Immunocytology Immunohistochemistry

In this cell surface antigen detection technique, mononuclear cells isolated from marrow aspirates and heparinized PB are incubated with a single or a panel of murine monoclonal antibodies directed against NB surface antigens. Monoclonal antibodies specific for the NB surface disialoganglioside GD2 are commonly used because this antigen is expressed homogeneously on NB cell surface in high density (Wu et al. 1986). The antibody of choice must have high affinity for tumor cells, with little or no cross reactivity to normal hematopoietic cells. Some examples of anti-GD2 monoclonal antibodies that have excellent specificity include 3F8 (Cheung et al. 1997; Faulkner et al. 1998), 14.18 (Mehes et al. 2001), 3A7 (Saarinen et al. 1996), and HSAN1.2 (Smith and Reynolds 1987). Its usual detection limit of 1 tumor cell in 105 normal hematopoietic cells may be improved to 1 in 106 or even lower by the use of a com

Figure 11.7.1 a,b a Bone marrow cells from a neuroblastoma patient stained with FITC labeled GD2 antibody. bThe same cells as in a were subsequently analyzed by fluorescence in situ hybridization using a MYCN-specific probe (FITC) and a chromosome-2 specific probe (TRITC). All three GD2 positive cells showed MYCN amplification.

Figure 11.7.1 a,b a Bone marrow cells from a neuroblastoma patient stained with FITC labeled GD2 antibody. bThe same cells as in a were subsequently analyzed by fluorescence in situ hybridization using a MYCN-specific probe (FITC) and a chromosome-2 specific probe (TRITC). All three GD2 positive cells showed MYCN amplification.

bined immunofluorescence and genetic approach (Mehes et al. 2001) and to 1 in 107-108 by the use of immuno-magnetic sorting (Faulkner et al. 2000).

One important advantage of this method is the possibility to quantify the exact number of tumor cells in a specimen. It can exploit a mixture of monoclonal antibodies of different specificities (Moss et al. 1991). More importantly, it allows the genetic confirmation of malignant features of individual cells, and provides insights into the biologic make up of disseminated tumor cells (DTCs);however,this assay requires freshly collected samples which must be processed right away. The technique is also labor intensive because it requires counting and analyzing cells under the microscope.

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