RU2818060C1 - Method of producing cell blocks for cytological and immunocytochemical diagnosis of malignant neoplasms - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a method of producing cell blocks for cytological and immunocytochemical (ICC) diagnostics of malignant neoplasms. Cell material obtained by fine-needle aspiration biopsy and endobronchial and endoesophageal ultrasonography with fine-needle aspiration biopsy and histological wiring, placed in eppendorf containing a transport medium consisting of a solution of neutral formalin 10% and isotonic saline 0.9% in ratio 1:1 in volume of 0.8–1.0 ml. Cell suspension is then centrifuged for 5 minutes at 4,000 rpm. Cell sediment is separated by draining supernatant, 1.5 ml of isopropyl alcohol and 5–6 drops of glycerine are added to the precipitate and centrifuged for 5 minutes at rate of 4,000 rpm. Formed cell clot is located in the eppendorf center at the boundary of the two-phase liquid. Supernatant is drained and transferred with a dissecting needle into a biopsy cartridge, at the bottom of which there is a sponge for carrying the biopsy material, and the clot is covered with the same sponge. Closing the cassette lid, placing it in a neutral buffered formalin solution for 12–18 hours, then the cassette with the cell clot is transferred into a histological processor for histological wiring for cytological and ICC studies.

EFFECT: invention provides preservation of morphological structure and antigenic properties of cell material, as well as simplification and acceleration of cell clot formation.

1 cl, 14 dwg, 2 ex

Description

The invention relates to the field of medicine, in particular to the diagnosis of physiological conditions of the human body by examining cellular material obtained from fine-needle aspiration biopsy and endobronchial and endoesophageal ultrasonography with fine-needle aspiration biopsy, etc. in vitro for cytological and immunocytochemical (ICC) studies. The invention can be used in oncology, hematology, therapy, phthisiology, obstetrics and gynecology, and clinical laboratory diagnostics.

It is known to use imported expensive media (Clear Prep from Resolusion Biomedical, USA; E-Prep from Celltrazone, South Korea; BD, USA; Novoprep Solution, UK, etc.) for transporting and preserving cellular material. Alcohols contained in the transport medium change the morphology of cells, thereby complicating cytological diagnostics and requiring special training of cytologists [1, p. 68].

There is a known method for producing cell blocks based on pouring cellular material with gelatin to make sections and microslides from it. Microslides obtained in this way cannot be used for ICC research, since gelatin cannot be removed from sections when fixing biological material in formaldehyde [2, p. 38].

There is a known method for preparing cell blocks for ICC research based on filling the cell sediment with agar-agar [3, p. 360]. This method has a disadvantage associated with the need to strictly adhere to the temperature regime, the violation of which leads to changes in the morphological characteristics of the cell: the formation of vacuoles, cell shrinkage, compaction or disappearance of the cytoplasm, which does not always allow an accurate assessment of cytoplasmic and membrane ICC reactions.

There is a known method for producing a cell clot using platelet mass [4, pp. 1-2, 4, 7]. The disadvantages of this method are:

- the technology is labor-intensive, since it is necessary to first prepare the platelet mass by constant stirring and heating to a temperature of 36-37°C, then adding it to the cell sediment, after clot retraction, centrifugation for 3-6 minutes at a speed of 1500 rpm and subsequent removal supernatant liquid;

- platelet mass must contain at least 50×10 ⁹ platelets in one bottle;

- the need to comply with the temperature regime for storing platelet mass (up to 20°C);

- limited shelf life of platelet mass (up to 3 days), since after this period its activity sharply decreases;

- the need to dispose of platelet mass residues as biological waste.

There is a known traditional method of histological processing, including dehydration, saturation with paraffin, embedding in a block, microtomy, making sections and microslides for morphological examination [5, pp. 53-83].

The technical task is to increase the accuracy of cytological and ICC diagnostics of malignant neoplasms.

To achieve this technical task, a method for obtaining cell blocks for cytological and ICC diagnostics of malignant neoplasms is proposed, characterized by the fact that the cellular material obtained by fine-needle aspiration biopsy and endobronchial and endoesophageal ultrasonography with fine-needle aspiration biopsy and histological testing, including dehydration, paraffin saturation, embedding into a block, microtomy, production of micropreparations for morphological research, characterized in that the cellular material is placed in an Eppendorf containing a transport medium consisting of a solution of neutral formaldehyde 10% and isotonic physiological solution of sodium chloride 0.9% in a ratio of 1:1 in a volume of 0 ,8-1.0 ml, centrifuge the cell suspension for 5 minutes at a speed of 4000 rpm, separate the cell sediment by draining the supernatant, add 1.5 ml of isopropyl alcohol and 5-6 drops of glycerol to the sediment, centrifuge for 5 minutes at a speed of 4000 rpm, while the formed cell clot is located in the center of the Eppendorf at the boundary of the two-phase liquid, the supernatant liquid is drained and with a dissecting needle it is transferred to a biopsy cassette, at the bottom of which there is a special sponge for guiding the biopsy material and the clot is covered with the same sponge , close the cassette lid, place it in a neutral buffered formaldehyde solution of 10% for 12-18 hours, then the cassette with the cell clot is transferred to a histological processor for histological processing, poured into a block, and microslides are made for cytological and ICC studies.

The claimed invention is aimed at solving the problem of obtaining optimal cell blocks from cellular material without changing its morphological structure and antigenic properties, obtained by fine-needle aspiration biopsy and endobronchial and endoesophageal ultrasonography with fine-needle aspiration biopsy for cytological and ICC diagnosis of malignant neoplasms.

The technical result is that the preservation of the morphological structure and antigenic properties of the cellular material is ensured, and the formation of a cellular clot is simplified and accelerated.

The essence of the claimed invention is that the use of a transport medium based on a solution of neutral formaldehyde 10% and isotonic physiological solution of sodium chloride 0.9%, in which the cellular material is placed, does not change the structure of the cells, unlike traditional imported transport media containing alcohols, formaldehyde, and the method of obtaining a cell clot based on the use of a two-phase liquid (isopropyl alcohol and glycerin) without the presence of protein fractions and polysaccharides, unlike methods using gelatin, platelet mass, paraffin, does not change the morphological and antigenic properties of cells, simplifies and accelerates the technology of cell formation clot. The resulting cell clot is used to prepare a cell block by carrying out histological testing, including dehydration, paraffin saturation, embedding in the block, microtomy, production of microslides for cytological and ICC diagnostics in complex diagnostic cases in order to determine the histogenesis and organ affiliation of the tumor. This is important for establishing an accurate clinical diagnosis, choosing special treatment methods, and subsequently affects the prognosis of the disease and the quality of life of patients.

The use of the proposed method in clinical oncological, hematological, therapeutic, phthisiatric, obstetric and gynecological practices and clinical laboratory diagnostics increases the accuracy of the morphological diagnosis of malignant neoplasms.

Application in the described method for obtaining cell blocks provides:

- preservation of morphological structures and antigenic properties of cells;

- simplification and acceleration of the technology for obtaining cell blocks;

- the possibility of using domestically produced reagents and consumables, which makes it possible for import substitution;

- economic feasibility due to the low cost of reagents and consumables;

- long-term preservation of cell blocks for subsequent cytological and ICC studies.

The advantage of the invention is that it preserves the morphological structure and antigenic properties of cells, simplifies and speeds up the technology for preparing cell blocks, is of economic importance, makes it possible for import substitution and increases the accuracy of cytological and ICC diagnostics of malignant neoplasms.

The method is implemented as follows. Cellular material for research is obtained by fine-needle aspiration biopsy and endobronchial and endoesophageal ultrasonography with fine-needle aspiration biopsy, placed in an eppendrf with 0.8-1.0 ml of transport medium consisting of a solution of neutral formaldehyde 10% and isotonic physiological solution of sodium chloride 0. 9%, intended for liquid cytology. The contents of the Eppendorf are gently mixed (without the formation of foam) to ensure greater contact of the cells with the medium. Biological material is immediately used for research or it can be stored at a temperature of 4-8°C for up to 24 hours. The faster the cellular material is formed into a cell block, the less likely it is that the ICC markers (especially nuclear ones) that are used to perform the ICC study will be destroyed. The cell suspension is centrifuged for 5 minutes at 4000 rpm, then the supernatant is drained. Add 1.5 ml of isopropyl alcohol and 5-6 drops of glycerin to the sediment. The cell suspension is centrifuged for 5 minutes at 4000 rpm. The resulting cell clot is located in the center of the two-phase liquid. The supernatant liquid is drained, the clot is transferred with a dissecting needle into a biopsy cassette, at the bottom of which there is a filter pad, the cell block is covered with it and the lid of the cassette is closed, which is placed in a neutral buffered formaldehyde solution of 10% for 12-18 hours, the cassette with the cell clot is transferred to the histological processor for histological processing, including dehydration, paraffin saturation, embedding in a block, microtomy, production of microslides for cytological and ICC studies.

The inventive method for obtaining cell blocks for cytological and ICC diagnostics of malignant neoplasms is optimal and meets all specified requirements.

The use of indifferent reagents for the transport medium based on a solution of neutral formaldehyde 10%, isotonic physiological solution of sodium chloride 0.9% and isopropyl alcohol, glycerin for the production of cell blocks, which do not contain protein fractions and polysaccharides do not change the morphological structure and the antigenic properties of cells represent a simple way to obtain a cell clot for cytological and ICC studies. The solutions used are produced by domestic industry and ensure that the proposed method meets the criterion of industrial applicability.

The essence of the claimed invention is illustrated by graphic materials, which are presented in:

FIG. 1. Traditional cytological preparation. Melanoma metastasis? Staining according to Romanovsky. Uv. about. 10x

FIG. 2. Traditional cytological preparation. Melanoma metastasis? Staining according to Romanovsky. About. uv. 10x

FIG. 3. ICC. Expression of Melan A. Uv. about. 20x

FIG. 4. ICC. Expression of HMB45. Uv. about. 100x

FIG. 5. Traditional cytological preparation. Low-grade adenocarcinoma. Staining according to Romanovsky. Uv. about. 10x

FIG. 6. Cell block. Adenocarcinoma. Hematoxylin-eosin staining. Uv. about. 20x

FIG. 7. ICC. CK7 expression. Uv. about. 20x

FIG. 8. ICC. Expression of CK19. Uv. about. 20x

FIG. 9. ICC. CK18 expression. Uv. about. 10x

FIG. 10. ICC. Expression of TTF-1. Uv. about. 20x

FIG. 11. Traditional cytological preparation. Small cell cancer. Staining according to Romanovsky. Uv. about. 10x

FIG. 12. Cell block. Among the fibrin are small clusters of dilapidated tumor cells with hyperchromic nuclei. Hematoxylin-eosin staining. Uv. about. 20x

FIG. 13. ICC. CD56 expression. Uv. about. 20x

FIG. 14. ICC. Expression of AE1/AE3. Uv. about. 100x

Examples of using the method

Example 1. Patient S., born in 1946. Complaints about the presence of a pigmented formation on the right shoulder for a year.

CT conclusion: Focal formations up to 8 mm are determined polysegmentally in both lungs. The right lung is reduced in volume due to atelectasis of the middle lobe. The middle lobe of the bronchus and its branches are “amputated” by pathological masses. In the mediastinal section of the right lung (from the lower edge of the right pulmonary artery to the right inferior pulmonary vein), pathological masses with signs of invasion are determined, narrowing the right superior pulmonary vein. Pathological masses merge with atelectasis parenchyma. Probably part of the pathological masses are enlarged bronchopulmonary lymph nodes. Intrathoracic lymph nodes - pre-, paratracheal, bifurcation and bronchopulmonary on both sides up to 10 mm. Axillary lymph nodes up to 15 mm on the right.

Fiberoptic bronchoscopy: The lumen of the middle lobe of the bronchus is obstructed by a tuberous white neoplasm. Infiltration spreads along the anterior wall of the lower lobe of the bronchus on the right, along the anterior, lateral and medial walls of the intermediate bronchus to a distance of 0.5 cm to the lower edge of the mouth of the upper lobe of the bronchus.

Conclusion: Tumor of the middle lobe of the bronchus with infiltration of the lower and intermediate lobes of the bronchus.

Cellular material for research was obtained by transesophageal ultrasonography with fine-needle aspiration biopsy. Part of the cellular material is transferred to a glass slide for traditional cytological examination, and the remaining part is transferred to an Eppendorf with 1.0 ml of transport medium consisting of a solution of neutral formaldehyde and isotonic physiological solution of sodium chloride 0.9% in a 1:1 ratio for liquid-based cytology and preparation cell block.

Preliminary diagnosis: Posterior mediastinal neoplasm.

Cytological conclusion: The material is represented by fields of scattered and extensive accumulations of malignant cells with tender, abundant cytoplasm, in which there is dust-like granularity, and there are also binucleate cells with large nucleoli and pigment inside the cytoplasm. The cytological picture allows one to suspect melanoma metastasis (FIG. 1, 2).

To clarify the morphological diagnosis, an ICC study was performed. The micropreparation was obtained from a cell block prepared using the technology of the proposed method.

Result of the ICC study: Melan A - positive reaction in tumor cells; HMB45 - positive reaction in tumor cells (FIG. 3, 4).

Pathological examination. Biomaterial - fragments of bronchus formation.

Microscopic description: In the studied samples, multiple foci of tumor growth were determined, consisting of polygonal cells with light cytoplasm and polymorphic hyperchromic nuclei.

Diagnosis: Malignant mesenchymal tumor? Amelanotic melanoma?

To verify the morphological diagnosis, an ICC study was performed.

IHC study results: Cytokeratin, clone AE1/AE3 - negative reaction in tumor cells; Vimentin, Clone V9, S-100, polyclonal, Melanosome, Clone HMB-45, Melan-A, Clone A103 - positive reaction in tumor cells.

The immunophenotype of tumor cells of the studied material corresponds to the immunophenotype of melanoma.

Conclusion: Metastases of non-pigmented melanoma.

Example 2. Patient K. born in 1941 Complains of cough, hemoptysis, shortness of breath with little physical exertion, and bubbling wheezing.

CT: Solitary focal formation of the lower lobe of the bronchus of the left lung. In the medial section of the right lung, a space-occupying formation with tuberous contours up to 39×38×52 mm is determined, not separated from the posterior wall of the right main bronchus, mediastinal pleura, or esophagus. In S7, areas of “ground glass” are located on the right perifocal to the formation. Intrathoracic lymph nodes - pre- and paratracheal up to 11 mm, bifurcation up to 14 mm.

Conclusion: Disseminated process of the lungs. Mediastinal lymphadenopathy.

Cellular material for research was obtained by endoesophageal ultrasonography with fine-needle aspiration biopsy. Part of the cellular material is transferred to a glass slide for traditional cytological examination, and the remaining part is transferred to an Eppendorf with 1.0 ml of transport medium consisting of a solution of neutral formaldehyde and isotonic physiological solution of sodium chloride 0.9% in a 1:1 ratio for liquid-based cytology and preparation cell block.

Cytological conclusion: Adenocarcinoma with a low degree of differentiation (FIG. 5, 6).

Considering the unclear histogenesis of the tumor, it was decided to conduct an ICC study. The micropreparation was obtained from a cell block prepared using the technology of the proposed method.

ICC result:

CK 7 - positive reaction in tumor cells;

CK 18 - positive reaction in tumor cells;

CK 19 - positive reaction in tumor cells;

TTF1 is a positive reaction in tumor cells.

The immunophenotype of tumor cells corresponds to lung adenocarcinoma (FIG. 7-10).

Example 3. Patient M., 68 years old. Complains of an increase in body temperature to 37.4°C in the evenings, a wet cough streaked with blood, and increased fatigue.

Computed tomography conclusion: Severe hilar lymphadenopathy with signs of invasion into the left brachiocephalic vein and the development of collaterals. Focal formations in the right lung, probably metastases. Mass formation of the right adrenal gland. Lymphadenopathy of the retroperitoneum.

Endosonography protocol: In the 2L projection of the group of mediastinal lymph nodes, a volumetric isoechoic neoplasm with a clear hyperechoic capsule is determined, occupying the entire echo window.

Preliminary diagnosis: Focal neoplasms of the right lung. Mediastinal lymphadenopathy.

For morphological verification of the diagnosis, biological material for research was obtained by endobronchial ultrasonography with fine-needle aspiration biopsy. Part of the cellular material was transferred to a glass slide for traditional cytological examination, part of it was transferred to Eppendorf with 1.0 ml of transport medium consisting of a solution of neutral formaldehyde 10% and isotonic physiological solution of sodium chloride 0.9% in a 1:1 ratio for liquid-based cytology and preparation cell block.

The resulting histological material was placed in a buffered neutral solution of formaldehyde 10% for pathological examination.

Cytological conclusion: the specimen contains blood elements, extensive accumulations of destroyed tumor cells, and in intact places there are fields with dystrophically altered, scatteredly lying similar cells of a malignant tumor. Undifferentiated cancer? Lymphoproliferative process? (FIG. 11).

To verify the morphological diagnosis, an ICC study was performed. The micropreparation is obtained from a cell block prepared using the technology of the proposed method.

Morphological study of the cell block: among the fibrin there are clusters of half-destroyed tumor cells with hyperchromatic nuclei; in individual cells a narrow rim of cytoplasm is discernible (FIG. 12).

Result of the ICC study:

Cytokeratin, clone AE1/AE3 - positive reaction in tumor cells;

CD56, clone MRQ-42 - positive reaction in tumor cells.

The immunophenotype of the surviving tumor cells corresponds to the immunophenotype of small cell carcinoma (FIG. 13-14).

Pathological conclusion: lung adenocarcinoma.

The given examples can serve as confirmation that the use of the method of obtaining cell blocks for cytological and ICC diagnostics of malignant neoplasms preserves the morphological structure and does not change the antigenic properties of cells, simplifies and simplifies the technology for preparing cell blocks, is of economic importance, creates conditions for import substitution, and preserves cell block for ICC research and increases the accuracy of cytological and ICC diagnostics of malignant neoplasms.

INFORMATION SOURCES

1. Gill G.W. Clinical cytology. Theory and practice of cytotechnology / G.U. Gill: trans. from English edited by A.V. Bezrukova. - M.: Practical Medicine, 2015, - 384 p.

2. Volchenko N.N. “Cell block” technology in cytological practice / N.N. Volchenko, O.V. Borisova, I.B. Baranova // Clinical laboratory diagnostics. - 2015. - No. 8. - pp. 39-41.

3. Jan D. Cell blocks in cytopathology: a review of preparative methods, utility in diagnosis and role in ancillary studies / D. Jan, S.R. Mathur, V.K. Iyer // Cytopathology. - 2014. - No. 25. - P. 356-371.

4. Tikhonov Ya.N. Method for manufacturing a cell block of cellular material / Ya.N. Tikhonov, I.V. Nazarova, I.V. Reva et al./ State patent for invention No. 2722661 dated 02/02/2020 // Official Bulletin of the FSPIS (Rospatent). - 2020. - No. 16.

5. Standard technological procedures when conducting a pathological examination. Clinical practice guideline RPS1.1. - M., 2016. - 119 p.

Claims (1)

A method for obtaining cell blocks for cytological and immunocytochemical (ICC) diagnosis of malignant neoplasms, characterized in that the cellular material obtained by fine-needle aspiration biopsy and endobronchial and endoesophageal ultrasonography with fine-needle aspiration biopsy and histological testing, including dehydration, paraffin saturation, embedding in the block , microtomy, production of micropreparations for morphological research, characterized in that the cellular material is placed in an Eppendorf containing a transport medium consisting of a solution of neutral formaldehyde 10% and isotonic physiological solution of sodium chloride 0.9% in a ratio of 1:1 in a volume of 0.8 -1.0 ml, centrifuge the cell suspension for 5 minutes at a speed of 4000 rpm, separate the cell sediment by draining the supernatant, add 1.5 ml of isopropyl alcohol and 5-6 drops of glycerol to the sediment, centrifuge for 5 minutes at speed of 4000 rpm, while the formed cell clot is located in the center of the Eppendorf at the boundary of the two-phase liquid, the supernatant liquid is drained and with a dissecting needle it is transferred to a biopsy cassette, at the bottom of which there is a sponge for guiding the biopsy material, and the clot is covered with the same sponge and closed cover the cassette, place it in a neutral buffered solution of formaldehyde 10% for 12-18 hours, then the cassette with the cell clot is transferred to a histological processor for histological processing, poured into the block, and microslides are made for cytological and ICC studies.