There has been great interest in telomere length in blood cells as a potential biomarker of cancer risk and other various aging-related phenotypes. Because many epidemiologic investigations involving telomere length rely on a one-time measurement of telomere length, reliability of the measurement over time should be established.
Using blood DNA samples from 27 women collected in 7 visits over a 9-month period, the present study reported a relatively high intraclass correlation coefficient of 0.64, suggesting that telomere length measurement at a single time point is a good representation of an individual's telomere length within a short period of time.
Telomere length measurement in blood cells might be influenced by a transient shift in the distribution of blood cell subpopulations caused by infection or acute inflammation, as blood cells comprise heterogeneous subpopulations with different replicative history and telomerase activity 
. The present data did not directly address the question, but suggests that such influence, if any, is far exceeded by between-individual variability in telomere length measurements.
Part of the variability in telomere length measurements can be attributed to the variation inherent in any quantitative PCR method 
. In our study, relative telomere length was determined using a recently-introduced single-tube, monochrome multiplexing quantitative PCR, which reduces pipetting-induced variation in telomere length measurements compared to the previously described qPCR method where telomeres and single copy genes are amplified independently in separate tubes that are usually located on separate plates 
. Even using the single tube method, we still observe appreciable plate-to-plate variability. To decrease the effect of inter-plate variability on the association between telomere length and outcomes of interest, it would be ideal to analyze all samples on a single plate, but even with 384 or newer high density 1,536 well plates, most epidemiologic studies will require multiple plates. The influence of possible differential misclassification bias can be minimized both by ensuring balance of cases and controls within plates and by placing technical replicates of samples on multiple plates. Inclusion of technical replicates within and across plates not only improves precision of an individual's measurement, but also allows investigators to generate reliable results even when an individual qPCR reaction fails 
Our analysis of quartile classification agreement between telomere length measurement at a single visit vs. the “gold standard” (i.e., average of multiple visits over a 9-month period) further supports the short-term reliability of telomere length measurement. It also provided a new way of examining the influences of the number and plate assignment of technical replicates on the classification accuracy of telomere length measurement at a single visit. Given that classification accuracy of women in the first and 4th quartiles is less subject to number of technical replicates compared to those in the middle quartiles, the disease association estimates for a comparison between the longest versus shortest quartiles of telomere length seem unlikely to be affected by poor precision of telomere length measurements. On the other hand, fewer number of technical replicates and the resulting loss of precision will lead to misclassification of subjects with telomere length in the middle categories, obscuring possible dose-response relationships between telomere length and the disease of interest.
Some aspects of our current investigation may limit the generalizability of our findings. First, our study is based on a small set of adult women volunteers. Although we observed a similar magnitude of good intraclass correlation coefficients for telomere length measurements within different subgroups by age, race and menopausal status, our findings may be less generalizable to children or male populations. For example, there have been reports suggesting accelerated telomere shortening in children 
, males 
and African Americans 
compared to adults, females and whites, respectively. Additionally, in this study we adopted a recently-introduced single-tube, monochrome multiplexing quantitative PCR 
for determination of telomere length. Despite increased efficiency in time and source DNA over the previously described method, this new multiplex qPCR might require more extensive optimization to be widely utilized 
In summary, our data demonstrated an overall good reliability of telomere length measurement within 1 year. However, some level of technical variability and significant plate-to-plate variation found in this study suggest the importance of technical replicates and balanced allocation of affected and unaffected samples within a single plate.