gms | German Medical Science

73. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC)
Joint Meeting mit der Griechischen Gesellschaft für Neurochirurgie

Deutsche Gesellschaft für Neurochirurgie (DGNC) e. V.

29.05. - 01.06.2022, Köln

Analysis of the glioblastoma invasion zone using 5-ALA guided Laser Microdissection for sampling single cells – a methodological approach

Analyse der Invasionszone des Glioblastoms mithilfe 5-ALA gestützter Laser Mikrodissektion zur Einzelzellentnahme – eine methodische Vorgehensweise

Meeting Abstract

  • presenting/speaker Johannes Falter - Universitätsklinikum Regensburg, Klinik und Poliklinik für Neurochirurgie, Regensburg, Deutschland
  • Anette Lohmeier - Universitätsklinikum Regensburg, Klinik und Poliklinik für Neurochirurgie, Regensburg, Deutschland
  • Nils-Ole Schmidt - Universitätsklinikum Regensburg, Klinik und Poliklinik für Neurochirurgie, Regensburg, Deutschland
  • Christoph Andreas Klein - Universitätsklinikum Regensburg, Experimental Medicine and Therapy Research, Regensburg, Deutschland
  • Martin Proescholdt - Universitätsklinikum Regensburg, Klinik und Poliklinik für Neurochirurgie, Regensburg, Deutschland

Deutsche Gesellschaft für Neurochirurgie. 73. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), Joint Meeting mit der Griechischen Gesellschaft für Neurochirurgie. Köln, 29.05.-01.06.2022. Düsseldorf: German Medical Science GMS Publishing House; 2022. DocV273

doi: 10.3205/22dgnc265, urn:nbn:de:0183-22dgnc2653

Veröffentlicht: 25. Mai 2022

© 2022 Falter et al.
Dieser Artikel ist ein Open-Access-Artikel und steht unter den Lizenzbedingungen der Creative Commons Attribution 4.0 License (Namensnennung). Lizenz-Angaben siehe http://creativecommons.org/licenses/by/4.0/.


Gliederung

Text

Objective: To specifically identify invasive glioblastoma (GBM) cells we used Laser Microdissection in GBM specimen acquired during 5-ALA supported resection. We addressed several obstacles to optimize the dissection protocol: a. stability of the Protoporphyin IX fluorescence after retrieval of the sample, b. detection and cutting of the single cells of interest, and c. the preservation of mRNA. To the best of our knowledge the method of 5-ALA based single cell Laser Microdissection has not been described, yet.

Methods: 5-ALA, applied immediately before surgical resection, specifically accumulates as Protoporphyrin IX in GBM cells. The tissue was frozen and cryosectioned immediately after resection and put on membrane slides (MMI, Eching). So far, we used variable slice thicknesses (5-30 µm), different fixations (100% methanol, 70% ethanol and 0.5% paraformaldehyde), variable counterstainings (Nissl, HE, Hoechst, Phalloidin) and the method of hyperosmotic shrinkage (protocols with 0.5M sucrose) to find the best setting for the morphological assessment of single cells and correspondingly the influence and possible interaction on the 5-ALA fluorescence.

Results: The perioperatively applied 5-ALA shows a good signal and facilitates the detection of single glioblastoma cells for guided single cell Laser Microdissection after resection. With the MMI Laser Microdissection Microscope single cells can be pinned and Whole Slide Scans can save the information on 5-ALA fluorescent cells, consequently the quick photobleaching poses no further problem. The cell density seems to be best with 25µm slices. Washing out of Protoporphyrin IX was observed in combination with HE; fluorescent counterstaining (nuclear/cell body) for better histological cell recognition seems recommendable. Hyperosmotic shrinkage shows little effect in enhancing the recognition of cell borders.

Conclusion: The tested protocols for RNA-protective, 5-ALA based single cell sampling from human glioblastoma tissue show morphologically satisfactory results and make single-cell Laser Microdissection technically feasible. The yield and quality of obtained RNA remains to be evaluated.