Simultaneous Phenotype-Specific Telomere Length Measured by Flow-FISH Using Thermally Stable Quantum Dots

Abstract The Flow-FISH technique used to measure telomere length by incorporation of fluorescein-labeled oligonucleotide probes complementary to telomeres repeat sequences has been extended to permit simultaneous multiple phenotypic analysis within a single sample. The necessary thermal stability of the fluorescent labeling needed for phenotyping cell populations can be achieved using quantum dots instead of conventional organic fluorophores, which are usually damaged during the high-temperature hybridization treatment and lose their fluorescence, limiting their usefulness for phenotypic analysis. In this study, quantum dots and conventional fluorophores were compared for their ability to survive the 82oC hybridization step necessary for PNA probe annealing. Quantum dots preserved their fluorescence following heat treatment and gave good signal-to-noise ratios when used to detect lymphocyte and monocyte markers (CD3, CD4 and CD14). This was in contrast to phycobiliproteins and low molecular weight fluorochromes such as fluorescein and the Alexa Fluor dyes, which decreased considerably in fluorescence following hybridization. Since addition of fluorescent immunophenotyping complicated an assay already heavily dependent on uniform technique, internal reference standards (previously reported by Lansdorp) were particularly critical in this technique. We used aliquoted frozen calf thymocytes (CT) as an internal standard, determining their telomere length and incorporating them into each sample to monitor tube to tube and day to day assay variability. The telomere length of the CTs and of CD3+CD4+, CD3+CD4− and CD14+, were determined and then corrected by normalization to the known CT telomere length. To validate the overall method, we then measured age dependence on telomere length for monocyte and lymphocyte subpopulations in young, middle-aged and older donors. Results were consistent with results previously reported using traditional Southern blotting and Flow-FISH with no immunphenotyping, suggesting that incorporation of immunolabeling did not adversely affect this technique. This method allows for the rapid, accurate and simultaneous determination of the telomere lengths of different cell populations within a single sample when little amount of sample is available.