Reproducibility of telomere length assessment: an international collaborative study

Abstract

Background: Telomere length is a putative biomarker of ageing, morbidity and mortality. Its application is hampered by lack of widely applicable reference ranges and uncertainty regarding the present limits of measurement reproducibility within and between laboratories.Methods: We instigated an international collaborative study of telomere length assessment: 10 different laboratories, employing 3 different techniques [Southern blotting, single telomere length analysis (STELA) and real-time quantitative PCR (qPCR)] performed two rounds of fully blinded measurements on 10 human DNA samples per round to enable unbiased assessment of intra- and inter-batch variation between laboratories and techniques.Results: Absolute results from different laboratories differed widely and could thus not be compared directly, but rankings of relative telomere lengths were highly correlated (correlation coefficients of 0.63–0.99). Intra-technique correlations were similar for Southern blotting and qPCR and were stronger than inter-technique ones. However, inter-laboratory coefficients of variation (CVs) averaged about 10% for Southern blotting and STELA and more than 20% for qPCR. This difference was compensated for by a higher dynamic range for the qPCR method as shown by equal variance after z-scoring. Technical variation per laboratory, measured as median of intra- and inter-batch CVs, ranged from 1.4% to 9.5%, with differences between laboratories only marginally significant (P = 0.06). Gel-based and PCR-based techniques were not different in accuracy.Conclusions: Intra- and inter-laboratory technical variation severely limits the usefulness of data pooling and excludes sharing of reference ranges between laboratories. We propose to establish a common set of physical telomere length standards to improve comparability of telomere length estimates between laboratories.

Telomere length and telomerase in a well-characterized sample of individuals with major depressive disorder compared to controls

“…Finally, the direct correlation between qPCR T/S ratio and Southern telomere kbp length results were modest, and non significant in controls; the confirmatory qPCR was also performed later at a different laboratory. It is worth noting that prior studies have also found wide variability in absolute results across laboratories, though standardized values examined as ranks were highly correlated, significant variance across labs remained and was largely explained by differences in qPCR and gel based methods (Martin-Ruiz et al, 2014)…”

Psychiatric disorders and leukocyte telomere length: Underlying mechanisms linking mental illness with cellular aging

“…Further, insufficient calibration of assay methods across labs and the lack of accepted “normal ranges” for LTL and PBMC basal TA make it premature for cell aging markers to enter clinical use at this time (Martin-Ruiz et al, In press). The relatively small effect sizes reported in reviewed positive studies, as well as the lack of diagnostic specificity of LTL and PBMC basal TA changes, also argue against the use of such markers as diagnostic tools, in isolation from other measures, at this time.…”

Leucocyte telomere length, genetic variants at theTERTgene region and risk of pancreatic cancer

“…Pooling data across studies and laboratories can be complex due to methodological differences in measuring telomere length. 37 In the current study, all samples were analysed together in the same laboratory using the same analytic protocol. Confounding by exposures such as smoking and obesity may affect associations.…”