sample of a patient is prepared for the
study. A blood sample of 10 ml (peripheral or venous) obtained from the patient
was mixed to prepare hemolysis with 100 ml of distilled water. The prepared sam-
Criteria and Method for Detection of Circulating Tumor Cells
ISSN 2663-2586 (Online), ISSN 2663-2578 (Print). Cyb. and comp. eng. 2019. № 1 (195) 87
ple was mixed in a haematological scanner-analyzer such as Vision Hema @ As-
sist. Next, we performed citation tests — the division of the blood sample into
plasma, red blood cells, lymphocytes, platelets with selective removal of pathol-
ogically altered cells using a centrifuge-generator of the scanner. The degree of
sedimentation of the particles in the solution (precipitation or expulsion, depend-
ing on the density of the solution) was estimated by the sedimentation factor (S):
(1 ) ,M VS
f
ρ−
=
where M — is the molecular weight of the particle; f — is a form of the particle
(coefficient of friction); V — is the partial volume of particle; ρ — is the
solution density.
The preparatory step ended with the procedure of disaggregation of cells and
lyse of red blood cells (within 20 minutes). The general view of the membrane
with the study areas is presented in Fig. 1.
In subsequent steps, the membranes were dried, washed with 96% alcohol or
phosphate-basic buffer. We performed cytomorphological, cytoimmunological
and molecular analysis on the slots of the received membranes. The painting of
slots was carried out using the Romanovskii-Giemsa methods or the modified
May-Grunwald-Giemsa method [30].
After filtration and appropriate treatment of the membrane, the doctor re-
ceives a sample of the cell preparation.
The study process involved the step-by-step obtaining of microscopic images
of the CTC, where we can see the lymphocyte (1), the CTC (2), the nuclear-free
CTC, i.e. the CTC death due to the courses of therapy or the body's immune re-
sponse (3), and filter pores (4) (Fig. 2).
Fig. 1. The general view of the membrane with the study areas
(1 — the site was treated with Cytokeratin clon AE /1 AE /3;
2 — the site was treated according to Romanovskii-Giemsa)
Chernyshova T.A.
ISSN 2663-2586 (Online), ISSN 2663-2578 (Print). Cyb. and comp. eng. 2019. № 1 (195) 88
Fig. 2. Results of the CTC detection
Thus, the first stage of the advanced method of the detection and allocation of
integral intact circulating tumor cells comprised the following steps: step 1 —
preparation of a patient’s blood sample for research; step 2 — the blood sample
division into plasma and uniform elements with the help of a centrifuge-generator
of the scanner; step 3 — sedimentation of particles in solution (sedimentating) or
(expulsion) and determination of degree of sedimentation.
The second stage II — confirmation of the tumor malignancy. This is the
key stage of the method because it is precisely at this stage we determine
whether there are CTCs in the patient’s blood. At the first steps of this stage,
confirmation of tumor malignancy is carried out using the developed set of crite-
ria described above. For this purpose, the criteria of the first group are sequen-
tially determined. If there are at least 4 criteria out of 5 in this group, the primary
conclusion about the tumor malignancy is drawn.
As a result of empirical and practical laboratory studies using the ISET method,
we found out that the NCR of two or even three cancer cells might have practically the
same meaning for essentially different sizes of CTCs and their nuclei. To avoid the
ambiguity of the results integration, we proposed to switch to the absolute sizes of
cells and their nuclei and introduce a blood contamination index, which was defined as
the amount of CTCs detection in 1 ml (or 10 ml) of blood.
For the final examination of the results of the tumor malignancy evaluation,
we compared them with the CTC template masks and benign tumor cells. The
algorithm for evaluation of tumor malignancy is presented in Fig. 3.
Automated CTC image formation. The developed method is the basis of the
automated system for the CTC digital images analysis [31, 32]. In order to automate
the analysis of a biomedical image, in particular blood cells, in the first stage imag-
ing procedures are performed that are associated with suppressing the distorting
factors of the image (filtering obstacles, eliminating lighting inequalities, etc.).
Criteria and Method for Detection of Circulating Tumor Cells
ISSN 2663-2586 (Online), ISSN 2663-2578 (Print). Cyb. and comp. eng. 2019. № 1 (195) 89
Detection and allocation of CTC
Anisonucleosis
(ratio > 0.5
Cell Nucleus size
>24µm
The presence of
nucleus
abnormalities
High NCR
The presence of
cellular
complexes
This is the circulating tumor cell
(min 4 criteria of 5)
Formation of template masks
This is benign cell
Formation of template masks
Determination of cell density
Determination of electric charge
Determination of the CTC number
in 1 (10) ml of blood
DB-3:
CTC
template
masks
DB-4:
parameters
and criterial
data for CTC
evaluation
DB5:
Template
masks of
benign cells
No
No
No
No
No
Yes
Yes
Yes
Yes
To microcontroller
start
Preparation stage
Fig 3. An algorithm for determining the tumor malignancy
Next, the process of scanning images of cells located on the surface of poly-
carbonate membranes is carried out. The size of the image which is scanned in
one step of the microscope's subject table should not exceed the microscope
monocular diameter. At the beginning of the process of scanning the 8 μm pores
Chernyshova T.A.
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membrane, there are images (scans) that lack any cells or their formation regard-
less their origin (malignant or benign) and they are excluded from further proc-
essing and analysis. Detected scans with existing cells or their entities are re-
corded and stored in the appropriate database (DB 1). After completion of the 8
μm pores membrane scanning, all of the above procedures are performed with
the 5 μm pores membrane (DB 2), and then with the 3 μm pores membrane (DB
3). The databases with CTC images scanning of various sizes provide a qualita-
tive and flexible classification of types of cancer and create the basis for on-
cological patients’ registration with their CTCs.
The further steps of this stage deal with the identification of disseminated
tumor cells and CTCs. Bringing the image to the required quality is based on
changing the contrast of the image, changing the narrowed or shifted range of
pixel brightness of the image, and its uneven filling, etc. [33].
Processing of histological sections images containing cellular structures is a
set of solutions of the following tasks: evaluation of visual quality of digital
images; linear image filtering; threshold processing; median filtration. Figure 4
shows the CTC image after calculating the brightness gradient (Fig. 4a) and the
CTC image after filling in the inner regions (Fig. 4b).
At the description stage we perform the definition and calculation of feature
characteristics of the object which are the basis for the third step connected with
the assignment of an object to a particular class. The description stage is the
main stage out of these three. The choice of signs and their informativity (the
possibility of attributing an object to a particular class due to its characteristics)
are crucial in determining the result of detection.
a b
Fig 4. CTC image after calculating the brightness gradient (a) and the CTC image after
filling in the inner regions (b)
Criteria and Method for Detection of Circulating Tumor Cells
ISSN 2663-2586 (Online), ISSN 2663-2578 (Print). Cyb. and comp. eng. 2019. № 1 (195) 91
RESEARCH USING THE PROPOSED METHOD
We performed the analysis of peripheral blood samples in patients with primary
diagnosis of colon, breast, lungs, prostate and skin cancer. Blood samples were
provided by oncology centers and clinics in Mariupol, Vinnitsa and Ternopil,
and were encoded by the name of the city and the serial number of the sample.
The studies were conducted at the specified clinical bases simultaneously with
other methods because of the need to adhere to certain requirements, such as:
blood sampling should be 10 ml in anticoagulant test tubes EDTA (blood from
the peripheral vein); storage time before the analysis beginning should be no
more than 90 minutes; storage temperature should be 4 0С; blood dilution in the
ratio of 1:10 (up to 100 ml) shoul be performed with a proprietary buffer solu-
tion (sanolin, paraformaldehyde, EDTA, pour albumin), which was used for red
blood cells lysis [34, 35].
The obtained results analysis showed that the use of the developed method
enabled to determine false-positive diagnoses in 6.2–8.9% of cases.
We also found out that in patients with colorectal cancer, CTC in the blood
was more often detected in the case of low-differentiated tumors. In patients with
breast cancer, the most cases of CTC detection fell on the third stage of the disease
(85% or 12 patients), other 15 % were on the second stage. It should be noted that
most patients with prostate cancer with detected CTC had metastases in the liver
and bones. The obtained data for small-cell lung cancer did not contradict the lit-
erary data indicating a decrease in the CTC incidence in non-small cell lung can-
cer than in the case of small-cell lung cancer. A similar conclusion can be made
about skin cancer. That is, the obtained results correlate with a high degree of sta-
tistic significance (> 95%) with the data suggested by the literature.
We sent the obtained microscopic CTC images and diagnosis made accord-
ing to them with unchanged codes to the appropriate clinics and conducted a
final discussion of these results. After a full-format analysis, we performed the
correction of range and treatment tactics in cases where it was necessary.
CONCLUSIONS
The proposed method for determining circulating tumor cells, which is an im-
provement of ISET technology, combines two stages. At the first stage we im-
proved the ISET technology by adding two additional polycarbonate filters with
5 and 3 micron diameter pores, and providing a mode of 100% sealing of the
chamber with hemolysis, and constant pressure throughout the filtration process.
At the second stage, we carried out the DTM of the isolated cells using the de-
veloped set of criteria. The application of the proposed method for processing
microscopic CTC images allows to detect smaller CTCs than in the case of using
traditional methods, and to ensure their integrity and intactness.
The use of the developed method in the automated system for the digital images
analysis of circulating tumor cells provides the detection and calculation of charac-
teristic features for assigning an object to a certain class of malignancy. Scanning of
images of the researched cells is carried out and detected scans with existing cells or
their entities are sequentially recorded and stored in the appropriate database. For the
final examination of the results of the tumor malignancy evaluation, comparisons are
made with the CTC template masks and benign tumor cells.
Chernyshova T.A.
ISSN 2663-2586 (Online), ISSN 2663-2578 (Print). Cyb. and comp. eng. 2019. № 1 (195) 92
Clinical and experimental study using the proposed method for CTC detect-
ing revealed an opportunity to clarify the diagnosis and identify false positive
diagnoses. The study proved that colorectal cancer was more often diagnosed
with low-differentiated tumors and, the highest breast cancer incidence occured
at the third stage of the disease. The effectiveness of the proposed method was
also confirmed in patients with other types of cancer.
REFERENCES
1. Krivacic R.. Ladanyi A., Curry D., Hsieh H., Kuhn P., Bergsrud D., Kepros J. A rare-cell
detector for cancer. Proceedings of the National Academy of Sciences of the United
States of America. 2004. V. 101, N 29. P. 10501–10504.
2. Harouaka R., Kang Z., Zheng S.Y., Cao L. Circulating tumor cells: advances in isolation
and analysis, and challenges for clinical applications. Pharmacol Ther. 2014. Vol. 141.
Р. 209–221.
3. Tewes M., Aktas В., Welt A., Mueller S., Hauch S., Kimmig R., Kasimir-Bauer S.
Molecular profiling and predictive value of circulating tumor cells in patients with
metastatic breast cancer: an option for monitoring response to breast cancer related
therapies. Breast cancer research and treatment. 2009. Vol. 115, N 3. P. 581–590.
4. Danila D.C., Heller G., Gignac G.A. et al. Circulating tumor cell number and prognosis
in progressive castration-resistant prostate cancer. Clin Cancer Res. 2007. Vol. 13(23).
Р. 7053–7058.
5. Krivacic R.. Ladanyi A., Curry D., Hsieh H., Kuhn P., Bergsrud D., Kepros J. A rare-
cell detector for cancer. Proceedings of the National Academy of Sciences of the United
States of America. 2004. Vol. 101, N 29. P. 10501–10504.
6. Ntouroupi Т., Ashraf S., McGregor S., Turney В., Seppo A., Kim Y., Wang X.
Detection of circulating tumour cells in peripheral blood with an automated scanning
fluorescence microscope. British journal of cancer. 2008. Vol. 99, N 5. P. 789–795.
7. Bauer K., de la Torre-Bueno J., Diel I., Hawes D., Decker W., Priddy C., Bossy B.
Reliable and sensitive analysis of occult bone marrow metastases using automated
cellular imaging. Clinical cancer research: an official journal of the American
Association for Cancer Research. 2000. Vol. 6, N 9. P. 3552–3559.
8. Kagan M., Howard D., Bendele Т., Mayes J., Silvia J., Repollet M., Doyle J. A Sample
Preparation and Analysis System for Identification of Circulating Tumor Cells. Journal
of Clinical Ligand Assay. 2002. Vol. 25, N 1. P. 104–110.
9. Andreopoulou E., Yang L.-Y., Rangel K., Reuben J., Hsu L., Krishnamurthy S., Valero
V. Comparison of assay methods for detection of circulating tumor cells in metastatic
breast cancer: AdnaGen AdnaTest BreastCancer Select. Detect™ versus Yeridex
CellSearch™ system. Int. journal of cancer. 2012. Vol. 130, N 7. P. 1590–1597.
10 Nezos A., Pissimisis N., Lembessis P., Sourla A., Dimopoulos P., Dimopoulos Т.,
Tzelepis K. Detection of circulating tumor cells in bladder cancer patients. Cancer
treatment reviews. 2009. V. 35, N 3. P. 272–279.
11. Tewes M., Aktas В., Welt A., Mueller S., Hauch S., Kimmig R., Kasimir-Bauer S.
Molecular profiling and predictive value of circulating tumor cells in patients with
metastatic breast cancer: an option for monitoring response to breast cancer related
therapies. Breast cancer research and treatment. 2009. V. 115, N 3. P. 581–590.
12. Ignatiadis M, Lee M, Jeffrey SS. Circulating Tumor Cells and Circulating Tumor DNA:
Challenges and Opportunities on the Path to Clinical Utility. Clin Cancer Res. 2015.
No 21(21) P. 4786–800.
13. Alix-Panabieres C, Pantel K. Technologies for detection of circulating tumor cells: facts
and vision. Lab Chip. 2014. No 14(1) P. 57–62.
14. Ferreira M.M, Ramani V.C, Jeffrey S.S. Circulating tumor cell technologies. Mol Oncol.
2016. 10(3) P. 374–94.
15. Gertler R., Rosenberg R., Fuehrer K., Dahm M., Nekarda H., Siewert J. Detection of
circulating tumor cells in blood using an optimized density gradient centrifugation.
Criteria and Method for Detection of Circulating Tumor Cells
ISSN 2663-2586 (Online), ISSN 2663-2578 (Print). Cyb. and comp. eng. 2019. № 1 (195) 93
Recent results in cancer research. Fortschritte der Krebsforschung. Progres dans les
recherches sur le cancer. 2003. Vol. 162. P. 149–155.
16. Tan S., Yobas L., Lee G., Ong C, Lira C. Microdevice for the isolation and enumeration
of cancer cells from blood. Biomedical microdevices. 2009. V. 11, N 4. P. 883–892.
17. Tan S., Yobas L., Lee G., Ong C, Lira C. Microdevice for the isolation and enumeration
of cancer cells from blood. Biomedical microdevices. 2009. V. 11, N 4. P. 883–892.
18. Kovalev A.A, Grudinskaya T.V., Kuznetsov T.P., Kovalev K.A. Heterogeneity of circu-
lating tumor cells. Oncology. 2012. V.4. No. 2 12.12-12.(in Russian)
19. Pavlov S.V, Kozhemyako V.P, Burdenyuk I.I. Rami Rebhi Hamdi. Optical and elec-
tronic technologies for the analysis of biomedical images. Vinnitsa: VNTU, 2011. 166 p.
(in Ukranian)
20. Nepomnyaschaya E.I., Kit O.I., Nistratova O.V., Novikova I.A. and others. Circulating
tumor cells and some morpho-immuno-histochemical indices at colorectal cancer.
Modern problems of science and education. 2016. No. 2. (in Russian)
21. G. Vona, C. Béroud, A. Benachi, A. Quenette, J.P. Bonnefont, Y. Dumez, B. Lacour, P.
Paterlini-Bréchot. Enrichment and genetic analyses of fetal cells circulating in the
maternal blood by the ISET technique and single cell microdissection: a non-invasive
tool for early prenatal diagnosis. Am. J. Pathol. 2002. No 160. P. 51–58.
22. Ismayilova G., Laget S., Paterlini-Brechot P. Diagnosis of circulating tumor cells using
ISET technology and their molecular characteristics for fluid biopsy. URL:
https://cyberleninka.ru/article/n/diagnostika-tsirkuliruyuschih-opuholevyh-kletok-s-
pomoschyu-tehnologii-iset-i-ih-molekulyarnaya-harakteristika-dlya-zhidkostnoy. (Last
accessed: 08.12.2018)
23. Ledov V.K., Skrinnikova MA, Popova O.P. Isolation of Circulating Tumor Cells by
Isolated Size (ISET) (overview). Voprosy Oncologii, 2014. №60 (5). P.548–552. (in
Russian)
24. Baikhenko A.K, Shaimardinova G. M., Popova N.V., Zhusinova B.T., Ismayilova G.N.
Circulating Cancer Cells: Molecular Characteristics and Monitoring of Cancer Treat-
ment. Clinical Medicine of Kazakhstan. 2013. Vol. 4 №30. (in Russian)
25. Volchenko N.N. Cytological diagnostics of tumors of the mammary gland. Mammology,
2006, No. 1, P. 35–39. (in Russian)
26. Paterlini-Bréchot P, Benali-Furet NL. Circulating tumor cells (CTC) detection : Clinical
impact and future directions. Cancer Letter. 2007. No 253. P. 180–204.
27. Zubtsov D.A., Zubtsova J.I., Lavrov A.V., Legchenko E.V. et al. Circulating tumor cells
(CAC) in breast cancer: prognostic significance and methods of excretion. Trudy MFTI.
2012. Volume 4. No. 3. P. 18–26. (in Russian)
28. Chimitov A.A.,.Ryantsantsev N.V, Dambayev G.T., Khitricheev V.E. and others. Filtra-
tion of venous blood of boluses with the aim of cytological diagnostics of malignant
neoplasms. Bulleten VSNI SO RAMN, 2010. No. 3 (73). (in Russian)
29. Laget S, Broncy L, Hormigos K, Dhingra DM, BenMohamed F, Capiod T, et al.
Technical Insights into Highly Sensitive Isolation and Molecular Characterization of
Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion. PLOS
ONE, 2017, 12 (1): e0169427. https://doi.org/10.1371/journal.pone.0169427
30. Ushakova G.O. Modern methods of clinical diagnostics. Guidelines. Dnipropetrovsk:
DNU. 2003. 52 p.
31. Zlepko S.M., Chernyshova T.A., Timchik S.V., Krivonosov V.E., Zlepko O.S. Informa-
tion system (IS) for receiving and processing microscopic images of circulating tumor
cells (CTC). Achievements of clinical and experimental medicine, 2017, № 4. P. 39–46.
(in Ukranian)
32. Azarhov O.Yu., Zlepko S.M., Timchik S.V., Chernyshova T.A.,. Danilkov S.O Methods
and tools for computer analysis of microscopic images of circulating tumor cells.
Bulletin of scientific researches. 2017. No. 4. P. 162–166. (in Ukranian)
33. Golovnya V.M., Zlepko S.М. The system of express diagnostics of formed blood ele-
ments. Measuring and computing engineering in technological processes. 2010, 2.
P. 163–168. (in Ukranian)
Chernyshova T.A.
ISSN 2663-2586 (Online), ISSN 2663-2578 (Print). Cyb. and comp. eng. 2019. № 1 (195) 94
34. Lyadov V.K., Ledin E.V., Skrypnikova M.A. Cytological diagnostics of pancreatic adenocarci-
noma by the method of isolating the size of circulating tumor cells from peripheral blood: a
message from practice. Clinical Laboratory Diagnostics, 2017. No. 11. P. 31–39. (in Russian)
35. Kit O.I., Novikova I.A, Selutina O.N., Dontsov V.A., Chernikova E.N., Samaneyeva
N.Yu., Nistratova O.V. Investigation of the level of the central ankylosing spondylitis in
epithelial tumors of various localizations. International Journal of Applied and Funda-
mental Researches. 2018. No. 12-5. P.817–820. (in Russian)
Resieved 26.12.2018
ЛИТЕРАТУРА
1. Krivacic R.. Ladanyi A., Curry D., Hsieh H., Kuhn P., Bergsrud D., Kepros J. A rare-cell
detector for cancer. Proceedings of the National Academy of Sciences of the United
States of America. 2004. V. 101, N 29. P. 10501–10504.
2. Harouaka R., Kang Z., Zheng S.Y., Cao L. Circulating tumor cells: advances in isolation and
analysis, and challenges for clinical applications. Pharmacol Ther. 2014. Vol. 141. Р. 209–221.
3. Tewes M., Aktas В., Welt A., Mueller S., Hauch S., Kimmig R., Kasimir-Bauer S.
Molecular profiling and predictive value of circulating tumor cells in patients with
metastatic breast cancer: an option for monitoring response to breast cancer related
therapies. Breast cancer research and treatment. 2009. Vol. 115, N 3. P. 581–590.
4. Danila D.C., Heller G., Gignac G.A. et al. Circulating tumor cell number and prognosis
in progressive castration-resistant prostate cancer. Clin Cancer Res. 2007. Vol. 13(23).
Р. 7053–7058.
5. Krivacic R.. Ladanyi A., Curry D., Hsieh H., Kuhn P., Bergsrud D., Kepros J. A rare-
cell detector for cancer. Proceedings of the National Academy of Sciences of the United
States of America. 2004. Vol. 101, N 29. P. 10501–10504.
6. Ntouroupi Т., Ashraf S., McGregor S., Turney В., Seppo A., Kim Y., Wang X.
Detection of circulating tumour cells in peripheral blood with an automated scanning
fluorescence microscope. British journal of cancer. 2008. Vol. 99, N 5. P. 789–795.
7. Bauer K., de la Torre-Bueno J., Diel I., Hawes D., Decker W., Priddy C., Bossy B.
Reliable and sensitive analysis of occult bone marrow metastases using automated
cellular imaging. Clinical cancer research: an official journal of the American
Association for Cancer Research. 2000. Vol. 6, N 9. P. 3552–3559.
8. Kagan M., Howard D., Bendele Т., Mayes J., Silvia J., Repollet M., Doyle J. A Sample
Preparation and Analysis System for Identification of Circulating Tumor Cells. Journal
of Clinical Ligand Assay. 2002. Vol. 25, N 1. P. 104–110.
9. Andreopoulou E., Yang L.-Y., Rangel K., Reuben J., Hsu L., Krishnamurthy S., Valero
V. Comparison of assay methods for detection of circulating tumor cells in metastatic
breast cancer: AdnaGen AdnaTest BreastCancer Select. Detect™ versus Yeridex
CellSearch™ system. Int. journal of cancer. 2012. Vol. 130, N 7. P. 1590–1597.
10 Nezos A., Pissimisis N., Lembessis P., Sourla A., Dimopoulos P., Dimopoulos Т.,
Tzelepis K. Detection of circulating tumor cells in bladder cancer patients. Cancer
treatment reviews. 2009. V. 35, N 3. P. 272–279.
11. Tewes M., Aktas В., Welt A., Mueller S., Hauch S., Kimmig R., Kasimir-Bauer S.
Molecular profiling and predictive value of circulating tumor cells in patients with
metastatic breast cancer: an option for monitoring response to breast cancer related
therapies. Breast cancer research and treatment. 2009. V. 115, N 3. P. 581–590.
12. Ignatiadis M, Lee M, Jeffrey SS. Circulating Tumor Cells and Circulating Tumor DNA:
Challenges and Opportunities on the Path to Clinical Utility. Clin Cancer Res. 2015.
No 21(21) P. 4786–800.
13. Alix-Panabieres C, Pantel K. Technologies for detection of circulating tumor cells: facts
and vision. Lab Chip. 2014. No 14(1) P. 57–62.
14. Ferreira M.M, Ramani V.C, Jeffrey S.S. Circulating tumor cell technologies. Mol Oncol.
2016. 10(3) P. 374–94.
15. Gertler R., Rosenberg R., Fuehrer K., Dahm M., Nekarda H., Siewert J. Detection of
circulating tumor cells in blood using an optimized density gradient centrifugation.
Criteria and Method for Detection of Circulating Tumor Cells
ISSN 2663-2586 (Online), ISSN 2663-2578 (Print). Cyb. and comp. eng. 2019. № 1 (195) 95
Recent results in cancer research. Fortschritte der Krebsforschung. Progres dans les
recherches sur le cancer. 2003. Vol. 162. P. 149–155.
16. Tan S., Yobas L., Lee G., Ong C, Lira C. Microdevice for the isolation and enumeration
of cancer cells from blood. Biomedical microdevices. 2009. V. 11, N 4. P. 883–892.
17. Tan S., Yobas L., Lee G., Ong C, Lira C. Microdevice for the isolation and enumeration
of cancer cells from blood. Biomedical microdevices. 2009. V. 11, N 4. P. 883–892.
18. А.А.Ковалев, Т.В. Грудинская, Т.П. Кузнецов, К.А. Ковалев. Гетерогенность цир-
кулирующих опухолевых клеток. Онкология. 2012. Т.4. №2. С.126–129.
19. Павлов С.В., Кожем’яко В.П., Бурденюк І.І. Рамі Ребхі Хамді. Оптико-електронні
технології аналізу біомедичних зображень. Вінниця: ВНТУ, 2011. 166 с.
20. Непомнящая Е.И., Кит О.И., Нистратова О.В., Новикова И.А. и др.. Циркулирую-
щие опухолевые клетки и некоторые морфо-иммуно-гистохимические показатели
при колоректальном раке. Современные проблемы науки и образования. 2016. №2.
21. G. Vona, C. Béroud, A. Benachi, A. Quenette, J.P. Bonnefont, Y. Dumez, B. Lacour, P.
Paterlini-Bréchot. Enrichment and genetic analyses of fetal cells circulating in the
maternal blood by the ISET technique and single cell microdissection: a non-invasive
tool for early prenatal diagnosis. Am. J. Pathol. 2002. No 160. P. 51–58.
22. Исмаилова Г. Laget S., Paterlini-Brechot P. Диагностика циркулирующих опухоле-
вых клеток с помощью технологии ISET и их молекулярная характеристика для
жидкостной биопсии. https://cyberleninka.ru/article/n/diagnostika-tsirkuliruyuschih-
opuholevyh-kletok-s-pomoschyu-tehnologii-iset-i-ih-molekulyarnaya-harakteristika-
dlya-zhidkostnoy.
23. Ледов В.К., Скринникова М.А., Попова О.П. Выделение циркулирующих опухоле-
вых клеток методом «изоляции по размеру» (ISET) (обзор). Вопр. Онкологи. 2014.
№60(5). С.548–552.
24. Байченко А.К., Шаймардинова Г. М., Попова Н.В., Жусинова Б.Т., Исмаилова Г.Н.
Циркулирующие раковые клетки : молекулярная характеристика и мониторинг ле-
чения рака. Клиническая медицина Казахстана. 2013. Vol.4. №30.
25. Волченко Н.Н. Цитологическая диагностика опухолей молочной железы. Маммо-
логия, 2006, №1, С. 35–39.
26. Paterlini-Bréchot P, Benali-Furet NL. Circulating tumor cells (CTC) detection : Clinical
impact and future directions. Cancer Letter. 2007. No 253. P. 180–204.
27. Д.А.Зубцов, Ж.И. Зубцова, А.В. Лавров, Е.В. Легченко и др. Циркулирующие
опухолевые клетки (ЦОК) при раке молочной железы: прогностическая значи-
мость и методы выделения. Труды МФТИ. 2012. Том 4. №3. С. 18–26.
28. А.А. Чимитов, Н.В.Рязанцева, Г.Ц.Дамбаев, В.Е.Хитрихеев и др. Фильтрация ве-
нозной крови болных с целью цитологической диагностики злокачественных но-
вообразований. Бюллетень ВСНИ СО РАМН, 2010. №3(73).
29. Laget S, Broncy L, Hormigos K, Dhingra DM, BenMohamed F, Capiod T, et al.
Technical Insights into Highly Sensitive Isolation and Molecular Characterization of
Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion. PLOS
ONE, 2017, 12 (1): e0169427. https://doi.org/10.1371/journal.pone.0169427
30. Ушакова Г.О. Сучасні методи клінічної діагностики. Методичні рекомендації.
Дніпропетровськ: ДНУ. 2003. 52 с.
31. Злепко С.М., Чернишова Т.А., Тимчик С.В., Кривоносов В.Є., Злепко О.С. Інфор-
маційна система (ШС) для отримання та оброблення мікроскопічних зображень
циркулюючих пухлинних клітин (ЦПК). Здобутки клінічної і експериментальної
медицини, 2017, № 4, С. 39–46.
32. О.Ю. Азархов, С.М. Злепко, С.В. Тимчик, Т.А. Чернишова, С.О. Данилков. Мето-
ди і засоби для комп’ютерного аналізу мікроскопічних зображень циркулюючих
пухлинних клітин. Вісник наукових досліджень. 2017. № 4. С. 162–166.
33. Головня В.М., Злепко С.М. Система експрес-діагностики формених елементів крові.
Вимірювальна та обчислювальна техніка в технологічних процесах. 2010, «2, с. 163–168.
34. Лядов В.К., Ледин Е.В., Скрипникова М.А. Цитологическая диагностика аденока-
рциномы поджелудочной железы методом выделения по размеру циркулирующих
Chernyshova T.A.
ISSN 2663-2586 (Online), ISSN 2663-2578 (Print). Cyb. and comp. eng. 2019. № 1 (195) 96
опухолевых клеток из периферической крови: сообщение из практики. Клиничес-
кая лабораторная диагностика, 2017. №11. С.31–39.
35. Кит О.И., Новикова И.А., Селютина О.Н., Донцов В.А., Черникова Е.Н., Саманеева
Н.Ю., Нистратова О.В. Исследование уровня ЦОК при эпителиальных опухолях
различных локализаций. Международный журнал прикладных и фундаменталь-
ных исследований. 2018. №12-5. С.817–820.
Получено 26.12.2018
Чернишова Т.А., лікар,
e-mail: tetyana.che@gmail.com
Авіаційний медичний центр
Національного авіаційного університету,
просп. Комарова, 1, 03058, Україна
КРИТЕРІЇ ТА МЕТОД ВИЯВЛЕННЯ ЦИРКУЛЮЮЧИХ ПУХЛИННИХ КЛІТИН
Вступ. Сучасні досягнення науки і техніки суттєво розширили можливості розпізна-
вання злоякісних новоутворень. Наявність великої групи методів виявлення і виділення
циркулюючих пухлинних клітин (ЦПК) явно вказує на інтерес, який проявляють дослід-
ники до ЦПК.
Мета роботи — формування комплексу критеріїв оцінювання злоякісності пухлини
та удосконалення методу виявлення циркулюючих пухлинних клітин в крові людини.
Результати. Запропонований метод визначення циркулюючих пухлинних клітин,
який є удосконаленням технології ISET, об’єднує два етапи. На першому етапі здійс-
нюється виділення ЦПК з удосконаленням за рахунок встановлення двох додаткових
полікарбонатних фільтрів з порами діаметром 5 і 3 мкм та забезпеченні режиму 100%
герметизації камери з гемолізатом і постійного тиску протягом всього процесу фільтра-
ції. На другому етапі за використання сформованого комплексу критеріїв здійснюється
визначення ступеня злоякісності виділених клітин. Використання розробленого методу
у автоматизованій системі аналізу цифрових мікроскопічних зображень циркулюючих
пухлинних клітин забезпечує визначення і розрахунок характерних ознак для відне-
сення об'єкта до певного класу злоякісності та формування бази даних сканованих
зображень з наявними клітинами або їх утвореннями, а також остаточну перевірку
результатів оцінювання злоякісності пухлини за шаблонами-масками ЦПК і доброякіс-
ної пухлинної клітини.
Висновки. Застосування запропонованого методу визначення циркулюючих пух-
линних клітин уможливлює виявлення цих клітин меншого розміру, ніж у разі викорис-
тання традиційних методів, а також забезпечення їх цілісності та неушкодженості.
Ключові слова: циркулюючі пухлинні клітини, критерії оцінювання злоякісності пух-
лин, метод визначення циркулюючих пухлинних клітин у крові людини.
Criteria and Method for Detection of Circulating Tumor Cells
ISSN 2663-2586 (Online), ISSN 2663-2578 (Print). Cyb. and comp. eng. 2019. № 1 (195) 97
Чернышева Т.А., врач,
e-mail: tetyana.che@gmail.com
Авиационный медицинский центр
Национального авиационного университета,
просп. Комарова,1, Киев, 03058, Украина
КРИТЕРИИ И МЕТОД ВЫЯВЛЕНИЯ
ЦИРКУЛИРУЮЩИХ ОПУХОЛЕВЫХ КЛЕТОК
Предложенный метод определения циркулирующих опухолевых клеток, который явля-
ется усовершенствованием технологии ISET, объединяет два этапа. На первом этапе
осуществляется выделение ЦОК с совершенствованием за счет установки двух допол-
нительных поликарбонатных фильтров с порами диаметром 5 и 3 мкм и обеспечении
режима 100% герметизации камеры гемолизации и постоянного давления в течение
всего процесса фильтрации. На втором этапе при использовании сформированного
комплекса критериев осуществляется определение степени злокачественности выде-
ленных клеток. Использование разработанного метода в автоматизированной системе
анализа цифровых микроскопических изображений циркулирующих опухолевых кле-
ток обеспечивает определение и расчет характерных признаков для отнесения объекта
к определенному классу злокачественности и формирования базы данных сканирован-
ных изображений с занесением имеющихся клеток или их образований, а также окон-
чательную проверку результатов оценки злокачественности опухоли по шаблонам-
маскам ЦОК и доброкачественной опухолевой клетки.
Ключевые слова: циркулирующие опухолевые клетки, критерии оценки злокачественности
опухолей, метод определения циркулирующих опухолевых клеток в крови человека.
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