Blood and dried blood spot telomere length measurement by qPCR: assay considerations.

Measurement of telomere length is crucial for the study of telomere maintenance and its role in molecular pathophysiology of diseases and in aging. Several methods are used to measure telomere length, the choice of which usually depends on the type and size of sample to be assayed, as well as cost and throughput considerations. The goal of this study was to investigate the factors that may influence the reliability of qPCR-based relative telomere length measurements in whole blood. Day to day intra-individual variability, types of blood anticoagulant, sample storage conditions, processing and site of blood draw were investigated. Two qPCR-based methods to measure telomere length (monoplex vs. multiplex) were also investigated and showed a strong correlation between them. Freezing and thawing of the blood and storage of the blood at 4°C for up to 4 days did not affect telomere length values. Telomere lengths in dried blood spots were significantly higher than both whole blood and peripheral mononuclear blood cells, and were highly correlated with both. We found that telomere length measurements were significantly higher in dried blood spots collected directly from fingertip prick compared to dried blood spots prepared with anticoagulated whole blood collected from the finger, and non-blotted whole blood taken from both finger and arm venipuncture. This suggests that DNA from cells blotted on paper is not equivalent to that collected from venipuncture whole blood, and caution should be taken when comparing between blood sample types

Relative T/S ratio in anti-coagulated whole blood, anticoagulated DBS and finger prick DBS samples over many days.

<p>Relative T/S ratios in whole blood collected into three anticoagulants from a single individual on 10 different days (panel A) and DBS created from the anticoagulated whole blood and from finger prick on the same days (panel B) were highly variable.</p

The effect of repeated freezing and thawing on T/S ratio measurements.

<p>DNA was extracted from whole blood aliquots obtained at each of seven consecutive freeze-thaw cycles and T/S ratios were determined.</p

Standard curves used to determine T/S ratio.

<p>Pooled blood DNA was serially diluted (1:2) to generate nine DNA standards for the ASPG accessory gene (S) (panel A) and the telomere region (T) (panel B). Stability of the T/S ratio within the range of DNA concentrations was tested (panel C). N = 1 for each point.</p

The effect of site of blood draw on T/S ratio measurements.

<p>T/S ratios were measured for DNA from arm EDTA whole blood, arm whole bloodDBS, finger EDTA WB, finger EDTADBS and direct finger prick blood made from a single blood draw from 12 distinct individuals. P values are shown, without adjustment for multiple comparisons. ns  =  not significant (p>0.05). The line represents the median and the + represents mean.</p

Correlation between monoplex and multiplex qPCR.

<p>ACD WB (N = 32) samples were measured by monoplex and multiplex qPCR. Correlations were done between S (panel A), T (panel B) and T/S (panel C). Pearson’s R<b>²</b> and p values are shown.</p

Stability of T/S measurements in anticoagulated whole blood samples in different storage conditions.

<p>ACD and EDTA anticoagulated whole blood was stored at 4°C and room temperature (RT) for nine days, and daily T/S ratios are shown.</p

Relative T/S ratio (n = 10) measured in WB collected by venipuncture in various anticoagulants and DBS samples (blotted with WB or directly from the fingertip) from a single individual.

a<p>Wilcoxon signed-rank test to compare between WB and WBDBS of the same anticoagulant</p>b<p>Friedman’s test to compare anticoagulants within WB and within DBS</p>c<p>DBS made from finger prick spotted directly onto paper (no anticoagulant)</p><p>Abbreviations: T/S  =  relative telomere length ratio, SD  =  standard deviation, CV  =  coefficient of variation, WB  =  whole blood, DBS  =  dried blood spot, ACD  =  acid citrate dextrose anticoagulated, EDTA  =  Ethylenediaminetetraacetic acid anticoagulated</p

Sample collection overview.

<p>Diagram outlining the sites of blood sample collection, anticoagulants used, sample processing, and parameters tested for each sample type.</p

Comparison of T/S measurements in DBS, PBMC and WB samples.

<p>T/S ratios were determined for DNA from DBS (n = 12), PBMC (n = 12) and WB (n = 12) samples made from a single blood draw from 12 distinct individuals. Pearson’s R<b>²</b> and p values are shown.</p