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Circulating tumor cells (CTCs) are indicative of metastatic disease in multiple types of solid tumors. Technologic advances in CTC enrichment have yielded profound variability in both quantity and phenotypic characteristics of CTCs. While size-based exclusion methods have improved the sensitivity of CTC capture, their diminished specificity requires subsequent robust cytopathologic identification of CTCs. In this study, we compared CTC counts from Isolation by Size of Epithelial/Trophoblastic Tumor cells (ISET®) filters sequentially stained by May-Grünwald/Giemsa (MGG), immunocytochemistry (ICC)/hematoxylin, and ICC/hematoxylin/eosin, followed by corresponding CTC criteria. An immune and endothelial cell cocktail of CD45/CD11b/CD31 antibodies adequately ruled out immune and endothelial cells, yet a substantial number of atypical morphologies with nuclear irregularity (i.e., circulating non-hematological/endothelial cells; CNHCs) were detected in both breast cancer cases and non-cancerous controls following hematoxylin nuclear counterstain. Cytoplasmic staining with eosin, significantly diminished CNHC counts. In conclusion, detection of CTCs from ISET filters using chromogenic ICC is feasible in conjunction with identification criteria of nuclear irregularity, negative reactivity to immune and endothelial cell markers, and presentation of visible cytoplasm. *Correspondence to: Takemi Tanaka, Ph.D., Associate Professor, University of Oklahoma Health Sciences Center, School of Medicine, Dept. of Pathology, Stephenson Cancer Center at 975 NE 10th, BRC-W, Rm 1415, Oklahoma City, OK, 73104, USA, Tel: Office (405)-271-8260, E-mail: takemi-tanaka@ouhsc.edu Roy Zhang, MD., Associate Professor, University of Oklahoma Health Sciences Center, School of Medicine, Dept. of Pathology, 940 SL Young Blvd, Oklahoma City, OK 73104, USA, Tel: Office (405)-271-1794, E-mail: roy-zhang@ouhsc.edu Special Issue: Recent Advancements in Clinical and Diagnostic Pathology Rawhi Omar University of Louisville School of Medicine USA

Cytopathologic identification of circulating tumor cells (CTCs) in breast cancer: Application of size-based enrichment