Transmission Electron Microscopy: A Surgical Pathology Tool for Neuroblastoma
Summary
Pediatric small round blue cell tumors are an intriguing and challenging collection of neoplasms. Therefore, transmission electron microscopy (TEM) and professional knowledge of pediatric tumors can be extremely valuable in surgical pathology. Here, we present a protocol to perform TEM for diagnosing neuroblastoma, one of the most common solid tumors in childhood.
Abstract
Pediatric small round blue cell tumors (PSRBCT) are an intriguing and challenging collection of neoplasms. Light microscopy of small round blue cell tumors identifies small round cells. They harbor a generally hyperchromatic nucleus and relatively scanty basophilic cytoplasm. Pediatric small round blue cell tumors include several entities. Usually, they incorporate Wilms tumor, neuroblastoma, rhabdomyosarcoma, Ewing sarcoma, retinoblastoma, lymphoma, and small cell osteosarcoma, among others. Even using immunohistochemistry, the differential diagnosis of these neoplasms may be controversial at light microscopy. A faint staining or an ambiguous background can deter pathologists from making the proper diagnostic decision. In addition, molecular biology may provide an overwhelming amount of data challenging to distinguish them, and some translocations may be seen in more than one category. Thus, transmission electron microscopy (TEM) can be extremely valuable. Here we emphasize the modern protocol for TEM data of the neuroblastoma. Tumor cells with tangles of cytoplasmic processes containing neurosecretory granules can diagnose neuroblastoma.
Introduction
The work of a pathologist may be pretty challenging both in clinical diagnostics and research fields. The evolution of light microscopy in the 18th and 19th century was remarkable. The power of an electron microscope relies primarily on the wavelength of the electrons, which is shorter than light1,2,3. Before the advent of the polyclonal and monoclonal antibodies and their application in immunohistochemistry, TEM played an influential role in diagnosing small round blue cell tumors.
Starting with the 90's of the last century, the immunohistochemical approach has substituted the morphologic tool in diagnostics4. Currently, there are thousands of new polyclonal and monoclonal antibodies directed to antigens of the small round blue cell tumor group4,6,7,8. In the last decade of the highly prolific 20th century and the first decade of the beginning of the 21st century, molecular biology, including fluorescence in situ hybridization, from genomic probes through next-generation sequencing, seems to have superseded the significant application role of immunohistochemistry in several laboratories4. The Food and Drug Administration (FDA) in the United States of America, the Canadian Food Inspection Agency (CFIA) of Canada, the Environmental Protection Agency (EPA), or similar governmental bodies in other countries do not always approve molecular biology protocols9. It seems that there is a lot of information quite challenging to insert in a pathology report that can be used for therapeutic purposes, and the oculate choice of a well-funded and running laboratory information system is critical10. In the meantime, immunohistochemistry has revealed numerous pitfalls, with epithelial tumors showing mesenchymal markers and vice versa11. The epithelial-mesenchymal transition has confused some borders in pathology groups12,13. In the last few years, it has become evident that electron microscopy flourished in several labs worldwide14. In particular, the tissue specimens' turnaround time has decreased from weeks to only 3 days or even less using several protocols approaching the staining with monoclonal or polyclonal antibodies4,10.
Moreover, applying an electronic camera coupled to the electron microscope helped provide the pathologists with a rapid image, which is versatile in different operating systems. Finally, some antibodies, even after antigen retrieval, are challenging to be revealed in some areas of necrosis or autophagy/ischemia-related changes. At the same time, electron microscopy in safe hands can still deliver excellent results and hints for the correct classification of unknown pathologic tumors15.
The pediatric small round blue cell tumor group includes several tumors, mainly neuroblastoma, Wilms tumor or nephroblastoma, rhabdomyosarcoma, and Ewing sarcoma. The molecular biology data relative to the pediatric group of small round blue cell tumors can be overwhelming because of the techniques applied. The small round blue cells may not differ much on routine stains (hematoxylin and eosin staining), and some tumors may have aberrant immunophenotypical features. Advances in molecular biology have been enormous since the discovery of TEM. In the group of small round blue cell tumors, some neoplasms may be more frequently encountered than others, but they need to be considered. Although the papillary renal cell carcinoma is not essentially a small round blue cell tumor but features papillae mostly, it may show some round cell areas that may need to be distinct from other well-known small round blue cell tumors (e.g., Wilms tumor) using several ancillary techniques16. Ultimately, metanephric stromal tumors may also need to be taken into differential diagnosis17. The rhabdoid tumor is a particularly malignant pediatric tumor distinct in the renal and extra-renal subtype18.
Neuroblastoma is one of the most common solid malignancies in infancy and childhood. Neuroblastoma cells are the malignant cells of this solid tumor that arise insidiously from derivatives of the primordial neural crest. Its diagnosis and differential diagnosis may be difficult. Its natural biology has seen remarkable advances in the last couple of decades. The forkhead family of transcription factors is characterized by a distinct "forkhead" domain (FOXO3/FKHRL1). These transcription factors function as a trigger for apoptosis (programmed cell death) through the expression of genes necessary for cell death. FOXO3/FKHRL1 is activated by 5-aza-2-deoxycytidine and induces silenced caspase-8, and this complex plays a crucial role in neuroblastoma. Nuclear FOXO3 predicts adverse clinical outcomes and promotes tumor angiogenesis in neuroblastoma7,19. Despite the molecular pathology advances, the Shimada classification remains the standard of practice for any pathologist and pediatric oncologist. It is critical in differentiating between favorable and unfavorable histology20,21,22,23.
The rationale for developing a straightforward protocol for the electron microscopy of tumors suspicious of neuroblastoma is linked to the feasibility and solidity of the ultrastructural examination of the tissue specimen. It is rarely altered by problems commonly encountered using immunohistochemistry. The rationale and protocol have been the basis of several textbooks and scientific contributions to pediatric pathology and electron microscopy4,24,25. This protocol spans the experience of three decades of the author and will focus on a few PSRBCT, emphasizing the personal experience and the literature review.
Protocol
Representative Results
Discussion
In this narrative review with associated protocol, we highlighted the distinctive ultrastructural features of the neuroblastoma. In the end, we suggest that electron microscopy is far from a "dead" or ancient technique, postulating the discovery of a new role if combined with single-cell omics technologies. This contribution would like to emphasize the never ancient role of electron microscopy in pediatric pathology4,27. The critical steps, the modificati…
Declarações
The authors have nothing to disclose.
Acknowledgements
We acknowledged the expertise and generous support of Dr. Richard Vriend, formerly an Alberta Health Services employee at the University of Alberta Hospital, and Steven Joy (1972-2019), also formerly an Alberta Health Services employee at the University of Alberta Hospital. We dedicate this work to the memory of Mr. Joy, a senior technologist expert in ultrastructural investigations who tragically and prematurely passed away a few years ago. Mr. Joy was a pillar for most electron microscopy studies in Alberta, Canada. Our thoughts and prayers for him and his family. We are also indebted to Ms. Lesley Burnet for her help and advice. Dr. C. Sergi's research has been funded by the generosity of the Children's Hospital of Eastern Ontario, Ottawa, Ontario, and the Stollery Children's Hospital Foundation and supporters of the Lois Hole Hospital for Women through the Women and Children's Health Research Institute (WCHRI, Grant ID #: 2096), Natural Science Foundation of Hubei Province for Hubei University of Technology (100-Talent Grant for Recruitment Program of Foreign Experts Total Funding: Digital PCR and NGS-based diagnosis for infection and oncology, 2017-2022), Österreichische Krebshilfe Tyrol (Krebsgesellschaft Tirol, Austrian Tyrolean Cancer Research Institute, 2007 and 2009 – "DMBTI and cholangiocellular carcinomas" and "Hsp70 and HSPBP1 in carcinomas of the pancreas"), Austrian Research Fund (Fonds zur Förderung der wissenschaftlichen Forschung, FWF, Grant ID L313-B13), Canadian Foundation for Women's Health ("Early Fetal Heart-RES0000928"), Cancer Research Society (von Willebrand factor gene expression in cancer cells), Canadian Institutes of Health Research (Omega-3 Fatty Acids for Treatment of Intestinal Failure Associated Liver Disease: A Translational Research Study, 2011-2014, CIHR 232514), and the Saudi Cultural Bureau, Ottawa, Canada. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Materials
| Acetone, ACS Reagent | Electron Microscopy Sciences | 10014 | Reagent as established by the American Chemical Society (ACS Reagent). |
| Automated tissue processor | Electron Microscopy Sciences | L12600 | LYNX II Automated Tissue Processor for Histology and Microscopy. |
| Digital camera software | Gatan | L12600 | Digital Micrograph |
| Spurr's resin | Electron Microscopy Sciences | 14300 | Embedding resin. It provides excellent penetration for embedding tissues and rapid infiltration. The blocks have excellent trimming and sectioning qualities, while thin sections reveal tough qualities under the electron beam. |
| Ethyl Alcohol | Electron Microscopy Sciences | 15058 | 100% Ethyl alcohol with molecular sieve, 50% and 70%. |
| Glutaraldehyde, 25% EM Grade Aqueous in Serum Vial | Electron Microscopy Sciences | 16214 | 2.5% gluteraldehyde in 0.1 M cacodylate buffer, pH 7.2-7.4 made from 25% gluteraldehyde, primary fixative for TEM tissue specimens. |
| Osmium tetroxide | Electron Microscopy Sciences | 19110 | Second fixative during TEM tissue processing used as OsO4 in distilled water |
| Polyethylene capsules | Electron Microscopy Sciences | 70021 | Flat Bottom Embedding Capsules, Size 00 |
| Scintillator | Electron Microscopy Sciences | 82010 | Phillip Quad Detector |
| Single Edge Razor Blade | Electron Microscopy Sciences | 71952-10 | Blade for Clean Rm., 10/Disp. . |
| Sodium Cacodylate Buffer | Electron Microscopy Sciences | 11655 | Sodium Cacodylate Buffer, 0.4M, pH 7.2, prepared from Sodium Cacodylate Trihydrate |
| Tannic Acid, Reagent, A.C.S., EM Grade | Electron Microscopy Sciences | 21700 | Reagent as established by the American Chemical Society (ACS Reagent). |
| Transmission Electron Microscope (1) | Hitachi | Hitachi 7100 | We use it at the HV 75 setting |
| Transmission Electron Microscope (2) | JEOL | JEM-1010 | We use it at the HV 38 setting |
| Toluene | Electron Microscopy Sciences | 22030 | Reagent as established by the American Chemical Society (ACS Reagent) |
| Ultracut microtome | Leica | 11865766 | Ultramicrotome |
| Uranyl acetate | Electron Microscopy Sciences | 22400 | Uranyless, substitute for uranyl acetate |
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