title: An immunomagnetic epithelial tumor cell enrichment model for minimal residual disease detection of cytokeratin 8+ malignancies.
abstract: Immunocytochemical detection of isolated tumor cells in peripheral blood and bone marrow is currently the most established method for monitoring early dissemination in epithelial cancer. In this study we used an immunomagnetic selection technique to develop an enrichment model for disseminated tumor cells in blood. Buffy coat cells spiked with varying numbers of BT-474 carcinoma cells were permeabilized and fixed, following which carcinoma cells were magnetically labelled with an anti-cytokeratin 8 mAb. Labelled cells were enriched by the use of magnetic columns. The eluted cytokeratin 8+ tumor cells were detected by flow cytometry and immunocytochemistry. Spiked samples were split and processed freshly in the immunomagnetic enrichment assay, as well as cryopreserved and processed in the assay after thawing. Enumeration of BT-474 cells demonstrated a detection limit of one BT-474 cell in 1.0 x 10(7) leukocytes in both fresh and cryopreserved-thawed samples. The pair wise comparison showed a significantly higher recovery of spiked BT-474 cells from freshly processed samples than from cryopreserved and thawed samples (57% vs 21%). Viability tests suggested that this outcome might be due to a greater susceptibility of BT-474 cells than buffy coat cells to the used cryopreservation and thawing technique. Altogether our findings show that the performance of the immunomagnetic enrichment assay on fresh samples is satisfactory with a recovery rate of almost 60% and a sensitivity of 10(-7). However, performance of the assay on cryopreserved and thawed cells needs to be improved.
authors.affiliation: Laboratory of Clinical & Tumor Immunology, Department of Medical Oncology, Erasmus MC, Daniel Den Hoed Cancer Center, Rotterdam, The Netherlands. R.Brooimans@erasmusmc.nl
authors.forename: R A
authors.initials: RA
authors.lastname: Brooimans
authors.forename: N
authors.initials: N
authors.lastname: de Leeuw
authors.forename: M
authors.initials: M
authors.lastname: Bontenbal
authors.forename: J W
authors.initials: JW
authors.lastname: Gratama
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis; D045744:Cell Line, Tumor; D002470:Cell Survival; D015925:Cryopreservation; D004847:Epithelial Cells / Q000166:cytology* / Q000473:pathology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D018189:Immunomagnetic Separation / Q000379:methods*; D007633:Keratins / Q000096:biosynthesis*; D008280:Magnetics; D009369:Neoplasms / Q000097:blood* / Q000473:pathology; D009375:Neoplasms, Glandular and Epithelial / Q000097:blood* / Q000473:pathology; D009360:Neoplastic Cells, Circulating / Q000378:metabolism; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D014408:Biomarkers, Tumor; D007633:Keratins