Comparison of different methods for telomere length measurement in whole blood and blood cell subsets: Recommendations for telomere length measurement in hematological diseases

Abstract

Different methods of telomere length measurement are used to identify patients with telomeropathies. In our lab, we established four different methods for telomere length measurement, terminal restriction fragment (TRF) analysis by Southern blot analysis, quantitative PCR (qPCR), quantitative telomere/centromere fluorescence in situ hybridization (T/C-FISH) and fluorescence in situ hybridization combined with flow cytometry (FlowFISH). The methods each have distinct properties and apart from this-according to our experience and data-may have an impact on the individual result. In this study, we therefore compared and validated these methods measuring 154 healthy individuals of different age groups (newborn-81 years). A linear decline was found for every method (Southern blotting 64 bp per year; qPCR 31 bp per year; T/C-FISH 36 bp per year; FlowFISH 50 bp per year). With the equation of the regression line the values of each method (T/S ratio, T/C value, RTL value) can be expressed in absolute kb. All methods showed acceptable accuracy. The analysis indicated good agreement between all methods, with the best agreement between T/C-FISH and FlowFISH. Here, FlowFISH was the most precise, accurate, and reproducible method compared to the other methods. Based on our data, we emphasize the influence of expertise and experience that is required to produce robust and reliable telomere length analyses. Furthermore, we want to provide the scientific community working in diagnostics and research with data-funded advice on how to choose the appropriate method to safely discriminate between natural variability and pathological telomere shortening in individual cases.

Comparison of flow‐FISH and MM–qPCR telomere length assessment techniques for the screening of telomeropathies

“…32 Overall, the range of correlation values was low in line with previous studies showing similar correlations between flow-FISH and MM-qPCR. [31][32][33][34][35] Bland-Altman analysis confirmed that the overall agreement between flow-FISH and MM-qPCR was poor. Mean ratios and limits of agreement (LoA) between flow-FISH and MM-qPCR were moderate, with an average of 0.28 kb (LoA, 3.71 to −3.42 kb; Fig.…”

The Importance of Telomere Shortening for Atherosclerosis and Mortality

“…Despite promising results from epidemiologic studies, pronounced intra- and interindividual variability hamper a wider application of LTL measurements in clinical practice [ 22 , 26 , 143 ]. Limited comparability between methods and laboratories further compromises the utility of LTL measurements on an individual basis [ 144 , 145 , 146 , 147 , 148 ].…”

Measuring biological aging in humans: A quest

“…Note that even in the state of robustness, damage can be already abnormally high (b) and resilience already abnormally low (c) al., 2014). Work is underway to establish an optimal "gold standard" assay for epidemiological studies (Behrens et al, 2017;Montpetit et al, 2014). At this stage, there is not enough evidence in the literature to consider measuring telomere shortening as a biological mechanism of aging or telomere length as a biomarker of biological aging.…”

“…Observational studies using these techniques have reported contrasting results, and longitudinal studies have revealed erratic changes over time, possibly due to large measurement error (Berglund et al, ; Bischoff et al, ; Eerola et al, ; Lin et al, ; Müezzinler, Zaineddin, & Brenner, ; Solomon et al, ). Work is underway to establish an optimal “gold standard” assay for epidemiological studies (Behrens et al, ; Montpetit et al, ). At this stage, there is not enough evidence in the literature to consider measuring telomere shortening as a biological mechanism of aging or telomere length as a biomarker of biological aging.…”