Immunocytochemical staining

Many different assays have been applied to detect DTC in breast cancer and other solid tumors. One major approach to identify DTC from BM includes density gradient centrifugation with subsequent immunocyto-chemical staining using monoclonal antibodies against epithelial or tumor-associated antigens (Figure 1). Different monoclonal and polyclonal antibodies or antibody cocktails were used for immunocytochemical identification of DTC in BM. Groups have used antibodies against EMA, directed against an epithelial cell-surface antigen (2), TAG 12, a tumor-associated glycoprotein (3), and cytokeratins (CK), the structural proteins of the epithelial cytoskeleton (4, 5). To date, cytokeratins have become the most widely accepted protein marker in such immunocyto-chemical assays. A combination of several antibodies to various CK antigens or an antibody against a common epitope present on various CK proteins (e.g., A45B/B3 directed among others against CK8, 18, 19) seems to be superior to monospecific antibodies directed against a single cytokeratin protein (e.g., CK2, against CK18) (5-7), because of the considerable antigenic heterogeneity of solid tumor cells. With this approach, one single DTC can be detected in the background of millions of hematopoetic cells. However, different staining techniques can result in specificity variations. Hematopoietic cells can be directly reactive to alkaline phosphatase (8) or produce endogenous peroxidase (9), conesquently resulting in false-positive staining in alkaline phosphatase-based or peroxi-dase-based methods, if these enzymes were not fully blocked. Several international organizations have recognized the need for standardization of the immunocytochemical assay and for its evaluation in prospective studies (10, 11).

The use of new automated devices for the microscopic screening of immunostained slides may help to read slides more rapidly and to increase reproducibility of the read-out (12-17). Another way to improve current detection assays for single tumor cells is to develop better tumor cell enrichment procedures using improved density gradients (18) and antibody-coupled magnetic particles (12, 19, 20). At present, it is unclear whether these new enrichment techniques provide more clinically relevant information than the standard density gradient procedure used to isolate the mononuclear cell fraction.

Figure 1. Immunocytochemical detection of DTC in patients with breast cancer. The process begins with a Ficoll density gradient centrifugation to isolate mononuclear cells and uses cytokeratins as markers of DTC. The detection of the stained DTC can be performed automatically.
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