An Assessment of Individual Preference for a Novel Capillary Blood Collection System.

Purpose: Typical barriers to venous blood collection for wellness testing include discomfort, time spent, and collection site accessibility. This study assessed individuals' experience, satisfaction, and preference associated with a FDA-cleared blood-collection device, the BD MiniDraw™ Capillary Blood Collection System (BD MiniDraw), in retail locations. Patients and Methods: A total of 113 individuals (≥18 years) with venous blood collection experience were enrolled; 107 completed the study. A pre-collection survey gathered information on demographics and past experiences with healthcare and venous blood collection settings. BD MiniDraw collection was conducted at three retail sites (two pharmacies and one grocery store) by trained healthcare workers using the Babson BetterWay blood testing service model. A follow up survey was performed two weeks later to determine experience with, and preference for, BD MiniDraw in terms of staff professionalism, blood collection location, blood collection time, and staff trustworthiness. Results: Among the 107 participants, 74 (69%) were female and 33 (31%) were male; the mean age was 49 years (range=18-71 years). Sixty-six (62%) participants viewed their prior venipuncture experience as "somewhat" or "very" positive. Following capillary collection, 96 (90%) participants expressed a "somewhat" or "very" positive experience with BD MiniDraw at a retail location. In particular, "very satisfied" responses were given for location (87/107; 81%) and collection time (78/1407; 73%). In a subset of respondents (n=89), those reasons (location and time savings) were most frequent for likelihood of future use. Ninety-nine participants (92%) rated the retail blood collection team as "very" or "extremely" trustworthy. Overall, 90 participants (84%) "strongly preferred" (56/107; 52%), "somewhat preferred" (14/107; 13%), or had "no preference" (20/107; 19%) for BD MiniDraw, compared to traditional venous blood collection. Conclusion: Most participants conveyed a preference for BD MiniDraw, primarily based on the blood collection retail location, perceived time savings, and professionalism and trustworthiness of the staff.

High-resolution forest carbon stocks and emissions in the Amazon.

Efforts to mitigate climate change through the Reduced Emissions from Deforestation and Degradation (REDD) depend on mapping and monitoring of tropical forest carbon stocks and emissions over large geographic areas. With a new integrated use of satellite imaging, airborne light detection and ranging, and field plots, we mapped aboveground carbon stocks and emissions at 0.1-ha resolution over 4.3 million ha of the Peruvian Amazon, an area twice that of all forests in Costa Rica, to reveal the determinants of forest carbon density and to demonstrate the feasibility of mapping carbon emissions for REDD. We discovered previously unknown variation in carbon storage at multiple scales based on geologic substrate and forest type. From 1999 to 2009, emissions from land use totaled 1.1% of the standing carbon throughout the region. Forest degradation, such as from selective logging, increased regional carbon emissions by 47% over deforestation alone, and secondary regrowth provided an 18% offset against total gross emissions. Very high-resolution monitoring reduces uncertainty in carbon emissions for REDD programs while uncovering fundamental environmental controls on forest carbon storage and their interactions with land-use change.

Large-scale impacts of herbivores on the structural diversity of African savannas.

African savannas are undergoing management intensification, and decision makers are increasingly challenged to balance the needs of large herbivore populations with the maintenance of vegetation and ecosystem diversity. Ensuring the sustainability of Africa's natural protected areas requires information on the efficacy of management decisions at large spatial scales, but often neither experimental treatments nor large-scale responses are available for analysis. Using a new airborne remote sensing system, we mapped the three-dimensional (3-D) structure of vegetation at a spatial resolution of 56 cm throughout 1640 ha of savanna after 6-, 22-, 35-, and 41-year exclusions of herbivores, as well as in unprotected areas, across Kruger National Park in South Africa. Areas in which herbivores were excluded over the short term (6 years) contained 38%-80% less bare ground compared with those that were exposed to mammalian herbivory. In the longer-term (> 22 years), the 3-D structure of woody vegetation differed significantly between protected and accessible landscapes, with up to 11-fold greater woody canopy cover in the areas without herbivores. Our maps revealed 2 scales of ecosystem response to herbivore consumption, one broadly mediated by geologic substrate and the other mediated by hillslope-scale variation in soil nutrient availability and moisture conditions. Our results are the first to quantitatively illustrate the extent to which herbivores can affect the 3-D structural diversity of vegetation across large savanna landscapes.

Programmable nano-bio-chip sensors: analytical meets clinical.

There have been many recent advances in the nano-bio-chip analysis methodology with implications for a number of high-morbidity diseases including HIV, cancer, and heart disease. (To listen to a podcast about this article, please go to the Analytical Chemistry multimedia page at pubs.acs.org/page/ancham/audio/index.html .).

Effects of fire on woody vegetation structure in African savanna.

Despite the importance of fire in shaping savannas, it remains poorly understood how the frequency, seasonality, and intensity of fire interact to influence woody vegetation structure, which is a key determinant of savanna biodiversity. We provide a comprehensive analysis of vertical and horizontal woody vegetation structure across one of the oldest savanna fire experiments, using new airborne Light Detection and Ranging (LiDAR) technology. We developed and compared high-resolution woody vegetation height surfaces for a series of large experimental burn plots in the Kruger National Park, South Africa. These 7-ha plots (total area approximately 1500 ha) have been subjected to fire in different seasons and at different frequencies, as well as no-burn areas, for 54 years. Long-term exposure to fire caused a reduction in woody vegetation up to the 5.0-7.5 m height class, although most reduction was observed up to 4 m. Average fire intensity was positively correlated with changes in woody vegetation structure. More frequent fires reduced woody vegetation cover more than less frequent fires, and dry-season fires reduced woody vegetation more than wet-season fires. Spring fires from the late dry season reduced woody vegetation cover the most, and summer fires from the wet season reduced it the least. Fire had a large effect on structure in the densely wooded granitic landscapes as compared to the more open basaltic landscapes, although proportionally, the woody vegetation was more reduced in the drier than in the wetter landscapes. We show that fire frequency and fire season influence patterns of vegetation three-dimensional structure, which may have cascading consequences for biodiversity. Managers of savannas can therefore use fire frequency and season in concert to achieve specific vegetation structural objectives.

Molecular detection of harmful algal blooms (HABs) using locked nucleic acids and bead array technology.

Harmful algal blooms (HABs) are a serious public health risk in coastal waters. As the intensity and frequency of HABs continue to rise, new methods of detection are needed for reliable identification. Herein, we developed a high-throughput, multiplex, bead array technique for the detection of the dinoflagellates Karenia brevis and Karenia mikimotoi. The method combined the Luminex detection system with two novel technologies: locked nucleic acid-modified oligonucleotides (LNA) and Mirus Label IT(®) nucleic acid technology. To study the feasibility of the method, we evaluated the performance of modified and unmodified LNA probes with amplicon targets that were biotin labeled with two different strategies: direct chemical labeling (Mirus Label IT) versus enzymatic end-labeling (single biotinylated primer). The results illustrated that LNA probes hybridized to complementary single-stranded DNA with better affinity and displayed higher fluorescence intensities than unmodified oligonucleotide DNA probes. The latter effect was more pronounced when the assay was carried out at temperatures above 53°C degree. As opposed to the enzymatic 5' terminal labeling technique, the chemical-labeling method enhanced the level of fluorescence by as much as ~83%. The detection limits of the assay, which were established with LNA probes and Mirus Label IT system, ranged from 0.05 to 46 copies of rRNA. This high-throughput method, which represents the first molecular detection strategy to integrate Luminex technology with LNA probes and Mirus Label IT, can be adapted for the detection of other HABs and is well suited for the monitoring of red tides at pre-blooming and blooming conditions.

Emerging concepts in biomarker discovery; the US-Japan Workshop on Immunological Molecular Markers in Oncology.

Supported by the Office of International Affairs, National Cancer Institute (NCI), the "US-Japan Workshop on Immunological Biomarkers in Oncology" was held in March 2009. The workshop was related to a task force launched by the International Society for the Biological Therapy of Cancer (iSBTc) and the United States Food and Drug Administration (FDA) to identify strategies for biomarker discovery and validation in the field of biotherapy. The effort will culminate on October 28th 2009 in the "iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", which will be held in Washington DC in association with the Annual Meeting. The purposes of the US-Japan workshop were a) to discuss novel approaches to enhance the discovery of predictive and/or prognostic markers in cancer immunotherapy; b) to define the state of the science in biomarker discovery and validation. The participation of Japanese and US scientists provided the opportunity to identify shared or discordant themes across the distinct immune genetic background and the diverse prevalence of disease between the two Nations. Converging concepts were identified: enhanced knowledge of interferon-related pathways was found to be central to the understanding of immune-mediated tissue-specific destruction (TSD) of which tumor rejection is a representative facet. Although the expression of interferon-stimulated genes (ISGs) likely mediates the inflammatory process leading to tumor rejection, it is insufficient by itself and the associated mechanisms need to be identified. It is likely that adaptive immune responses play a broader role in tumor rejection than those strictly related to their antigen-specificity; likely, their primary role is to trigger an acute and tissue-specific inflammatory response at the tumor site that leads to rejection upon recruitment of additional innate and adaptive immune mechanisms. Other candidate systemic and/or tissue-specific biomarkers were recognized that might be added to the list of known entities applicable in immunotherapy trials. The need for a systematic approach to biomarker discovery that takes advantage of powerful high-throughput technologies was recognized; it was clear from the current state of the science that immunotherapy is still in a discovery phase and only a few of the current biomarkers warrant extensive validation. It was, finally, clear that, while current technologies have almost limitless potential, inadequate study design, limited standardization and cross-validation among laboratories and suboptimal comparability of data remain major road blocks. The institution of an interactive consortium for high throughput molecular monitoring of clinical trials with voluntary participation might provide cost-effective solutions.

Multiplex microsphere immuno-detection of potato virus Y, X and PLRV.

To monitor seed potatoes for potato virus X, Y and PLRV, a multiplex microsphere immunoassay (MIA) was developed based on the Luminex xMAP technology, as an alternative to ELISA. The xMAP technology allowed detection of a number of antigens simultaneously whereas ELISA only allowed simplex detection of antigens. The use of paramagnetic beads in the MIA procedure allowed efficient removal of excess sample compounds and reagents. This resulted in lower background values and a higher specificity than a non-wash MIA procedure using conventional beads. In a simplex MIA detection, levels for PVY and PLRV in potato leaf extracts were 10 times lower than ELISA but for PVX 10 timers higher, whereas the specificity was similar. Results of a multiplex assay performed on viruses added to potato leaf extracts were largely similar to those of ELISA for individual viruses. Results of samples infected naturally with PVX, PVY or PLRV were comparable with ELISA.

Quantitative, multiplexed detection of Salmonella and other pathogens by Luminex xMAP suspension array.

We describe a suspension array hybridization assay for rapid detection and identification of Salmonella and other bacterial pathogens using Luminex xMAP technology. The Luminex xMAP system allows simultaneous detection of up to 100 different targets in a single multiplexed reaction. Included in the method are the procedures for (1) design of species-specific oligonucleotide capture probes and PCR amplification primers, (2) coupling oligonucleotide capture probes to carboxylated microspheres, (3) hybridization of coupled microspheres to oligonucleotide targets, (4) production of targets from DNA samples by PCR amplification, and (5) detection of PCR-amplified targets by direct hybridization to probe-coupled microspheres. The Luminex xMAP suspension array hybridization assay is rapid, requires few sample manipulations, and provides adequate sensitivity and specificity to detect and differentiate Salmonella and nine other test organisms through direct detection of species-specific DNA sequences.

A Quick Test of Cognitive Speed: patterns of age groups 15 to 95 years.

A Quick Test of Cognitive Speed color, form, and color-form naming were administered to 300 normal participants (ages 15-95 years) to explore the effects of age on perceptual (single-dimension naming) and cognitive speed (dual-dimension naming). Naming time means (sec.) were consistent with previous findings. Correlations between age and naming time were low, but significant. Linear regression with age as a factor indicated time increases of 1 sec. per decade for colors and color-form combination naming and of 6 sec. per decade for form naming. Participants were divided into age cohorts, each covering a decade, and naming times were transformed to normalized z scores. The normalized means were similar for color, form, and color-form naming and increased by about 1 SD between ages 15-25 and 75-85 years. The ranges were similar across cohorts, about 2 SD. The findings concur with age patterns for visual-pattern comparison speed, fluid intelligence, and working memory reported by Salthouse in 2004.

Testis and antler dysgenesis in sitka black-tailed deer on Kodiak Island, Alaska: Sequela of environmental endocrine disruption?

It had been observed that many male Sitka black-tailed deer (Odocoileus hemionus sitkensis) on Kodiak Island, Alaska, had abnormal antlers, were cryptorchid, and presented no evidence of hypospadias. We sought to better understand the problem and investigated 171 male deer for phenotypic aberrations and 12 for detailed testicular histopathology. For the low-lying Aliulik Peninsula (AP), 61 of 94 deer were bilateral cryptorchids (BCOs); 70% of these had abnormal antlers. Elsewhere on the Kodiak Archipelago, only 5 of 65 deer were BCOs. All 11 abdominal testes examined had no spermatogenesis but contained abnormalities including carcinoma in situ-like cells, possible precursors of seminoma; Sertoli cell, Leydig cell, and stromal cell tumors; carcinoma and adenoma of rete testis; and microlithiasis or calcifications. Cysts also were evident within the excurrent ducts. Two of 10 scrotal testes contained similar abnormalities, although spermatogenesis was ongoing. We cannot rule out that these abnormalities are linked sequelae of a mutation(s) in a founder animal, followed by transmission over many years and causing high prevalence only on the AP. However, based on lesions observed, we hypothesize that it is more likely that this testis-antler dysgenesis resulted from continuing exposure of pregnant females to an estrogenic environmental agent(s), thereby transforming testicular cells, affecting development of primordial antler pedicles, and blocking transabdominal descent of fetal testes. A browse (e.g., kelp) favored by deer in this locale might carry the putative estrogenic agent(s).

Development of biosensors for cancer clinical testing.

Biosensors are devices that combine a biochemical recognition/binding element (ligand) with a signal conversion unit (transducer). Biosensors are already used for several clinical applications, for example for electrochemical measurement of blood glucose concentrations. Application of biosensors in cancer clinical testing has several potential advantages over other clinical analysis methods including increased assay speed and flexibility, capability for multi-target analyses, automation, reduced costs of diagnostic testing and a potential to bring molecular diagnostic assays to community health care systems and to underserved populations. They have the potential for facilitating Point of Care Testing (POCT), where state-of-the-art molecular analysis is carried out without requiring a state-of-the-art laboratory. However, not many biosensors have been developed for cancer-related testing. One major challenge in harnessing the potential of biosensors is that cancer is a very complex set of diseases. Tumors vary widely in etiology and pathogenesis. Oncologists rely heavily on histological characterization of tumors and a few biomarkers that have demonstrated clinical utility to aid in patient management decisions. New genomic and proteomic molecular tools are being used to profile tumors and produce "molecular signatures." These signatures include genetic and epigenetic signatures, changes in gene expression, protein profiles and post-translational modifications of proteins. These molecular signatures provide new opportunities for utilizing biosensors. Biosensors have enormous potential to deliver the promise of new molecular diagnostic strategies to patients. This article describes some of the basic elements of cancer biology and cancer biomarkers relevant for the development of biosensors for cancer clinical testing, along with the challenges in using this approach.

Interlaboratory comparability study of cancer gene expression analysis using oligonucleotide microarrays.

A key step in bringing gene expression data into clinical practice is the conduct of large studies to confirm preliminary models. The performance of such confirmatory studies and the transition to clinical practice requires that microarray data from different laboratories are comparable and reproducible. We designed a study to assess the comparability of data from four laboratories that will conduct a larger microarray profiling confirmation project in lung adenocarcinomas. To test the feasibility of combining data across laboratories, frozen tumor tissues, cell line pellets, and purified RNA samples were analyzed at each of the four laboratories. Samples of each type and several subsamples from each tumor and each cell line were blinded before being distributed. The laboratories followed a common protocol for all steps of tissue processing, RNA extraction, and microarray analysis using Affymetrix Human Genome U133A arrays. High within-laboratory and between-laboratory correlations were observed on the purified RNA samples, the cell lines, and the frozen tumor tissues. Intraclass correlation within laboratories was only slightly stronger than between laboratories, and the intraclass correlation tended to be weakest for genes expressed at low levels and showing small variation. Finally, hierarchical cluster analysis revealed that the repeated samples clustered together regardless of the laboratory in which the experiments were done. The findings indicate that under properly controlled conditions it is feasible to perform complete tumor microarray analysis, from tissue processing to hybridization and scanning, at multiple independent laboratories for a single study.

Cancer diagnostics: decision criteria for marker utilization in the clinic.

A new diagnostic tool must pass three major tests before it is adopted for routine clinical use. First, the tool must be robust and reproducible; second, the clinical value of the tool must be proven, i.e. the tool should reliably trigger a clinical decision that results in patient benefit; and, third, the clinical community has to be convinced of the need for this tool and the benefits it affords. Another factor that can influence the adoption of new tools relates to the cost and the vagaries of insurance reimbursement. The Cancer Diagnosis Program (CDP) of the US National Cancer Institute (NCI) launched the Program for the Assessment of Clinical Cancer Tests (PACCT) in 2000 to develop a process for moving the results of new technologies and new understanding of cancer biology more efficiently and effectively into clinical practice. PACCT has developed an algorithm that incorporates the iterative nature of assay development into an evaluation process that includes developers and end users. The effective introduction of new tests into clinical practice has been hampered by a series of common problems that are best described using examples of successes and failures. The successful application of the PACCT algorithm is described in the discussion of the recent development of the OncotypeDX assay and plan for a prospective trial of this assay by the NCI-supported Clinical Trials Cooperative Groups. The assay uses reverse transcription (RT)-PCR evaluation of a set of 16 genes that were shown to strongly associate with the risk of recurrence of breast cancer in women who presented with early stage disease (hormone responsive, and no involvement of the auxiliary lymph nodes). The test is highly reproducible. It provides information to aid the physician and patient in making important clinical decisions, including the aggressiveness of the therapy that should be recommended. A trial is planned to test whether OncotypeDX can be used as a standalone trigger for specific treatment decisions. The problems that have been encountered and have delayed the development of other diagnostic tools are exemplified in the development of tests for human epidermal growth factor receptor (HER2) overexpression, for predictors of response to epidermal growth factor receptor inhibitors, and for the detection of residual disease following chemotherapy.

Rapid screening for 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator gene by Lminex xMAP suspension array.

A suspension array hybridization assay is described for the detection of 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using Luminex xMAP technology. The Luminex xMAP system allows simultaneous detection of up to 100 different targets in a single multiplexed reaction. Included in the method are the procedures for design of oligonucleotide capture probes and PCR amplification primers, coupling oligonucleotide capture probes to carboxylated microspheres, hybridization of coupled microspheres to oligonucleotide targets, production of targets from DNA samples by multiplexed PCR amplification, and detection of PCR-amplified targets by direct hybridization to probe-coupled microspheres. Mutation screening with the system is rapid, requires relatively few sample manipulations, and provides adequate resolution to reliably genotype the 25 CFTR mutations and 6 CFTR polymorphisms contained in the ACMG/ACOG/NIH-recommended core mutation panel for general population CF carrier screening.

Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system.

Escherichia coli, Salmonella, Listeria monocytogenes and Campylobacter jejuni are bacterial pathogens commonly implicated in foodborne illnesses. Generally used detection methods (i.e., culture, biochemical testing, ELISA and nucleic acid amplification) can be laborious, time-consuming and require multiple tests to detect all of the pathogens. Our objective was to develop rapid assays to simultaneously detect these four organisms through the presence of antigen or DNA using the Luminex LabMAP system. For nucleic acid detection, organism-specific capture probes corresponding to the 23S ribosomal RNA gene (rrl) were coupled covalently to LabMAP microspheres. Target molecules included synthetic complementary oligonucleotides and genomic DNA isolated from ATCC type strains or other well-characterized strains of each organism. Universal PCR primers were designed to amplify variable regions of bacterial 23S ribosomal DNA, yielding biotinylated amplicons of 86 to 109 bp in length. Varying quantities of targets were hybridized to the combined microsphere sets, labeled with streptavidin-R-phycoerythrin and analyzed on the Luminex(100) system. Results of nucleic acid detection assays, obtained in 30 to 40 min following amplification, correctly and specifically identified each bacterial species with a detection sensitivity of 10(3) to 10(5) genome copies. Capture-sandwich immunoassays were developed with organism-specific antibodies coupled to different microsphere sets. Microspheres were incubated with organism-specific standards and reactivity was assessed with biotinylated detection antibodies and streptavidin-R-phycoerythrin. In the immunoassays, microsphere-associated fluorescence was organism concentration dependent with detectable response at < or = 1000 organisms/ml and with no apparent cross-reactivity. We have demonstrated that the Luminex LabMAP system is a rapid, flexible platform capable of simultaneous, sensitive and specific detection of pathogens. The practical significance of this multiplexing approach would be to provide more timely, economical and comprehensive information than is available with conventional isolation and identification methodologies.

Multiple rapid automatic naming measures of cognition: normal performance and effects of aging.

Rapid automatic naming tasks are clinical tools for probing brain functions that underlie normal cognition. To compare performance for various stimuli in normal subjects and assess the effect of aging, we administered six single-dimension stimuli (color, form, number, letter, animal, and object) and five dual-dimension stimuli (color-form, color-number, color-letter, color-animal, and color-object) to 144 normal volunteers who ranged in age from 15 to 85 years. Rapid automatic naming times for letters and numbers were significantly less than for forms, animals, and objects. Rapid automatic naming times for color-number and color-letter stimuli were significantly less than for color-form, color-animal, or color-object stimuli. Age correlated significantly with rapid automatic naming time for each single-dimension stimulus and for color-form, color-number, color-animal, and color-object stimuli. Linear regression showed that rapid automatic naming times increased with age for aggregated color stimuli, aggregated single-dimension stimuli, and aggregated dual-dimension stimuli. This age effect persisted in subgroups less than 60 years of age and greater than 60 years of age. We conclude that normal performance time is dependent on the task, with letter and number stimuli eliciting most rapid responses, and that most rapid automatic naming times increase with age.