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.

Keywords: Ageing; biomarker; human; telomeres; variation.

Figure 1.

Telomere length ratios (TLRs) by laboratory, round and sample. TLRs are normalized to sample G, first round. Symbols indicate laboratories and techniques: ▪ Lab 1 South; ▴ Lab 2 South; ✖ Lab 3 STELA; ▴ Lab 4 qPCR; ◆ Lab 5 qPCR; ✻ Lab 6 qPCR; ▪ Lab 7 qPCR; Δ Lab 8 qPCR; ◇ Lab 9 qPCR; • Lab 10 qPCR duplex; ○ Lab 10-2 qPCR monoplex .

Figure 2.

Correlation between TLRs measured by Southern blotting/STELA vs qPCR. Data are scatterplots of means (± SD) of sample TLRs per technique. Results from rounds 1 and 2 are combined. Linear regression (solid line) and 95% confidence intervals (dotted) are shown. The correlation coefficient is r²= 0.676.

Figure 3.

Coefficients of variation by technique and laboratory. Box plots indicate median (central line), upper and lower quartiles (boxes), upper and lower centiles (whiskers) and outliers (dots). (a) Intra-batch CVs per technique. (b) Inter-batch CVs per technique. (c) Intra-laboratory CVs (both intra- and inter-batch CVs combined).