Dried blood spot collection, sample quality, and fieldwork conditions: Structural validations for conversion into standard values.

OBJECTIVES: SHARE, a pan-European panel study in 27 European countries and Israel, has collected dried blood spot (DBS) samples from approximately 27 000 respondents in 13 countries. We aim to obtain factors to convert analyte values between DBS and venous blood samples (VBS) taking account of adverse fieldwork conditions such as small spot size, high temperature and humidity, short drying time and long shipment times. METHODS: We obtained VBS and DBS from a set of 20 donors in a laboratory setting, and treated the DBS in a systematic and controlled fashion simulating SHARE fieldwork conditions. We used the 3420 outcomes to estimate from DBS analyte values the values that we would have obtained had it been feasible to collect and analyze the donors' venous blood samples. RESULTS: The influence of field conditions and sample quality on DBS analyte values is significant and differs among assays. Varying spot size is the main challenge and affects all markers except HbA1c. Smaller spots lead to overly high measured levels. A missing desiccant is detrimental for all markers except CRP and tHb. The temperature to which the samples are exposed plays a significant role for HDL and CysC, while too brief a drying time affects CRP and CysC. Lab-based adjustment formulae only accounting for the differences between re-liquefied DBS and venous blood do not address these fieldwork conditions. CONCLUSIONS: By simulating adverse fieldwork conditions in the lab, we were able to validate DBS collected under such conditions and established conversion formulae with high prediction accuracy.

The metabolic syndrome and mammographic breast density in a racially diverse and predominantly immigrant sample of women.

PURPOSE: The metabolic syndrome [MetS, clustering of elevated blood pressure, triglycerides and glucose, reduced high-density lipoprotein cholesterol (HDL-C), abdominal obesity] has been associated with increased breast cancer risk, but less is known about its association with mammographic breast density, a strong risk factor for breast cancer. METHODS: We collected data on risk factors, body size, and blood pressure via in-person interviews and examinations and measured glucose, triglycerides, and HDL-C from dried blood spots from women recruited through a mammography screening clinic (n = 373; 68 % Hispanic, 17 % African-American, 63 % foreign born). We performed linear regression models to examine the associations of each MetS component and the MetS cluster (≥3 components) with percent density and dense breast area, measured using a computer-assisted technique and Cumulus software. RESULTS: About 45 % of women had the MetS, with the prevalence of the individual components ranging from 68 % for abdominal obesity to 33 % for elevated triglycerides. The prevalence of the MetS increased with higher body mass index (BMI) and postmenopausal status, but did not vary substantially by ethnicity, immigrant generational status, parity, age at menarche, or alcohol consumption. Low HDL-C (<50 mg/dL), but not the MetS cluster or the other MetS components, was associated with larger dense breast area after adjusting for age, BMI, fasting time, and educational attainment (ß = 8.77, 95 % CI 2.39, 15.14). The MetS and its individual components were not associated with BMI-adjusted percent density. CONCLUSIONS: HDL-C alone may have an influence on dense breast tissue that is independent of BMI, and may be in the same direction as its association with breast cancer risk.

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.

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.

BACKGROUND: 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. METHODS: Peripheral blood mononuclear cells (PBMC) were stained with the cell division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated with phytohemagglutinin (PHA), grown for 5-6 days, hybridized with a telomere sequence-specific peptide nucleic acid fluorescent probe (PNA-Cy5), counterstained with DAPI, and analyzed by flow cytometry. The cell cycle distribution and cell division generations were respectively identified by analysis of DAPI emission and deconvolution of CFSE emission, and Cy5 emission was used to determine telomere-specific fluorescence, an indicator of telomere length, in each cell. RESULTS: In stimulated PBMC, in each cell cycle phase, the telomere-specific fluorescence diminished with increasing cell generation. The rate of decline of the telomere-specific fluorescence per cell generation did not significantly differ between cell cycle phases. CONCLUSIONS: Application of flow-FISH-DDD to measure mean telomere length and the rate of telomere attrition in proliferating cells may find use in studies of ageing and disease, the effects of telomere-modifying agents, and variability between individuals.

The cell cycle phases of DNA damage and repair initiated by topoisomerase II-targeting chemotherapeutic drugs.

Although cytostasis and cytotoxicity induced by cancer chemotherapy drugs targeting topoisomerase II (topoII) arise in specific cell cycle phases, it is unknown whether the drug-initiated DNA damage triggering these responses, or the repair (reversal) of this damage, differs between cell cycle phases or between drug classes. Accordingly, we used a flow cytometric alkaline unwinding assay to measure DNA damage (strand breakage (SB)) and SB repair in each cell cycle compartment of human cancer cell lines treated with clinically relevant concentrations of doxorubicin, daunomycin, etoposide, and mitoxantrone. We found that treated HeLa and A549 cells exhibited the greatest SB in G2/M phase, the least in G1 phase, and generally an intermediate amount in S phase. The cell cycle phase specificity of the DNA damage appeared to be predictive of the cell cycle phase of growth arrest. Furthermore, it appeared to be dependent on topoIIalpha expression as the extent of SB did not differ between cell cycle compartments in topoIIalpha-diminished A549(VP)28 cells. HeLa cells were apparently unable to repair doxorubicin-initiated SB. The rate of repair of etoposide-initiated SB in HeLa cells and of mitoxantrone-initiated SB in HeLa and A549 cells was similar in each cell cycle compartment. In A549 cells, the rate of repair of doxorubicin and etoposide-initiated SB differed between cell cycle phases. Overall, these results indicate that the cell cycle phase specificity of cytostasis and cytotoxicity induced in tumor cells by topoII-targeting drugs may be directly related to the cell cycle phase specificity of the drug-initiated DNA damage. Analysis by cell cycle compartment appears to clarify some of the intercellular heterogeneity in the extent of drug-initiated DNA damage and cytotoxicity previously observed in cancer cells analyzed as a single population; this approach might be useful in resolving inconsistent results reported in investigations of tumor cell topoII content versus response to topoII-targeting drugs.

Flow cytometric analysis of the cell cycle phase specificity of DNA damage induced by radiation, hydrogen peroxide and doxorubicin.

We have optimized a flow cytometric DNA alkaline unwinding assay to increase the sensitivity in detecting low levels of DNA damage (strand breaks and alkali-labile sites) and to permit the measurement of the extent of DNA damage within each cell cycle compartment. The lowest gamma radiation dose that induced detectable DNA damage in each cell cycle phase of HeLa and CEM cells was 10 cGy. The lowest H(2)O(2) concentration that induced detectable DNA damage in each cell cycle phase was 0.5 microM in HeLa cells, and 1-2.5 TmicroM in CEM cells. For both HeLa cells and CEM cells, DNA damage in each cell cycle compartment increased approximately linearly with increasing doses of gamma radiation and H(2)O(2). Although untreated HeLa and CEM cells in S phase consistently exhibited greater DNA unwinding than did G(1) or G(2) cells (presumably due to DNA strand breaks associated with replication forks), there was no difference between the susceptibility of G(0)/G(1), S and G(2)/M phase cells to DNA damage induced by gamma radiation or H(2)O(2), or in the rate of repair of this damage. In each cell cycle phase, the susceptibility to gamma radiation-induced DNA damage was greater in CEM cells than in HeLa cells. In contrast to the lack of cell cycle phase-specific DNA damage induced by exposure to gamma radiation or H(2)O(2), the cancer chemotherapeutic drug doxorubicin (adriamycin) predominantly induced DNA damage in G(2) phase cells.