Analytical Validation of Telomere Analysis Technology® for the High-Throughput Analysis of Multiple Telomere-Associated Variables

Abstract

Background A large number of studies have suggested a correlation between the status of telomeres and disease risk. High-throughput quantitative fluorescence in situ hybridization (HT Q-FISH) is a highly accurate telomere measurement technique that can be applied to the study of large cell populations. Here we describe the analytical performance testing and validation of Telomere Analysis Technology (TAT®), a laboratory-developed HT Q-FISH-based methodology that includes HT imaging and software workflows that provide a highly detailed view of telomere populations. Methods TAT was developed for the analysis of telomeres in peripheral blood mononuclear cells (PBMCs). TAT was compared with Terminal Restriction Fragment (TRF) length analysis, and tested for accuracy, precision, limits of detection (LOD) and specificity, reportable range and reference range. Results Using 6 different lymphocyte cell lines, we found a high correlation between TAT and TRF for telomere length (R2 ≥ 0.99). The standard variation (assay error) of TAT was 454 base pairs, and the limit of detection of 800 base pairs. A standard curve was constructed to cover human median reportable range values and defined its lower limit at 4700 bp and upper limits at 14,400 bp. Using TAT, up to 223 telomere associated variables (TAVs) can be obtained from a single sample. A pilot, population study, of telomere analysis using TAT revealed high accuracy and reliability of the methodology. Conclusions Analytical validation of TAT shows that is a robust and reliable technique for the characterization of a detailed telomere profile in large cell populations. The combination of high-throughput imaging and software workflows allows for the collection of a large number of telomere-associated variables from each sample, which can then be used in epidemiological and clinical studies.

Ergothioneine Mitigates Telomere Shortening under Oxidative Stress Conditions

“…All telomere length measurements were performed using Life Length's proprietary Telomere Analysis Technology (TATV R ). TATV R measures telomere length using a high-throughput (HT) Q-FISH (quantitative fluorescent in situ hybridization) technique as previously described (de Pedro et al 2020). This method is based on a quantitative fluorescence in situ hybridization method modified for measuring individual chromosomes of cells in interphase.…”

Mathematical Connection Between Short Telomere Induced Senescence Calculation and Mortality Rate Data

“…Since the discovery of telomeres, hundreds of papers have been published reporting measurements of telomere lengths and their correlation with lifestyle factors and with longevity, building the evidence for using telomere length as a cellular marker of aging [3][4][5][6] [16] and one company [17]. An example of this measurement is shown in Fig 2a for a 44 year-old USA male with average measured telomere length [18].…”

“…An example of this measurement is shown in Fig 2a for a 44 year-old USA male with average measured telomere length [18]. This typical individual has a very asymmetric distribution of widely different telomere lengths (Fig 2a), ranging from several hundred base pairs to tens of thousands [17]. Because of the asymmetry, the Median and Average Telomere Lengths (MTL and ATL) are much longer than the peak of the distribution.…”

“…We shall arbitrarily estimate that cellular This difference we call ∆ which is defined as ∆ = TL m -TL 10 . The STLIS model then can be described with the use of Fig 2: as this individual ages, his entire telomere length distribution (Fig 2a) will shift to lower lengths, making the important assumption (for simplicity) that it will maintain approximately the same shape [17]. Thus, both TL m and TL 10 also shift together to lower lengths, with the difference in TL between them (∆) remaining constant.…”

Method comparison studies of telomere length measurement using qPCR approaches: A critical appraisal of the literature

“…These assays vary in the information they yield on TL. While most focus on the measurement of the average TL within the sample, assays also measure chromosome-specific TL [ 6 , 17 ], the complete distribution of TL in a cell population [ 33 ], or the shortest TL [ 26 ]. The shortest TL has received considerable attention, given evidence from in vitro and preclinical models suggesting that the shortest TL is most predictive of cellular senescence [ 36 , 37 ].…”