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

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

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

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

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

Sample collection overview.

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

Daily Oral Supplementation with 60 mg of Elemental Iron for 12 Weeks Alters Blood Mitochondrial DNA Content, but Not Leukocyte Telomere Length in Cambodian Women

There is limited evidence regarding the potential risk of untargeted iron supplementation, especially among individuals who are iron-replete or have genetic hemoglobinopathies. Excess iron exposure can increase the production of reactive oxygen species, which can lead to cellular damage. We evaluated the effect of daily oral supplementation on relative leukocyte telomere length (rLTL) and blood mitochondrial DNA (mtDNA) content in non-pregnant Cambodian women (18–45 years) who received 60 mg of elemental iron as ferrous sulfate (n = 190) or a placebo (n = 186) for 12 weeks. Buffy coat rLTL and mtDNA content were quantified by monochrome multiplex quantitative polymerase chain reaction. Generalized linear mixed-effects models were used to predict the absolute and percent change in rLTL and mtDNA content after 12 weeks. Iron supplementation was not associated with an absolute or percent change in rLTL after 12 weeks compared with placebo (ß-coefficient: −0.04 [95% CI: −0.16, 0.08]; p = 0.50 and ß-coefficient: −0.96 [95% CI: −2.69, 0.77]; p = 0.28, respectively). However, iron supplementation was associated with a smaller absolute and percent increase in mtDNA content after 12 weeks compared with placebo (ß-coefficient: −11 [95% CI: −20, −2]; p = 0.02 and ß-coefficient: −11 [95% CI: −20, −1]; p= 0.02, respectively). Thus, daily oral iron supplementation for 12 weeks was associated with altered mitochondrial homeostasis in our study sample. More research is needed to understand the risk of iron exposure and the biological consequences of altered mitochondrial homeostasis in order to inform the safety of the current global supplementation policy.Land and Food Systems, Faculty ofMedicine, Faculty ofOther UBCNon UBCPathology and Laboratory Medicine, Department ofPediatrics, Department ofReviewedFacult

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