Automated systems for creating tissue microarrays in oncomorphological studies

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INTRODUCTION

The relevance of implementing automated systems for constructing tissue microarrays (TMAs) in oncomorphological research is driven by the need to improve the accuracy and efficiency of high-volume tissue sample analysis. TMAs enable simultaneous evaluation of dozens to hundreds of samples on a single slide, which significantly accelerates diagnostic and investigative processes in oncology [1]. The TMA technique proposed by Battifora in 1986 is widely used for validation of diagnostic and prognostic biomarkers, quality control in immunohistochemical (IHC) analysis, and other scientific applications [2, 3].

The challenges of traditional manual TMA construction are largely related to the labor-intensive nature of the process, high operator skill requirements, and considerable time consumption [4]. Automation of TMA construction overcomes these challenges owing to high precision, substantial time savings, and reduced risk of cross-contamination [5, 6]. However, despite the clear advantages of automated systems, their implementation in clinical practice in Russia remains limited due to the absence of regulatory certification [7].

Published studies have confirmed the effectiveness of automated platforms for TMA construction. For example, automated systems have been successfully applied for biomarker evaluation across multiple tumor types [3], ensuring high accuracy. Documented savings of time and resources make automated systems attractive for laboratories with a high sample load and substantial workload demands [5].

Thus, the introduction of automated systems for TMA construction is a major step forward in improving efficiency and standardization within oncomorphological research. Nevertheless, full integration of these systems into clinical practice requires resolution of issues related to registration and standardization of their use in Russia [7].

This review summarizes the capabilities and advantages of automated systems for TMA construction compared with manual methods, with emphasis on their application in the analysis of Ewing sarcoma and other undifferentiated round cell sarcomas.

EXPERIENCE IN THE APPLICATION OF AUTOMATED SYSTEMS FOR TISSUE MICROARRAY CONSTRUCTION

Automated systems for TMA construction are high-technology devices that enable precise and rapid fabrication of microarrays from tens to hundreds of tissue samples [4]. The main modules of such systems include: a unit for automated extraction of tissue cores from donor paraffin blocks and their transfer into recipient blocks (see Fig. 1); a high-precision micromanipulator for positioning of tissue cores; a tissue core depth-control system (laser sensor or mechanical probe); and dedicated software for building a virtual TMA layout [8].

Fig. 1. Device for automatic punch needle positioning: A, punch needle; B, histological slide with marked area of interest, stained with hematoxylin and eosin, placed in the center of the rotator for positioning; C, recipient block; D, donor block 1; E, donor block 2.

The automated TMA fabrication process begins with uploading the virtual layout of core placement into the recipient block. Modern TMA design software allows specifying diameter of tissue cores, distance between cores, their number and arrangement, and assigning individual annotation to each sample [4]. Based on the virtual layout, the device automatically forms holes of a predetermined diameter in the recipient block for placement of each sample (see Fig. 2).

Fig. 2. Software-guided punch needle positioning.

The operator then sequentially loads donor paraffin blocks into the instrument and, using the micromanipulator, positions the punch needle over the region of interest selected in each sample based on the corresponding hematoxylin–eosin–stained control section (see Fig. 2) [9]. The device automatically extracts a tissue core of the predefined diameter (typically 0.6–2 mm) from the donor block and transfers it into the corresponding hole of the recipient block. The depth-control system ensures precise placement of each tissue core at a uniform level, which is particularly important for obtaining high-quality sections [10].

After completion of core collection, the recipient block is remelted to secure the cores in place and then cooled. The resulting TMA block is sectioned at 4–5 µm thickness and can be used for histological and IHC studies, as well as for fluorescence in situ hybridization (FISH) [11]. Examples of micropreparations produced using this technology are shown in Fig. 3.

Fig. 3. Overview of the microslides: a–c, microslides with marked areas of interest, stained with hematoxylin and eosin; d, microslide with tissue microarray, stained with hematoxylin and eosin; e, f, microslides stained by immunohistochemistry for CD99 and transcription factor NKX2.2, respectively.

Fig. 4. Findings in patients with Ewing sarcoma: H&E, slides stained with hematoxylin and eosin, ×40; CD99, slides stained with immunohistochemistry for CD99, ×40; NKX2.2, slides stained with immunohistochemistry for transcription factor NKX2.2, ×40. Only slides positive for antibodies against CD99 and NKX2.2 markers are presented, ×40.

An important aspect of TMA construction is the height of tissue cores. To preserve the original diagnostic material, additional paraffin blocks from the same case are often used for TMA preparation. This is standard practice that allows studies to be conducted without compromising primary diagnostics.

In our laboratory, an automated TMA construction system is used for both research and clinical-diagnostic purposes¹. In particular, we constructed a TMA from 106 Ewing sarcoma cases [12] and other undifferentiated round cell sarcomas (BCOR-altered sarcoma and CIC-rearranged sarcoma). For each case, two representative tumor tissue cores 1 mm in diameter were selected and transferred into recipient paraffin blocks. TMA sections were stained with hematoxylin and eosin to assess morphological features. For differential diagnostic algorithm development, IHC studies were performed on an automated immunostainer Ventana BenchMark ULTRA (Roche, Switzerland) using antibodies to markers relevant for phenotypic characterization of Ewing sarcoma and other undifferentiated round cell sarcomas: CD99 (Cell Marque, US), NKX2.2 (BioSB, US), BCOR (Cell Marque, US), SATB2 (Santa Cruz, US), TLE1 (Cell Marque, US), WT1 (Cell Marque, US), ETV4 (Invitrogen, US), desmin (Cell Marque, US), myogenin (Cell Marque, US), MyoD1 (Cell Marque, US), and pancytokeratin (Ventana, US). Since the study was retrospective, the immunohistochemical antibody panel was selected considering that the most common variants of round cell sarcomas were included. The quality of TMA sections and staining results was independently evaluated by two pathologists.

The use of an automated TMA construction system in our study enabled efficient analysis of a large cohort (106 cases) of Ewing sarcoma and other undifferentiated round cell sarcomas, and made it possible to identify novel combinations of diagnostic markers. IHC results demonstrated differences in marker expression between Ewing sarcoma, BCOR-altered sarcoma, and CIC-rearranged sarcoma. Co-expression of CD99 and NKX2.2, characteristic of Ewing sarcoma, was observed in 94% of cases, whereas this combination was not detected in other tumor types. BCOR-altered sarcoma was distinguished by co-expression of BCOR, TLE1, and SATB2 in 80% of cases. CIC-rearranged sarcoma was characterized by co-expression of ETV4 and WT1, although this was identified in only 25% of cases. Based on these results, a differential diagnostic algorithm was developed for tumors both within this group and in comparison with other round cell neoplasms [12].

ADVANTAGES AND DISADVANTAGES OF AUTOMATED SYSTEMS

The implementation of automated systems for TMA construction opens new opportunities for standardization and acceleration of oncomorphological studies. Compared with manual TMA fabrication, automated production offers several advantages [6]:

• Higher accuracy of tissue core sampling due to high-precision mechanics and depth-control systems [3];
• Substantial time savings for highly trained personnel, especially when processing large sample volumes [5];
• Reduced risk of cross-contamination between samples and tissue damage due to minimization of manual manipulation [7];
• Flexible TMA design with the ability to select various core parameters and spatial layout using specialized software [4];
• Individual labeling and annotation of tissue cores allowing subsequent correlation of morphologic features, IHC, and FISH results [13].

Notably, TMAs serve as a universal control. Although not all samples will yield a positive reaction with antibodies against markers relevant to a specific diagnostic case, the value of TMA lies in the ability to simultaneously analyze numerous specimens, which is particularly important for large-scale studies.

The use of automated systems requires consideration of additional time expenditures for programming and instrument maintenance. Nevertheless, our experience shows that with appropriate workflow organization and regular utilization, automated systems allow substantial optimization of laboratory processes, particularly when handling large volumes of samples.

Despite the clear advantages of using automated systems for TMA construction, obtaining valid results requires adherence to several methodological considerations: mapping should be performed using tissues structurally distinct from the target material [3]; tissue cores must be selected with attention to tumor heterogeneity to ensure representativeness [2]; a sufficient number of tissue cylinders per case (at least 2–3) should be included [14]; staining and interpretation protocols must be standardized [5, 15].

Experience in our laboratory demonstrates that implementation of automated systems for TMA construction substantially optimizes the simultaneous production of high-quality sections from a large number of specimens for all major types of morphological studies.

CONCLUSION

Tissue microarrays represent a modern oncomorphology tool that enables efficient assessment of large archival sample series for validation of diagnostic and prognostic biomarkers, both in research settings and for immunohistochemistry quality control.

Automation of TMA construction using dedicated systems ensures standardization of the pre-analytical phase and increases laboratory throughput compared with manual methods.

Further development and wider implementation of automated systems for TMA construction will enhance the efficiency of oncomorphological research and accelerate translation of scientific advances into clinical practice.

ADDITIONAL INFORMATION

Author contributions: I.A. Parfenova: conceptualization, methodology, writing—original draft, writing—review & editing; S.A. Eryshova: conceptualization, methodology, formal analysis, writing—original draft. All the authors approved the version of the manuscript to be published and agreed to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Acknowledgements: The authors express their gratitude to Dr. Ilya V. Sidorov, pathologist at the Pathology Department of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology, and Dr. Dmitry M. Konovalov, Head of the Pathology Department at the same institution, for providing clinical material and valuable consultations during the research in patients with undifferentiated round cell sarcomas. Thanks to their expert knowledge and support, this work was successfully completed.

Funding sources: No funding.

Disclosure of interests: The authors have no relationships, activities, or interests for the last three years related to for-profit or not-for-profit third parties whose interests may be affected by the content of the article.

Statement of originality: No previously obtained or published material (text, images, or data) was used in this study or article.

Data availability statement: All data obtained in this study are available in this article.

Generative AI: No generative artificial intelligence technologies were used to prepare this article.

Provenance and peer review: This paper was submitted unsolicited and reviewed following the standard procedure. The peer review process involved two external reviewers, a member of the Editorial Board, and the in-house scientific editor.

¹ At the time of preparing this publication, automated tissue microarray construction instruments do not have regulatory registration in Russia.

About the authors

Inna A. Parfenova

Author for correspondence.
Email: pathmorf@mail.ru
ORCID iD: 0009-0002-0667-9352
Russian Federation, Moscow

Sofia A. Eryshova

Email: sofya.eryshova@dgoi.ru
ORCID iD: 0009-0008-8862-2619
Russian Federation, Moscow

References

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2. Fig. 2. Software-guided punch needle positioning.

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3. Fig. 3. Overview of the microslides: a–c, microslides with marked areas of interest, stained with hematoxylin and eosin; d, microslide with tissue microarray, stained with hematoxylin and eosin; e, f, microslides stained by immunohistochemistry for CD99 and transcription factor NKX2.2, respectively.

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4. Fig. 4. Findings in patients with Ewing sarcoma: H&E, slides stained with hematoxylin and eosin, ×40; CD99, slides stained with immunohistochemistry for CD99, ×40; NKX2.2, slides stained with immunohistochemistry for transcription factor NKX2.2, ×40. Only slides positive for antibodies against CD99 and NKX2.2 markers are presented, ×40.

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5. Fig. 1. Device for automatic punch needle positioning: A, punch needle; B, histological slide with marked area of interest, stained with hematoxylin and eosin, placed in the center of the rotator for positioning; C, recipient block; D, donor block 1; E, donor block 2.

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6. Fig. 4. Findings in patients with Ewing sarcoma: H&E, slides stained with hematoxylin and eosin, ×40; CD99, slides stained with immunohistochemistry for CD99, ×40; NKX2.2, slides stained with immunohistochemistry for transcription factor NKX2.2, ×40. Only slides positive for antibodies against CD99 and NKX2.2 markers are presented, ×40.

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