A Novel Manual “Centrifuged-Enhanced” Cytosmear Technique for Improving Hypocellular Cytology in the Diagnosis of Vitreoretinal Lymphoma
Year: 2025
Type: article
Abstract: Purpose: To develop a manual "centrifuged-enhanced cytosmear" technique for cytologic and immunohistochemical analysis of hypocellular vitreous biopsy specimens in vitreoretinal lymphoma (VRL). Methods: Diffuse large B-cell lymphoma cells were diluted to simulate vitreous biopsy yields (1000-50,000 cells/100 µL) and fixed in PreservCyt. Samples were centrifuged in a standard laboratory centrifuge and deposited into 1000 cells/100 µL PAP pen-defined circles on charged slides. Smears were air-dried, methanol-fixed, and stained with toluidine blue and CD20 immunohistochemistry (IHC). Cell density per high-power field (40×) was quantified and compared with noncentrifuged smears. Reproducibility was assessed across replicates and between two operators. Genomic DNA concentration was measured. Results: Centrifuged-enhanced cytosmears demonstrated significantly higher cellular densities than standard smears across all concentrations (P < 0.05), especially at 1000 to 2000 cells/100 µL, where standard smears failed to detect cells. CD20 IHC was successful without cell dropout. Genomic DNA yields ranged from 5.25 ng (1000 cells) to 143 ng (50,000 cells). Interuser variability was not significant. Comparison with cytospin clinical cases showed that centrifuged-enhanced cytosmears are of comparable cellularity to clinical cases. Conclusions: This centrifuged-enhanced cytosmear technique reliably concentrates sparse cells for cytology and IHC. It is reproducible and cost-effective, requiring no specialized equipment. Genomic DNA yields at very low cell counts of 1000 cells/100 µL is potentially sufficient for adjuvant MYD88 mutation analysis and can be used to support cytology diagnosis. Translational Relevance: The centrifuged-enhanced cytosmear offers an accessible alternative to cytospin preparations, enabling VRL diagnosis and IHC analysis in settings lacking cytology infrastructure or where cell blocks cannot be performed.
Authors: Candice Ee Hua Ho, Thaddeus Luke Shen, Mona Meng Wang, Meihui Wu, Zhen Wei Neo, Felicia Wee, Joe Yeong, Anita Sook Yee Chan
Institutions: Singapore Eye Research Institute, University of Turku, Agency for Science, Technology and Research, Singapore General Hospital, Institute of Molecular and Cell Biology, National University of Singapore, Cancer Science Institute of Singapore, Singapore National Eye Center, Duke-NUS Medical School
Language: English
Subfield: Pathology and Forensic Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $1,850
OpenAlex ID: W4416144528