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Their role in disease monitoring of advanced melanoma patients treated with immune checkpoint inhibitors (ICI) is unknown. CTC protein expression of human leukocyte antigen class\u2010I (HLA I) and programmed death ligand\u20101 (PD\u2010L1) may give insight into how a patient's disease evolves over the course of treatment. In our study, we utilize microfluidic Exclusion\u2010based Sample Preparation (ESP) technology to isolate and characterize CTCs from patients with advanced\u2010stage melanoma. CTC samples from melanoma patients are collected, captured, and stained. A range of 2 to 35 CTCs is observed in a cohort of 16 samples from 10 advanced\u2010stage melanoma patients treated with ICI therapy. Single\u2010cell protein expression data is generated from image cytometry analysis and used to calculate mean HLA I and PD\u2010L1 expression. Using our ESP capture approach, we successfully detect phenotypic and numerical heterogeneity in CTCs from melanoma patients. Our assay shows sufficient capture sensitivity and promising prognostic and predictive information, as we illustrate in our case example. A greater clinical sample size will be necessary to confirm the diagnostic sensitivity and specificity of the assay in predicting clinical outcomes for patients with advanced\u2010stage melanoma.<\/jats:p>","DOI":"10.1111\/pcmr.70030","type":"journal-article","created":{"date-parts":[[2025,6,3]],"date-time":"2025-06-03T02:09:05Z","timestamp":1748916545000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Microfluidic Characterization and Analysis of Circulating Tumor Cells From Patients With Metastatic Melanoma"],"prefix":"10.1111","volume":"38","author":[{"given":"Matthew C.","family":"Mannino","sequence":"first","affiliation":[{"name":"University of Wisconsin\u2010Madison  Madison Wisconsin USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuang G.","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Human Oncology University of Wisconsin\u2010Madison  Madison 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