Alan J. Potter

Flow cytometric analysis of fluorescence in situ hybridization with dye dilution and DNA staining (flow‐FISH‐DDD) to determine telomere length dynamics in proliferating cells

Telomeres shorten during DNA replication; extensive erosion of telomeres likely promotes replicative senescence and chromosomal instability. Telomere length in individual cells has been quantified by flow cytometric analysis of fluorescence in situ hybridization (flow‐FISH). To determine the rate of telomere attrition (telomere erosion per cell division), we combined flow‐FISH with dye dilution and DNA staining (flow‐FISH‐DDD) and measured telomere‐specific fluorescence in proliferating cells identified by cell generation and cell cycle phase.

Use of a commercial ELISA for the detection of measles-specific immunoglobulin G (IgG) in dried blood spots collected from children living in low-resource settings.

Seroepidemiological monitoring of population immunity to vaccine‐preventable diseases is critical to prevent future outbreaks. Dried blood spots (DBS), drops of capillary blood dried on filter paper, are an affordable, minimally invasive alternative to venipuncture for collecting blood in field settings. However, few proven methods exist to analyze DBS for the presence of protective antibodies. This study validates a novel technique for measuring measles‐specific immunoglobulin G (IgG) in capillary DBS using a commercial ELISA. The predictive performance of a new method for analyzing DBS was tested by comparing matched serum and DBS samples from 50 children. The accuracy, precision, and reliability of the procedure were evaluated, and the optimal cut points to classify positive and negative samples were determined. The method was then applied to 1,588 DBS collected during a large survey of children in Mexico and Nicaragua. Measles‐specific IgG in serum samples were 62% negative, 10% equivocal, and 28% positive. In comparisons with matched serum, DBS results were 100% sensitive and 96 · 8% specific, and agreed in 46 of 50 (92%) cases. The inter‐assay and intra‐assay coefficients of variation from kit‐provided controls were greater than desired (24.8% and 8.4%, respectively). However, in predictive simulations the average misclassification was only 3.9%. Procedures were found to be acceptable to surveyors and participants. Analyzing DBS collected in low‐resources settings is a feasible and accurate means of measuring population immunity to measles and should be used to generate objective measures of health status and health system performance. J. Med. Virol. 87:1491–1499, 2015.

Blood spot-based measures of glucose homeostasis and diabetes prevalence in a nationally representative population of young US adults

PURPOSE We investigated understudied biomarker-based diabetes among young US adults, traditionally characterized by low cardiovascular disease risk. METHODS We examined 15,701 participants aged 24 to 32 years at Wave IV of the National Longitudinal Study of Adolescent Health (Add Health, 2008). The study used innovative and relatively noninvasive methods to collect capillary whole blood via finger prick at in-home examinations in all 50 states. RESULTS Assays of dried blood spots produced reliable and accurate values of HbA1c. Reliability was lower for fasting glucose and lowest for random glucose. Mean (SD) HbA1c was 5.6% (0.8%). More than a quarter (27.4%) had HbA1c-defined prediabetes. HbA1c was highest in the black, non-Hispanic race/ethnic group, inversely associated with education, and more common among the overweight/obese and physically inactive. The prevalence of diabetes defined by previous diagnosis or use of antidiabetic medication was 2.9%. Further incorporating HbA1c and glucose values, the prevalence increased to 6.8%, and among these participants, 38.9% had a previous diagnosis of diabetes (i.e., aware). Among those aware, 37.6% were treated and 64.0% were controlled (i.e., HbA1c < 7%). CONCLUSIONS A contemporary cohort of young adults faces a historically high risk of diabetes but there is ample opportunity for early detection and intervention.

Validation and modification of dried blood spot-based glycosylated hemoglobin assay for the longitudinal aging study in India.

This study aims to validate a modified dried blood spot (DBS)‐based glycosylated hemoglobin (HbA1c) assay protocol, after a pretest in India showed poor correlation between the original DBS‐based protocol and venous results.

HPLC-based Measurement of Glycated Hemoglobin using Dried Blood Spots Collected under Adverse Field Conditions

ABSTRACT Glycated hemoglobin (HbA1c) measured using high-performance liquid chromatography (HPLC) assays with venous blood and dried blood spots (DBS) are compared for 143 paired samples collected in Aceh, Indonesia. Relative to gold-standard venous-blood values, DBS-based values reported by the HPLC are systematically upward biased for HbA1c<8% and the fraction diabetic (HbA1c ≥ 6.5%) is overstated almost five-fold. Inspection of chromatograms from DBS assays indicates the % glycosylated calculated by the HPLC excludes part of the hemoglobin A which is misidentified as a hemoglobin variant. Taking this into account, unbiased DBS-based values are computed using data from the machine-generated chromatograms. When the DBS are collected in a clinic-like setting, under controlled humidity/temperature conditions, the recalculated values are almost identical to venous-based values. When DBS are collected under field conditions, the recalculated values are unbiased, but only about half the HbA1c values are measured reliably, calling into question the validity of the other half. The results suggest that collection conditions, particularly humidity, affect the quality of the DBS-based measures. Cross-validating DBS-based HbA1c values with venous samples collected under exactly the same environmental conditions is a prudent investment in population-based studies.