High-throughput immunoassay for the biochemical diagnosis of Friedreich ataxia in dried blood spots and whole blood.

BACKGROUND: Friedreich ataxia (FRDA) is caused by reduced frataxin (FXN) concentrations. A clinical diagnosis is typically confirmed by DNA-based assays for GAA-repeat expansions or mutations in the FXN (frataxin) gene; however, these assays are not applicable to therapeutic monitoring and population screening. To facilitate the diagnosis and monitoring of FRDA patients, we developed an immunoassay for measuring FXN. METHODS: Antibody pairs were used to capture FXN and an internal control protein, ceruloplasmin (CP), in 15 µL of whole blood (WB) or one 3-mm punch of a dried blood spot (DBS). Samples were assayed on a Luminex LX200 analyzer and validated according to standard criteria. RESULTS: The mean recovery of FXN from WB and DBS samples was 99%. Intraassay and interassay imprecision (CV) values were 4.9%-13% and 9.8%-16%, respectively. The FXN limit of detection was 0.07 ng/mL, and the reportable range of concentrations was 2-200 ng/mL. Reference adult and pediatric FXN concentrations ranged from 15 to 82 ng/mL (median, 33 ng/mL) for DBS and WB. The FXN concentration range was 12-22 ng/mL (median, 15 ng/mL) for FRDA carriers and 1-26 ng/mL (median 5 ng/mL) for FRDA patients. Measurement of the FXN/CP ratio increased the ability to distinguish between patients, carriers, and the reference population. CONCLUSIONS: This assay is applicable to the diagnosis and therapeutic monitoring of FRDA. This assay can measure FXN and the control protein CP in both WB and DBS specimens with minimal sample requirements, creating the potential for high-throughput population screening of FRDA.

A Comparative Effectiveness Study of Newborn Screening Methods for Four Lysosomal Storage Disorders.

Newborn screening for one or more lysosomal disorders has been implemented in several US states, Japan and Taiwan by multiplexed enzyme assays using either tandem mass spectrometry or digital microfluidics. Another multiplex assay making use of immunocapture technology has also been proposed. To investigate the potential variability in performance of these analytical approaches, we implemented three high-throughput screening assays for the simultaneous screening for four lysosomal disorders: Fabry disease, Gaucher disease, mucopolysaccharidosis type I, and Pompe disease. These assays were tested in a prospective comparative effectiveness study using nearly 100,000 residual newborn dried blood spot specimens. In addition, 2nd tier enzyme assays and confirmatory molecular genetic testing were employed. Post-analytical interpretive tools were created using the software Collaborative Laboratory Integrated Reports (CLIR) to determine its ability to improve the performance of each assay vs. the traditional result interpretation based on analyte-specific reference ranges and cutoffs. This study showed that all three platforms have high sensitivity, and the application of CLIR tools markedly improves the performance of each platform while reducing the need for 2nd tier testing by 66% to 95%. Moreover, the addition of disease-specific biochemical 2nd tier tests ensures the lowest false positive rates and the highest positive predictive values for any platform.

Influences of wood preservation, lumber size, and weather on field leaching of red pine lumber.

Alkaline copper quaternary (ACQ) is a widely used wood preservative. This study evaluated leachate volume generation and contaminant leaching from ACQ-treated lumber during rainfall events in comparison to untreated lumber. The influences of wood preservation with ACQ, lumber size, and weather on leachate generation ratio and contaminant concentrations in wood leachate were investigated with four red pine lumber piles exposed to natural weather conditions. The average volumetric ratio of leachate to rainfall was significantly higher for the large-lumber piles (0.62) compared with the small-lumber piles (0.35). Less leachate was generated in the ACQ-treated lumber piles (0.42) than the untreated lumber piles (0.55). Leachate volume could be predicted with rainfall depth, air temperature, and wetted lumber surface area. Lumber size did not make a statistically significant difference in leachate quality except for zinc concentration. The average copper concentrations were 4034 µg/L in the leachate from the ACQ-treated lumber piles and 87 µg/L in the leachate from the untreated lumber piles. Moreover, ACQ treatment significantly increased leaching of arsenic and total dissolved solids. Copper concentration in leachate from ACQ-treated lumber can be predicted with rainfall intensity, the time interval between two consecutive leachate-generating events, rain copper concentration, and rain pH.

Modeling of air pollutant removal by dry deposition to urban trees using a WRF/CMAQ/i-Tree Eco coupled system.

A distributed adaptation of i-Tree Eco was used to simulate dry deposition in an urban area. This investigation focused on the effects of varying temperature, LAI, and NO2 concentration inputs on estimated NO2 dry deposition to trees in Baltimore, MD. A coupled modeling system is described, wherein WRF provided temperature and LAI fields, and CMAQ provided NO2 concentrations. A base case simulation was conducted using built-in distributed i-Tree Eco tools, and simulations using different inputs were compared against this base case. Differences in land cover classification and tree cover between the distributed i-Tree Eco and WRF resulted in changes in estimated LAI, which in turn resulted in variations in simulated NO2 dry deposition. Estimated NO2 removal decreased when CMAQ-derived concentration was applied to the distributed i-Tree Eco simulation. Discrepancies in temperature inputs did little to affect estimates of NO2 removal by dry deposition to trees in Baltimore.

Development of a distributed air pollutant dry deposition modeling framework.

A distributed air pollutant dry deposition modeling system was developed with a geographic information system (GIS) to enhance the functionality of i-Tree Eco (i-Tree, 2011). With the developed system, temperature, leaf area index (LAI) and air pollutant concentration in a spatially distributed form can be estimated, and based on these and other input variables, dry deposition of carbon monoxide (CO), nitrogen dioxide (NO(2)), sulfur dioxide (SO(2)), and particulate matter less than 10 microns (PM10) to trees can be spatially quantified. Employing nationally available road network, traffic volume, air pollutant emission/measurement and meteorological data, the developed system provides a framework for the U.S. city managers to identify spatial patterns of urban forest and locate potential areas for future urban forest planting and protection to improve air quality. To exhibit the usability of the framework, a case study was performed for July and August of 2005 in Baltimore, MD.

Retrospective determination of ceruloplasmin in newborn screening blood spots of patients with Wilson disease.

Wilson disease is an autosomal recessive disorder of copper transport, caused by the reduced or absent function of the Wilson disease gene ATP7B on chromosome 13. The disease is characterized by reduced incorporation of copper into the ceruloplasmin protein and reduced excretion of copper into the bile. Wilson disease is effectively treated if detected early. Our study goals were to determine the feasibility of a population screening for Wilson disease using dried blood spots and to characterize the base-line ceruloplasmin concentration in newborn blood spots of patients with Wilson disease. Ceruloplasmin was analyzed in dried blood spots obtained from 353 Mayo Clinic pediatric volunteers aged from 3 months to 18 years and from 1045 anonymous newborn screening specimens using a sandwich enzyme-linked immunosorbent assay. The original newborn screening blood spots were retrieved from two patients with Wilson disease along with age-matched controls for ceruloplasmin determination. The mean (+/-SD) concentration of ceruloplasmin in the pediatric blood spots was 40.0+/-14.4 mg/dL (range 13.1 to >60 mg/dL) and newborn blood spots was 47.2+/-15.5mg/dL (range 6.5 to >60 mg/dL). Ceruloplasmin in the newborn blood spots from two Wilson disease patients were 2.6 and 2.8 mg/dL, respectively. The newborns affected with Wilson disease had significantly lower ceruloplasmin levels in blood spots than unaffected newborns. These findings support that presymptomatic screening for Wilson disease using dried blood spots could be possible, even in the newborn period.