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 (1–4). 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. RCAmediated signal amplification has been successfully applied to the detection of cell surface antigens (e.g., CD4 and CD28) on PBMCs. This technical report describes the technology and protocol for flow cytometric analysis of lymphocyte surface markers. We have achieved a 10- fold increase in median fluorescent intensity (MFI) with RCA compared with conventional detection methods. Human PBMCs were separated from whole blood by density gradient centrifugation using Vacutainer CPT tubes (Becton Dickinson). Two low-speed (300–400g) washes were performed in 1 phosphate-buffered saline (PBS; pH 7.4) to minimize platelet contamination. After centrifugation, cells were diluted to obtain 5 106 cells so that 100
L would give 500 000 cells/sample. Although this may be a limitation in studies requiring recovery of viable cells, we found that cell fixation and permeabilization play an important role in cell staining and RCA. Although fixation was not absolutely necessary for amplification of signals, a light fixation substantially enhanced the efficiency of the subsequent RCA reaction. Therefore, for the studies described here, 500 000 cells were fixed with 5 g/L paraformaldehyde for 2 min at room temperature. The cells were then washed and blocked with 50 mL/L normal goat serum (NGS) in PBS, pH 7.4. An equal volume of 50 mL/L NGS, isotype controls (negative control samples), or 2 mg/L primary antibody (phycoerythrin-conjugated anti-CD4/CD28) diluted in 50 mL/L NGS was added to the cells and incubated in the dark for 30 min at room temperature. Alternatively, samples to be detected by second-step reagents were incubated with a biotinylated anti-CD4/ CD28 antibody (30 min at room temperature), washed in PBS, and resuspended in a 1:1000 dilution of phycoerythrin-conjugated streptavidin (cat. no. S-866; Molecular Probes) for 15 min at room temperature (in the dark). After this incubation, cells were washed, centrifuged, and kept on ice until analysis by flow cytometry. RCA. The blocked PBMCs were incubated with a preannealed complex of an anti-CD4–primer 4.2 conjugate [details of conjugates and circle are described in Gusev et al. (4)] and circle 4.2 in the conjugate-circle-complex diluent [C3-diluent; containing 150 mmol/L potassium glutamate, 10 mmol/L HEPES (pH 7.4), and 5 mL/L polyvinyl alcohol] for 30 min at room temperature. The negative controls were incubated with circle only (without conjugate) in the C3-diluent. Preannealing was done with 100 mg/L antibody conjugate and 2000 nmol/L DNA circle in the C3-diluent for 15 min at 37 °C, and the mixture was diluted 50-fold (in C3-diluent) before addition to cells. Alternatively, for indirect detection followed by RCAT analysis, the preannealed complex was prepared with an anti-biotin–primer 4.2 conjugate and circle 4.2, diluted 50-fold in C3-diluent, and added for 5 min at room temperature directly to PBS-washed cells that were incubated with a biotinylated primary antibody. After the preannealed conjugate/circle incubations, the cells were washed and subjected to RCAT in the presence of 30 U of 29 polymerase per 25-
L reaction volume. The reaction mixture contained 150 mmol/L potassium glutamate, 35 mmol/L HEPES (pH 7.4), 10 mmol/L magnesium acetate, 7 mmol/L dithiothreitol, 70 mg/L bovine serum albumin, and 400
mol/L each deoxynucleotide triphosphate. The reaction was incubated at 31 °C for 15 min. The cells were then centrifuged with an addition of 100
L of 50 mL/L NGS in PBS, pH 7.4, and decorated in 50
L of an 8 mg/L phycoerythrin–decorator complex in 50 mL/L NGS. The decorator used was an oligonucleotide complementary to the DNA circle 4.2 and had a biotinylated thymidine at the 3 end with an eight-nucleotide spacer of deoxyinosine and deoxyuridine. This decorator was incubated with phycoerythrin-conjugated streptavidin overnight at 4 °C in PBS, pH 7.4, and purified on a sizeexclusion column to remove the free constituents,