Improvement of breast cancer cell detection by immunomagnetic enrichment W Kru ¨ ger, F To ¨ gel, S Ro ¨ ssing, N Kro ¨ ger and AR Zander Bone Marrow Transplantation Center, University-Hospital Eppendorf, Martinistraße 52, 20246 Hamburg, Germany Background Contaminating breast cancer cells in leukapheresis harvested for reinfusion to rebuild hemopoiesis after high-dose therapy have been described by several investigators. Methods for tumor cell detection are conventional immunocytochemistry, culture techniques and reverse transcriptase PCR. The percentage of tumor cell positive leukaphereses shows a wide variation. An approach to clarify if these cells can induce systemic relapse is to characterize them molecular-genetically and immunologically, but these techniques require a sufficient cell count. Methods We compared conventional immunocytochemistry with immuno- cytochemistry after immunomagnetic enrichment of cancer cells by HEA-125 magnetic microbeads for the detection of micro- metastatic tumor cells. A total of 25 samples, consisting of 16 samples from G-CSF-mobilized peripheral stem cell harvests, eight BM aspirations and one peripheral blood sample were investigated without [median 2, range 1–3  10 6 MNCs] and after [median 5, range 1–10  10 7 , MNCs] HEA-microbead selection. Additionally 10 buffy coat samples from healthy subjects were investigated. Results Using conventional immunocytochemistry, tumor cells could be detected in nine stem cell samples. Two BM samples and the blood sample (48%) were positive, with a median tumor cell load of 0 (0–12) cells per sample (mean: 2.4). By HEA-bead selection the rate of positivity could be increased to 88% (13 stem cell samples, eight marrow samples and one blood sample) with a median load of 6 (0–47) (mean 10.6) suspected cells (p < 0.007). However, calculation of recovery revealed tumor cell losses by immunobead selection. False positive results were not seen. Discussion We conclude first that immunomagnetic selection is an excellent and highly sensitive tool to enrich contaminating cancer cells from marrow and stem cell samples; second that the existence of real tumor cell negative stem cell harvests is doubtful; and third that immunobead selection delivers sufficient tumor cell counts for their further characterization by molecular and immunological methods. Keywords circulating tumor cells, bone marrow, blood stem cells, cell selection, breast cancer. Introduction The presence of micrometastatic tumor cells in the BM of breast cancer patients at the time of diagnosis worsens the prognosis of these women significantly [1]. High-dose chemotherapy, with autologous blood stem cell reinfusion, is a promising therapeutic approach for women with high- risk and metastatic breast cancer and is currently being investigated by several clinical trials [2,3]. Cancer cell contamination of leukapheresis harvests, reinfused for hemopoietic reconstitution after high-dose therapy, has been described by several investigators [4–6]. The percentage of positive harvests shows a wide variation, but the number of tumor cells in leukaphereses is generally low in well-conducted trials [7]. The methods used for tumor cell detection are immunocytochemistry, culture techniques and reverse transcriptase PCR (rtPCR) [8–11]. Whilst conventional immunocytochemistry is accepted as the ‘gold standard’ for tumor-cell detection, cancer cell detection by rtPCR is discussed controversially in the literature [10–13]. However, the prognostic impact of accidentally-reinfused breast cancer cells after completion of high-dose therapy and stem-cell grafting is still unclear. Correspondence to: William Kru ¨ ger, Einrichtung fu ¨ r Knochenmarktransplantation, Abteilung Ha ¨matologie/Onkologie, Universita ¨ts- Krankenhaus Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany Cytotherapy (1999) Vol. 1, No. 2, 135–139 ß 1999 ISHAGE 135