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Advanced Collection and Preparation Techniques
APPENDIX
5
Advanced Collection and Preparation Techniques CELL BLOCK
CELL TRANSFER
The main advantage of the cell block technique is retention of architectural patterns, such as acinar, pavement, honeycomb, papillary, and perivascular. Aspirate biopsy material is fixed in 70% ethanol in 0.6-mL Eppendorf tubes and centrifuged at 2800 × g for 10 minutes. The supernatant is removed, and 2% liquid agarose is added. The tube is again centrifuged at 2800 × g for 10 minutes to obtain a solid pellet. Finally, the pellet is paraffin-embedded and routinely processed for histopathologic evaluation. Further discussion of the method can be found in Zanoni et al., 2012. An alternate method uses HistoGel™ to embed specimens has been recently described (Joiner and Spangler, 2012). Furthermore, a commercial embedding system is available that requires longer incubation with a non-formalin fixative in a proprietary unit for a cytocentrifuge.*
Cell transfer of archived cytologic smears previously stained by an aqueous Romanowsky method is demonstrated in Appendix Fig. 5-1. This method (Stone and Gan, 2014) allows for a variety of additional diagnostic tests such as special histochemical stains, immunocytochemical staining, and molecular testing for infectious agents. Transfer of cells was found to be easier when archived slides had been previously coverslipped. Staining of media-coated sections requires removal the hardened Pertex by soaking in xylene for 20 to 30 minutes. Each slide is then briefly dried in a 55°C oven to ensure optimal adhesion of the cells to the slides. Routine coverslipping is performed prior to microscopic examination.
Joiner KS, Spangler EA: Evaluation of HistoGel™-embedded specimens for use in veterinary diagnostic pathology, J Vet Diagn Invest 24:710–715, 2012. * Shandon™ Cytoblock™ Cell Block Preparation System (Cheshire, UK) Stone BM, Gan D: Application of the tissue transfer technique in veterinary cytopathology, Vet Clin Pathol 43(2):295-302, 2014. Zanoni DS, Grandi F, Rocha NS: Agarose cell block technique as a complementary method in the diagnosis of fungal osteomyelitis in a dog, Vet Clin Pathol 41:307–308, 2012.
509
510
Canine and Feline Cytology
A
B
C
E
D n APPENDIX FIGURE 5-1 Cell transfer method. Lymph node aspirate from a dog. (Images are courtesy of Brett Stone, Australia.) A, Archived aqueous Romanowsky coverslipped slide. B, Coverslip removal by xylene immersion followed by coating of cytologic material with rapid mounting medium (e.g., Pertex). The slides are then baked in an oven at 55°C for 3 hours until the media hardens. The up direction is labelled on the strip where separate sections will be created. C, Pertex and attached cells are removed by scraping the surface of the slide and simultaneously lifting the cell-containing hardened media from the slide using a disposable microtome blade. D, The hardened Pertex layer is cut into multiple sections. E, The individual pieces are pressed firmly onto positively charged slides that are baked in an oven at 55°C for 1 hour. Slides are stored at room temperature until processed further.
5
Advanced Collection and Preparation Techniques CELL BLOCK
CELL TRANSFER
The main advantage of the cell block technique is retention of architectural patterns, such as acinar, pavement, honeycomb, papillary, and perivascular. Aspirate biopsy material is fixed in 70% ethanol in 0.6-mL Eppendorf tubes and centrifuged at 2800 × g for 10 minutes. The supernatant is removed, and 2% liquid agarose is added. The tube is again centrifuged at 2800 × g for 10 minutes to obtain a solid pellet. Finally, the pellet is paraffin-embedded and routinely processed for histopathologic evaluation. Further discussion of the method can be found in Zanoni et al., 2012. An alternate method uses HistoGel™ to embed specimens has been recently described (Joiner and Spangler, 2012). Furthermore, a commercial embedding system is available that requires longer incubation with a non-formalin fixative in a proprietary unit for a cytocentrifuge.*
Cell transfer of archived cytologic smears previously stained by an aqueous Romanowsky method is demonstrated in Appendix Fig. 5-1. This method (Stone and Gan, 2014) allows for a variety of additional diagnostic tests such as special histochemical stains, immunocytochemical staining, and molecular testing for infectious agents. Transfer of cells was found to be easier when archived slides had been previously coverslipped. Staining of media-coated sections requires removal the hardened Pertex by soaking in xylene for 20 to 30 minutes. Each slide is then briefly dried in a 55°C oven to ensure optimal adhesion of the cells to the slides. Routine coverslipping is performed prior to microscopic examination.
Joiner KS, Spangler EA: Evaluation of HistoGel™-embedded specimens for use in veterinary diagnostic pathology, J Vet Diagn Invest 24:710–715, 2012. * Shandon™ Cytoblock™ Cell Block Preparation System (Cheshire, UK) Stone BM, Gan D: Application of the tissue transfer technique in veterinary cytopathology, Vet Clin Pathol 43(2):295-302, 2014. Zanoni DS, Grandi F, Rocha NS: Agarose cell block technique as a complementary method in the diagnosis of fungal osteomyelitis in a dog, Vet Clin Pathol 41:307–308, 2012.
509
510
Canine and Feline Cytology
A
B
C
E
D n APPENDIX FIGURE 5-1 Cell transfer method. Lymph node aspirate from a dog. (Images are courtesy of Brett Stone, Australia.) A, Archived aqueous Romanowsky coverslipped slide. B, Coverslip removal by xylene immersion followed by coating of cytologic material with rapid mounting medium (e.g., Pertex). The slides are then baked in an oven at 55°C for 3 hours until the media hardens. The up direction is labelled on the strip where separate sections will be created. C, Pertex and attached cells are removed by scraping the surface of the slide and simultaneously lifting the cell-containing hardened media from the slide using a disposable microtome blade. D, The hardened Pertex layer is cut into multiple sections. E, The individual pieces are pressed firmly onto positively charged slides that are baked in an oven at 55°C for 1 hour. Slides are stored at room temperature until processed further.