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 18^th and 19^th century was remarkable. The power of an electron microscope relies primarily on the wavelength of the electrons, which is shorter than light^1,2,3. 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 group^4,6,7,8. In the last decade of the highly prolific 20^th century and the first decade of the beginning of the 21^st 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 groups^12,13. 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 antibodies^4,10.

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 neuroblastoma^7,19. 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 histology^20,21,22,23.

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 microscopy^4,24,25. 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

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The electron microscopy study is part of the normal routine for microscopic investigation of the samples received for diagnostic purposes and does not require approval by the Bioethics Committee. This study is retrospective and respects the complete anonymity of the samples.

1. TEM protocol for FFPE (formalin-fixed and paraffin-embedded) retrieval and glutaraldehyde-fixed tissue specimens

1. Retrieve the last hematoxylin- and eosin-stained slides and the FFPE tissue block.
2. With a permanent marker, select the area of interest on the tissue slide and match it to the area on the tissue block.
3. Cut out the selected area with a new razor blade from the block and accurately cut it into 5 mm cubes on filter paper.
4. Place the filter paper with the retrieved tissue in a labelled petri dish. Lodge the petri dish in a 60-70 °C oven for 15 min, i.e., until the paraffin melts (paraffin wax consists of a mixture of solid straight-chain hydrocarbons ranging in melting point of 48 °C-66 °C (120 to 150 °F)) and gets absorbed by the filter paper.
5. Transfer the retrieved tissue free of paraffin into a scintillation vial filled with 4 mL of 100% toluene (methylbenzene) v/v [100% toluene means 100 mL of toluene]. Ensure that the samples continuously rotate through the deparaffinization process (Rotation speed: 24 rotations/min).
6. Go through two changes of toluene, 1 h each, and one change of 4 mL of 100% toluene 30 min each, using an angled rotating device.
7. Proceed through one change in 4 mL of 100% absolute ethanol (C₂H₅OH) for 1 h, and then two changes of 4 mL of 100% absolute ethanol for 30 min each. Then, another change of 30 min in 4 mL of 70% ethanol.
8. Proceed through one change of 4 mL of 0.1 M sodium cacodylate buffer, 30 min. Transfer the specimens in 4 mL of 2.5% glutaraldehyde and keep at 4 °C until processing.
9. Use the following steps in an automated tissue processor to standardize the processing: 0.1 M sodium cacodylate buffer for 5 min, 2% osmium tetroxide for 1 h and 50 min, dH₂O for 5 min, 50% ethanol for 15 min, 70% ethanol for 30 min, 100% ethanol for 15 min (three times), 100% ethanol for 30 min (three times), 100% ethanol for 45 min, acetone for 50 min, resin for 1 h (two times), and fresh resin overnight. The volume of all solutions is 15 mL.
10. Embed with fresh resin right out of the tissue processor.
    1. Position polyethylene capsules in a holder with numbered strips of paper. Drop fresh resin in the capsules, transfer the specimen to the appropriate blocks and keep the blocks overnight in a 60 °C (140 °F) oven.
11. Stain semi-thin (500 nm) sections with 1% toluidine blue on the hot plate, minimum heat setting for 30 s, rinse in dH₂O, and then place a coverslip.
12. Here, the Ultracut microtome (UCM) is used for sectioning semi-thin sections.
    1. Take resin embedded block and install it in the block holder of the UCM. Then, install the block holder on the stage of the UCM and tighten it.
    2. Cut excess resin around the sample using a single edge coated razor blade using the oculars. Cut the face of the block in a trapezoidal shape.
    3. Remove the block holder from the stage and install it in the UCM arm, tight. Next, install the blade holder on the UCM stage. Finally, tighten a glass knife on the blade holder.
    4. Using the oculars and the light provided by the UCM, slide the edge of the glass knife slowly, as close as possible, to the surface of the resin block to cut. Tighten the blade holder in place on the stage.
    5. Set the cutting thickness of the UCM at 500 nm and trim the resin block until 75% full-faced.
    6. Change the glass knife to a new one. Add sterile water to the glass knife's reservoir.
    7. Label a microscope slide with the case number and put four separate drops of sterile water on the glass slide.
    8. Still at 500 nm setting, cut four sections from the block. Using fine forceps, transfer 1 section at a time in one of the drops of water on the glass slide. Each drop on the slide will contain one section. Dry the slide on a hot plate at the lowest heat setting until dry (30 s).
13. Stain with the Toluidine blue method. Rinse in water and place a coverslip. Examine toluidine blue sections under a light microscope. If desired tissue structures are visible, cut ultra-thin sections. If desired tissue structures are not visible, cut deeper sections and stain them using the same steps until desired structures are exposed and shown on the toluidine blue slide.
14. When the desired area for electron microscopy is identified from the toluidine blue sections, cut the ultra-thin sections on the copper grids. Cut ultrathin slices using an ultramicrotome (samples destined for viewing in the TEM must be cut into very thin slices (80 nm)).
    1. Take resin embedded block and install it in the block holder of the UCM. Then, install the block holder on the stage of the UCM and tighten it.
    2. Using the oculars, cut the resin around the desired area for electron microscopy using a single-edge coated razor blade. Cut the face of the block in a trapeze shape, no bigger than 1 mm square. If the desired area for electron microscopy is challenging to see because of the glare of the light, use a dab of chloroform to highlight the structures in the resin block.
    3. Remove the block holder from the stage and install it in the UCM arm, tight. Next, install the blade holder on the UCM stage. Finally, tighten a diamond knife on the blade holder.
    4. Using the oculars and the light provided by the UCM, slide the edge of the diamond knife slowly, as close as possible, to the surface of the resin block to cut. Tighten the blade holder in place on the stage.
    5. Adjust the different angles of the diamond knife using the screws of the UCM. Ensure that the edge of the diamond knife is aligned as perfectly as possible with the surface of the block to cut. Fill the diamond knife's reservoir with sterile water.
    6. Set the UCM at 80 nm thickness and 1 mm/s speed. Here, the automated function is used for ultra-thin sectioning. Set the window of cutting on the UCM (top and bottom of the block's face).
    7. Press the Start button of the UCM to start sectioning. The sections will float on the sterile reservoir of the diamond knife.
    8. When enough sections are cut, use chloroform fumes to straighten the sections. Dip a cotton swab in chloroform and pass it above the sections without touching them.
15. Dip a copper grid in 100% alcohol and then sterile water using fine forceps. Clean each grid before being used. Pick the sections on a few grids.
16. Leave the grids with sections on labeled filter paper. Place this filter paper in a Petri dish and put it in the 60 °C oven for 30 min to allow the sections to dry on the grids.
17. Then, perform the contrast staining.
    1. Stain the grids in uranyl acetate or any environment-friendly alternative substitutive for 1 min. Then, rinse them in sterile water for injection (40 dips in four different water containers for each rinse after uranyl acetate and lead citrate). Lead citrate step is also 1 min.
18. Insert the grid in the TEM, allowing a high voltage electron beam to be emitted by a cathode and created by magnetic lenses. The beam that has been partially transmitted through the thin (electron-semitransparent) specimen forms the TEM information on the screen (see below).

2. TEM protocol for scanning and photographing the specimens

1. After the sections on the grids are stained, screen them with the electron microscope. At the electron microscope, turn on the ACC voltage to a pre-established setting (200-300 kV), which depends on the electron microscope used.
2. At the computer, turn on the digital camera software and enter the case number. This is important because the microphotographs are saved in the corresponding case file.
3. Load the grid in the electron microscope, pull the gun sample halfway out and flip the switch to AIR to allow the vacuum from the specimen gun chamber to fill with ambient (room) air.
4. Once it is full of air, pull out the gun sample. Place the gun sample on the designed holder. Then, flip open the grid holder at the tip of the gun.
5. Place the grid in place. Close the grid holder and ensure that the grid is secured.
6. Put the gun sample back in the specimen chamber halfway through and flip the switch to EVAC to create a vacuum in the specimen chamber.
7. The green light indicator will turn on when the vacuum is achieved in the specimen chamber. Then, push the gun sample carefully into the electron microscope column and make sure not to allow it to go too fast. Otherwise, it would damage the object at the tip of the gun sample.
8. Once the grid is well in place in the electron microscope column, turn on the filament to the intermediate (pre-established) saturation setting.
9. Turn on the camera through the ON/OFF switch. On the computer, click on Live Image in the imaging software.
10. On the electron microscope, push the Low Magnification button and open the objective aperture. This allows for choosing the desired screening area. Next, position the desired area of the section to the middle of the computer screen, using the two controllers on either side of the electron microscope's column.
11. On the electron microscope, push the Zoom button and close the objective aperture to obtain better contrast.
12. Start the screening of the section by taking microphotographs at 2,500x magnification. Then, adjust the focus using the wobbler button and the brightness button on the electron microscope to ensure the quality of the microphotograph.
13. Click on Final Image on the computer to take the microphotograph. If the colors are not even, perform a background correction on the camera software. Also, adjust the contrast on the final image using the software before saving the microphotograph.
14. When the microphotograph is being saved, double-check the case number and magnification to ensure proper identification of the microphotographs taken.
    NOTE: In some cases, the user can also enter extra captions, and the electron microscopy technologist's initials are also entered.
15. Perform the micrograph process through the whole area of the section to be screened. After enough 2,500x magnification, microphotographs are saved to cover the area of interest, capture 4,000x and 8,000x magnification microphotographs and save them on the computer with the appropriate case number identification.
16. Make a close-up on the section for specific ultrastructural details if very specific screening instructions are given. Then, save the measurements of specific ultrastructural details in the computer considering higher magnifications if needed.
17. When the specimen screening is complete, turn off the filament, the camera, the high voltage, and the camera software in sequence. Next, remove the grid from the electron microscope column, allowing air in the gun sample chamber.
18. File the grid in a gelatin capsule and store the capsule in a properly labeled box, along with the resin blocks of the same case. Put the gun sample back in the gun chamber and store it in a cool and dry environment to preserve the material used.
19. Transfer the digital microphotographs on common collaborative platforms (e.g., SharePoint) or internal servers to allow the pathologist to view them.
20. Enter the work units in the Laboratory Information System (LIS).
21. If more specific microphotographs are required, pull out the field grid and reuse them for additional screening or specific questions.

Results

The distinctive TEM Features of the neuroblastoma are displayed here. Here we will illustrate the distinctive TEM features of the neuroblastoma.

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. This histogenesis explains some biochemical and morphological characteristics of this blue tumor. However, neuroblastomas' deter...

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 pathology^4,27. The critical steps, the modificati...

Materials

Name	Company	Catalog Number	Comments
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