Isolation and analysis of rare cells in the blood of cancer patients using a negative depletion methodology.
A variety of enrichment/isolation technologies exist for the characterization of rare cells in the blood of cancer patients. In this article, a negative depletion process is presented and discussed which consists of red blood cell (RBC) lysis and the subsequent removal of CD45 expressing cells through immunomagnetic depletion. Using this optimized assembly on 120 whole blood specimens, from 71 metastatic breast cancer patients, after RBC lysis, the average nucleated cell log depletion was 2.56 with a 77% recovery of the nucleated cells. The necessity of exploring different anti-CD45 antibody clones to label CD45 expressing cells in this enrichment scheme is also presented and discussed. An optimized, four-color immunofluorescence staining is conducted on the cells retained after the CD45-based immunomagnetic depletion process. Different types of rare non-hematopoietic cells are found in these enriched peripheral blood samples and a wide range of external and internal markers have been characterized, which demonstrates the range and heterogeneity of the rare cells.
William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, United States.
Yongqi
Y
Wu
Clayton J
CJ
Deighan
Brandon L
BL
Miller
Priya
P
Balasubramanian
Maryam B
MB
Lustberg
Maciej
M
Zborowski
Jeffrey J
JJ
Chalmers
D014408:Biomarkers, Tumor / Q000032:analysis; D001943:Breast Neoplasms / Q000276:immunology; D002452:Cell Count; D045744:Cell Line, Tumor; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D017493:Leukocyte Common Antigens / Q000276:immunology*; D009360:Neoplastic Cells, Circulating / Q000276:immunology*
D014408:Biomarkers, Tumor; D017493:Leukocyte Common Antigens
Circulating tumor cells; Flow cytometry; Immunocytochemistry; Immunofluorescence; Immunomagnetic separation; Tumor-related cell marker