Immunomagnetic Techniques for the Enrichment and Detection of Isolated Breast Carcinoma Cells in Bone Marrow and Peripheral Blood

Abstract

Detection of isolated tumor cells (TC) in bone marrow (BM) from patients with breast cancer is usually accomplished by immunocytochemical (ICC) analysis of up to 2 X 10(6) mononuclear cells (MNC). However, this method is cumbersome if large numbers of BM cells (i.e. > 1 X 10(7) cells) are to be analyzed. This emphasizes the need for TC enrichment strategies. This report describes immunomagnetic separation (IMS) techniques for enrichment and detection of viable breast carcinoma cells in BM and peripheral blood (PB). The positive IMS technique was performed by incubation of MNC with 2.8 microns magnetic particles (rat antimouse IgG1 M280-Dynabeads) coated with monoclonal antibody (mAb) against epithelial surface antigens. The rosetted tumor cells were then visualized by ICC staining using alkaline phosphatase-conjugated A45-B/B3 anticytokeratin mAb (Fab). The negative IMS technique was performed by incubation of MNC with anti-CD45-coated M450-Dynabeads (4.5 microns), followed by ICC staining of the nonrosetted cells. When 1000, 100, and 10 breast carcinoma cells were mixed with 1 X 10(7) MNC, an average of 748 (n = 9), 70 (n = 10), and 7.8 TC (n = 8), respectively, were detected with the positive IMS technique. With the negative IMS technique, 648 (n = 8), 57.8 (n = 6), and 7.3 TC (n = 6), respectively, were detected. The analysis of 1 X 10(7) MNC with the IMS techniques was compared with the ICC analysis of 2 X 10(6) unseparated MNC. A mean 3.7-fold (range 1.5-6.4) to 4.2-fold (2.5-8.2) (positive IMS) and 3.1-fold (range 2.0-5.0) to 3.8-fold (2.0-6.0) (negative IMS) higher TC detection frequency was achieved after enrichment by IMS in experiments with 100 and 1000 TC/10(7) MNC. The IMS techniques were used for examination of BM samples from locally advanced breast cancer patients. A 5.3-fold mean increase (range 2.1-13.3) in the number of TC detected was obtained when the use of positive and negative IMS together was compared with the direct ICC analysis of unseparated MNC (n = 11). Enrichment of TC by IMS techniques enables us to examine large numbers of MNC from BM or PB, which can result in the detection and characterization of minimal residual disease with increased sensitivity and specificity.

Detection of circulating tumor cells in breast cancer patients utilizing multiparameter flow cytometry and assessment of the prognosis of patients in different CTCs levels

“…To avoid possible false-negative, we also chose another epithelial-specific marker: Ep-CAM (25). To detect the tumor cells that come from epithelium tissue, a monoclonal antibody directed against CD45 for negative selection of leukocytes was used (7)(8)(9). Therefore, the detection of dual-positive cells (CD45-Ep-CAM1CK1) is expected (such as in the peripheral blood) and has been proposed as a surrogate marker for epithelial tumor cells.…”

Detection of circulating tumour cells in patients with breast or ovarian cancer by molecular cytogenetics

“…In immunocytochemical assays, epithelial specific antibodies have been used to detect isolated tumour cells in bone marrow (BM) and blood (Cote et al, 1991(Cote et al, , 1995(Cote et al, , 1996Diel et al, 1996;Franklin et al, 1996).In an effort to obtain greater sensitivity, several investigators have developed techniques for the enrichment of tumour cells before their identification by immunocytochemistry, PCR or FISH (Hardingham et al, 1993(Hardingham et al, , 1995Berois et al, 1997;Eaton et al, 1997;Hildebrandt et al, 1997;Naume et al, 1997;Martin et al, 1998).Therefore, the aim of our study was to analyse the presence and frequency of circulating tumour cells in the peripheral blood of patients with breast or ovarian cancer by using a combination of magnetic activated cell sorting (MACS) and interphase FISH. …”

“…In an effort to obtain greater sensitivity, several investigators have developed techniques for the enrichment of tumour cells before their identification by immunocytochemistry, PCR or FISH (Hardingham et al, 1993(Hardingham et al, , 1995Berois et al, 1997;Eaton et al, 1997;Hildebrandt et al, 1997;Naume et al, 1997;Martin et al, 1998).…”

Magnetic Microspheres: A Novel Drug Delivery System

“…[50] 12-Magnetic Dynabeads have been used in immune-magnetic techniques for the enrichment and detection of isolated breast carcinoma cells in bone marrow and peripheral blood. [51] 13-Magnetic microspheres carriers of contraceptive has been designed responsive to the changes in steroid secretions during menstrual cycle. [52] www.wjpps.com Vol 6, Issue 9, 2017.…”