Rolling Circle Amplification Technology as a Potential Tool in Detection and Monitoring of Cancer by Flow Cytometry

Abstract

Rolling circle amplification (RCA) generates a localized signal via isothermal amplification of an oligonucleotide circle reporter sequence. The application of this approach to flow cytometry could extend the utility of existing methods by enhancing the sensitivities and specificities for various applications, including early diagnosis of cancer and of hematologic and other abnormalities. RCA technology is applicable to a variety of platforms for the simultaneous detection of molecules as a function of either antigenicity or nucleic acid sequence (1)(2)(3)(4). In flow cytometry, cells of interest are characterized based on population gating. Efficient gating strategies are crucial for accurate immunophenotyping, more so in a heterogeneous cell suspension such as peripheral blood mononuclear cells (PBMCs). Usually a combination of light scatter (forward and side) and immunophenotypic markers is critical in identifying the specific cells of interest. A panel of antibodies is usually used to characterize a subset of cells based on their surface markers. However, cells can be best characterized only when the staining for each of these markers is bright enough to clearly differentiate them from unstained cells. This requires specific antibodies and intense detection signals. Abundantly expressed cell surface markers are not difficult to stain and identify compared with rare surface antigens, which are currently gaining importance in diagnostics and clinical studies. Therefore, the common challenge in clinical or diagnostic flow analysis is insufficient signal (low-intensity signals) leading to inefficient use of the existing antibody library. RCA technology can help overcome these problems. The RCA technology (RCATTM) also allows for multiplexing or multiparametric analysis of various markers simultaneously, supporting the expanding use of complex marker panels for disease diagnosis and prognosis. RCA-mediated signal amplification has been successfully applied to the detection of cell surface antigens (e.g., …