A significant percentage of women with primary breast cancer have micrometastatic disease in the bone marrow. Bone marrow involvement may be an adverse prognostic factor, and more aggressive therapy may be indicated for these patients. There are a number of different techniques and antibodies used to detect tumor cells in the bone marrow. We used the same panels of four antibreast cancer antibodies and compared three immunodetection techniques: two-color immunofluorescence, immunohistochemical staining, and fluorescence-activated cell sorting with cytologic examination of the sorted cells. The two-color immunofluorescence technique was superior and consistently detected one tumor cell contaminating one million normal bone marrow cells and had no reactivity with normal bone marrow.

Background: Flow cytometric immunophenotyping (FCI) is recognized as a rapid, sensitive, and accurate method for diagnosis of B-cell lymphomas. We observed that FCI failed to identify the clonal B-cell population in several cases of large B-cell lymphoma (DLBCL) when tissue samples were prepared by a commercially available mechanical tissue disaggregation method. We tested a manual tissue disaggregation method and compared it with the mechanical method.Methods: FCI findings from 51 cases of DLBCL processed with the mechanical tissue disaggregation method, 27 cases processed using the manual method, and 15 cases processed using a combination of both methods were compared. The histological and immunohistochemical findings in each case were reviewed.Results

Summary:High-dose chemotherapy (HDC) with hematopoietic support appears promising in the treatment of breast cancer, although reinfusion of contaminating tumor cells may contribute to disease relapse. Ex vivo expansion may reduce tumor cell content through use of a small inoculum volume and by passive purging during culture. We assessed the ex vivo expansion potential of tumor cell positive bone marrow (BM) from breast cancer patients and the effect of ex vivo expansion on tumor cell content. Cryopreserved/thawed mononuclear cell (C/T MNC) BM harvests with known tumor cell contamination (n = 7) were assessed for tumor cells preand post-expansion using immunocytochemical (ICC) staining. Pre-expansion inoculum samples contained a range of 6-2128 tumor cells per 5.0 × 10 6 nucleated cells. Ex vivo expansion resulted in fold expansions of 6.67 and 11.37 for total cells and CFU-GM, respectively. Tumor cells were undetectable in four of the seven postexpansion samples and were reduced in the remaining three samples. The data demonstrate passive purging of breast cancer cells during ex vivo expansion, with hematopoietic progenitor cell expansion comparable to that of normal BM. Reduction in tumor cell number contained in the small volume culture inoculum combined with passive purging during the ex vivo expansion process suggest a potential 2-4+ log reduction in tumor cell content in the reinfused cell product. Keywords: bone marrow transplantation; ex vivo expansion; tumor cell contamination Immunologic techniques have revealed tumor cell infiltration of the BM in as many as 48% of early stage breast cancer patients at the time of diagnosis. [1][2][3][4] The presence of tumor cells in the BM has been shown to be predictive of relapse in early stage 5-7 and advanced stage 8,9 breast cancer patients. In addition, the number of tumor cells identified in the BM has been correlated with shorter disease-free and overall survival. 6 Although the prognostic significance of tumor cells in PBPC collections requires further evaluation,