RU2432575C2 - Method for preparing sections of fresh frozen lymph node tissue - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: sections are prepared by freezing a biopsy material plate in n-hexane cooled to temperature -95°C, and making sections in thickness no more than 7 mcm in a cryostat. The section is placed on a slide, drowned in an alcohol-aldehyde solution at room temperature (20-25°C) for 3 minutes, then washed in flowing water from a fixative for 30 seconds, stained with histological stains and enclosed by a cover glass consists under integumentary glass in the usual way. The preparations produced as described above preserve completely the structure of tissues and separate cells and allow describing lymphatic tumours according to the existing classifications. The method can be used in urgent histological examinations (intraoperative biopsies) as allows drawing up a conclusion decision for 20 minutes.

EFFECT: use of the method allows reducing working hours for the histological examinations required for diagnostics, in comparison with a conventional method, does not deteriorate the performed operations, and accelerates considerably drawing up the conclusion decisions.

3 dwg, 3 ex

Description

The invention relates to the field of medical diagnosis and can be used in the diagnosis of tumor diseases.

A standard study of the structure of tumor tissue for diagnostic purposes is carried out on sections obtained from biopsy material fixed with a formalin solution, dehydrated and embedded in paraffin or other synthetic media (for example, paraplast, histoplast, etc.). The process of tissue processing, ending with the manufacture of slices from paraffin blocks, is long and takes more than 42 hours [1].

Different approaches are used to speed up slicing. One of these approaches is the processing of tissue in a vacuum, which reduces the time of dehydration and impregnation of the material [2]. Another approach is microwave exposure to tissue. It can significantly reduce the time at the stage of tissue fixation and dehydration [3]. A combination of these two methods is also used [4].

On the basis of the described methods, devices have been developed that make it possible to produce paraffin blocks and obtain slices from them in 4-5 hours, but in general practice these methods of distribution have not been obtained.

In pathoanatomical practice, urgent histological studies are carried out along with planned ones. This is, first of all, intraoperative biopsies, allowing in difficult cases to decide on the volume of surgical intervention. The time taken to obtain an opinion in these cases should be no more than 20 minutes. Paraffin sections do not meet this requirement. To quickly obtain a histological preparation in biopsy diagnostics, rapid tissue freezing is used, followed by the manufacture of sections using a cryostat or a freezing attachment to the microtome. Such sections are useful for solving the problem of the presence or absence of tumor growth for solid tumors (cancer), and also allow determining the nature of the formation removed by the surgeon (parathyroid gland, lymph node, fatty tissue, metastasis, etc.) [5]. These sections are not used to verify the final diagnosis due to the large (> 10 μm) thickness of the sections and the distortion of the fine structure of tissues and cells as a result of the formation of relatively large ice crystals [6, 7].

About the way.

Only sections with a thickness of less than 7 μm with a maximum degree of preservation of the tissue and structure of individual cells are suitable for studying the structure of the lymph nodes, since the analysis of the pathological state of the lymph node (reactive changes, lymphoma, metastasis) is carried out at the level of details of the cellular structure.

To obtain a high-quality frozen slice, a necessary condition is rapid tissue freezing, in which water does not crystallize, but passes into a state of amorphous ice [8].

The speed of freezing is primarily limited by the rate of heat transfer. The tissue biopsy material itself has low thermal conductivity. Therefore, it is impossible to quickly and uniformly cool a large sample without damage. To ensure maximum cooling rate, the biopsy specimen should be frozen in a liquid medium. This liquid (refrigerant) should have a melting point below the temperature of initiation of crystallization of water - 70 ° C [9]. A number of alkanes satisfies this requirement: pentane, 2-methylbutane (isopentane), n-hexane, heptane.

As noted above, the closest technical solution to the issue of significant acceleration of the slice is a method of freezing tissue in isopentane [8]. However, relatively small pieces of tissue (less than 1 cm ³ ) are frozen in isopentane, the volume of which may be insufficient for diagnostic studies. The use of a frozen biopsy specimen in the form of a plate, which provides a large surface area of the tissue with a minimum volume, is unacceptable due to cracking in isopentane.

The solution to the problem was found in freezing a biopsy sample in the form of a plate of not more than 3 mm and using n-hexane as the freezing medium. In this case, the plate does not crack. Therefore, when developing the method, the authors chose the form of a biopsy sample in the form of a plate. As a result, slices with a thickness of less than 7 μm of high quality were obtained from quick-frozen material, which made it possible to carry out microscopic studies of biopsy material in the framework of both planned and cytobiopsies. In addition, the application of the described method with planned biopsies significantly reduces labor costs and reduces the time for the manufacture of histological sections.

The goal set in this work is thus achieved by freezing tissue in n-hexane, cooled to a melting point (-95 ° C), in the form of a plate with a thickness of not more than 3 mm and making sections of it with a thickness of less than 7 μm from it in a cryostat according to the standard method , followed by quick fixation and coloring.

Technical description of the method.

Making slices from freshly frozen lymph node tissue involves 3 steps:

- obtaining a block of frozen tissue in the form of a plate with a thickness of not more than 3 mm with a minimum of ice crystals;

- obtaining a slice with a thickness of less than 7 μm from the block and quick fixation of the slice on a glass slide, staining with a standard dye (hematoxylin-eosin, azure-eosin, etc.);

- conclusion under a coverslip. The result is a long-term storage drug (permanent drug).

Freezing the biopsy sample. A plastic cup, with a volume of at least 50 ml, is filled with n-hexane, at least 30 ml, and then placed in a foam base with a wall thickness of at least 4 cm to increase thermal insulation. Liquid nitrogen is added to the glass to bring n-hexane to a mushy state (mixture of liquid and solid). From a biopsy sample delivered from the operating room, a tissue plate 2-3 mm thick is cut, which is quickly immersed in a glass with chilled n-hexane. Next, the glass with the material is transferred to the working chamber of the cryostat (chamber temperature - 20 ° C) and after 30 s. the frozen piece is removed from the glass and transferred to filter paper in the working area of the cryostat chamber to remove excess n-hexane.

The production of a slice with a thickness of less than 7 μm from a frozen plate is carried out by the standard method. The slice is transferred onto a glass slide and immediately placed in an alcohol-aldehyde solution at room temperature (20-25 ° C) for 3 minutes, then washed with running water from the fixative for 30 s and stained with histological stains in the usual way. The stained sections are enclosed under a coverslip in a water-soluble medium, or dehydrated in an organic medium, and enclosed in a synthetic organic medium. Finished sections are viewed under a light microscope.

Slices obtained in this way allow one to study the general pattern of biopsy tissue, as well as the details of the cellular structure, allowing the identification of individual tumor cells in the lymphoid tissue.

Examples of specific research on the described method.

Example 1. Patient G.

At the SSC RAMS, the patient underwent thoracoscopic cytobiopsy of the mediastinal neoplasm. Frozen sections were made from one biopsy fragment using the method described above. The goal of cytobiopsy was to establish a differential diagnosis between lymphoma and a tumor of non-lymphoid origin.

On a frozen section, connective tissue was presented, against which the sites of lymphoid tissue consisting of small lymphocytes, pro-lymphocytes, as well as large cells of an atypical type were determined. Most of these cells had large nuclei in which large round nucleoli were present (comparable to the size of the nucleus of a small lymphocyte). According to the description, they fully corresponded to the Hodgkin and Berezovsky-Sternberg cells observed with lymphogranulomatosis (Hodgkin's lymphoma according to the WHO classification) (Fig. 1). Thus, a diagnosis of lymphogranulomatosis was established on a frozen section.

The time spent on the diagnosis of lymphogranulomatosis by a frozen section was 20 minutes.

The same biopsy, but another tissue fragment, was placed in a 4% formalin solution for the subsequent manufacture of paraffin sections according to the standard method.

In a histological examination, a paraffin section contained connective tissue with areas of lymphoid tissue represented by small lymphocytes and pro-lymphocytes. Against the background, typical Hodgkin and Berezovsky-Sternberg cells were determined.

The time taken to obtain a diagnosis of lymphogranulomatosis by paraffin section was 42 hours.

Example 2. Patient I.

The patient had enlarged cervical and axillary lymph nodes. In the clinical analysis of blood leukocytes - 14.5 thousand, lymphocytes - 71.5%. For the diagnosis, a cervical lymph node biopsy was performed.

A histological examination was performed on a frozen slice obtained by the method described above. Part of the biopsy material was used for freezing. Another part was sent for additional research.

The frozen section was used to determine the type of tumor and the alleged diagnosis. At low magnification, the pattern characteristic of normal lymph node tissue was erased (figure 2). Instead, diffuse proliferation of lymphoid cells with a small number of vessels was observed. At a larger magnification, the cells had the same structure. All of them were small, with a narrow cytoplasm and rounded dark nuclei. Nuclear chromatin is packaged in large lumps. Nucleoli in most of these nuclei were not visualized. There were a few prolymphocytes.

According to this description of the tissue, a diagnosis of mature cell lymphoma with a low proliferative potential of possible forms was suggested: B-cell chronic lymphocytic leukemia (B-CLL), lymphoma from mantle zone cells, lymphoma from marginal zone cells. With the help of studies clarifying the diagnosis, a diagnosis of B-CLL was established in the lymph node.

The time taken to make a preliminary diagnosis of a frozen slice was 30 minutes. To clarify the diagnosis, the total elapsed time was 24 hours.

Paraffin sections were made from a part of the lymph node using a standard technique. On the obtained slice at low magnification, the picture completely corresponded to that observed on the frozen slice (Fig. 2). With a large increase in the structure of lymphoid cells, there were no significant discrepancies with the frozen slice. The difference was the presence of gaps between the cells. On the frozen section, the cells were tightly adjacent to each other. The preliminary diagnosis, made by paraffin section, is identical to the diagnosis made by frozen section.

When making a paraffin section, a preliminary diagnosis was made 42 hours after taking the biopsy.

Example 3. Patient N.

The patient had generalized lymphadenopathy for two months. To verify the diagnosis, a biopsy of the cervical lymph node was performed.

For the diagnosis, a histological examination was performed on a frozen section.

Frozen sections were made from biopsy material as described above. A frozen section established a preliminary diagnosis. On a frozen slice at low magnification, the elements of the capsule and marginal sinus were not visualized (Fig. 3). There was moderate vascular parenchyma hyperplasia. The structures characteristic of a normal lymph node were absent in the section - the entire section area was filled with diffuse proliferate of large and medium-sized atypical lymphoid cells with a fairly wide cytoplasm and roundish light nuclei. In the majority of the described cells, large nucleoli were determined, round, represented in the amount of 1-2 per nucleus. Single nucleoli were located centrally. The chromatin of most nuclei had a granular or granular structure (small granules). There was an admixture of small lymphocytes lying diffusely between pathological cells. There were single macrophages. Among large cells, figures of mitosis and apoptosis were revealed. According to this study, the diagnosis was suggested: a large cell lymphoid tumor.

Based on the results of the studies, the final diagnosis of DBCCL (diffuse large B-cell lymphoma) was established.

In this case, the time taken to make a preliminary diagnosis of a frozen section was 25 minutes. The total time spent on the diagnosis of DBKKL was 24 hours.

Part of the biopsy specimen was used to prepare paraffin sections using a standard technique. On the paraffin section at low magnification, as well as on the frozen section, a pattern of diffuse growth of lymphoid tissue was observed. With a large increase, medium and large cells of atypical type and small lymphocytes were revealed. The structure of the pathological cells looked the same as on a frozen section, but with the following differences:

(1) The cytoplasm had a brighter pink color;

(2) The chromatin of these cells was very diverse in structure: nuclei with single nucleoli contained chromatin in the form of strands and granules;

(3) On a frozen slice, most large cells contained only chromatin granules.

These differences did not affect the diagnosis of large cell lymphoma, because the main signs of this disease, such as diffuse tissue growth, polymorphism of pathological cells, structural features of their nucleoli, the presence of a large number of cells in mitosis, were sufficient for diagnosis on a frozen section.

The time taken to obtain a preliminary diagnosis for the paraffin section was 42 hours. Subsequent additional studies would take another 8-24 hours.

Conclusion

In the period from 2003 to the present, studies have been performed with frozen sections of more than 500 biopsy specimens (lymph nodes).

The time required to obtain an accurate diagnostic report is no more than 24 hours. In the case of paraffin sections - from 50 hours to several days.

The proposed method reduces the labor costs of making a preliminary diagnosis, which is necessary for directing the search for an accurate diagnosis, in comparison with the standard method, does not impair the quality of the work performed, increases labor productivity in the field of diagnosing lymphoproliferative diseases, and significantly speeds up the issuance of an opinion.

Accelerated (by freezing) obtaining a slice allows already in cytodiagnostics to give an accurate diagnosis, in contrast to a paraffin slice.

Bibliography.

1. Yu.A. Krivolapov, E.E. Leenman. Morphological diagnosis of lymphomas, pp. 6-9, 2006.

2. Krivonosov L.K. Simple method of embedding in paraffin under vacuum Arkh Patol .; 40 (11): 75-76, 1978.

3. Anthony S-Y Leong, M.D., Microwave thechnology for light microscopy and ultrastructural studies, first edition, 2005.

4. Kok L.P., Boon ME Nonchemical dehydration of fixed tissue combining microwaves and vacuum, Eur JMorphol; 32 (1): 86-94, Mar, 1994.

5. M.A.Paltsev, I.A. Kazantseva, O.V. Zairatyants, L.V. Kaktursky. The procedure for the pathoanatomical study of biopsy (diagnostic and surgical) material Federal Service for Supervision of Health and Social Development. The system of voluntary certification of processes for the implementation of pathological studies and pathological services in healthcare 291-297, Moscow, 2006.

6. Juan Lechago MD. The frozen section Pathology in the trenches. Arch PatholLabMed-Vol 129, Desember 2005.

7. Edvard B. Desciak, Mary E. Maloney. Artifacts in frozen section preparation J Dermatol Surg 26: 5; May 2000.

8. A.H. Zinkin, N.G. Zingilevskaya, B. B. Muselyan. Cryotherapy in otorhinolaryngology (guidelines), the mechanism of action of low temperatures on biological tissues. Krasnodar, 1997.

9. D.A. Davis, D.M. Pellowski, and C.William Hanke. Preparation of Frozen Sections Dermatol Surg; 30: 1479-1485, 2004.

Claims (1)

A method of manufacturing slices from freshly frozen tissue of the lymph nodes, including freezing the biopsy material of the lymph node, obtaining a slice for examination, fixing it, washing, staining, placing it under a cover glass using a water-soluble medium or through dehydration, characterized in that the plate of the biopsy material is frozen in thickness not more than 3 mm in n-hexane cooled to -95 ° C and a section with a thickness of less than 7 μm is obtained for investigation.

Images