A novel microchip-based imaged CIEF-MS system for comprehensive characterization and identification of biopharmaceutical charge variants.

A microfluidic system has been designed that integrates both imaged capillary isoelectric focusing (iCIEF) separations and downstream MS detection into a single assay. Along with the construction of novel instrumentation and an innovative microfluidic chip, conversion to MS-compatible separation reagents has also been established. Incorporation of 280 nm absorbance iCIEF-MS analysis not only permits photometric quantitation of separated charge isoforms but also facilitates the direct monitoring of analyte focusing and mobilization in real-time. The outcome of this effort is a device with the unique ability to allow for both the characterization and identification of protein charge and mass isoforms in under 15 min. Acquisition, quantitation, and identification of highly resolved intact mAb charge isoforms along with their critical N-linked glycan pairs clearly demonstrate analytical utility of our innovative system. In total, 33 separate molecular features were characterized by the iCIEF-MS system representing a dramatic increase in the ability to monitor multiple intact mAb critical quality attributes in a single comprehensive assay. Unlike previously reported CIEF-MS results, relatively high ampholyte concentrations, of up to 4% v/v, were employed without impacting MS sensitivity, observed to be on the order of 1% composition.

Developmental validation of a fully integrated sample-to-profile rapid human identification system for processing single-source reference buccal samples.

UNLABELLED: Short tandem repeat (STR) DNA typing is a global standard for human identification. Current practice involves highly trained forensic analysts, operating in a laboratory setting, using multiple instruments to process samples and analyze the data. Here, we report the developmental validation of a fully integrated and automated DNA profiling system, the RapidHIT® System, capable of producing up to five high quality STR profiles with full controls in approximately 90min using PowerPlex®16 HS RapidHIT chemistry. The system integrates all sample handling steps: starting from lysis of cells on buccal swabs or other buccal sample types through DNA extraction, normalization, amplification,capillary array electrophoresis, detection, and integrated software analysis. The results describe the developmental validation of the RapidHIT™ System for buccal samples processed with the DNA IQ™ extraction chemistry using a guandinium chaotropic agent and paramagnetic beads followed by amplification using a modified version of PowerPlex 16 HS chemistry (PowerPlex 16 HS RapidHIT chemistry), and capillary electrophoresis with manual review of genotyping data following interpretation guidelines. All processing from the buccal swab to generation and processing of the profile occurs on the RapidHIT platform. RESULT: are concordant with traditional methods, with 88% first pass success rates for both the CODIS and PowerPlex 16 loci. Average peak height ratios were 0.89 for buccal swabs. The system produces full profiles from swabs with at least 176 ng of saliva DNA. Rapid DNA identification systems will significantly enhance capabilities for forensic labs, intelligence, defense, law enforcement, refugee and immigration applications, and kinship analysis.

Prevalence of diabetes and associated obesity in Pennsylvania adults, 1995-2010.

INTRODUCTION: This study examined trends in the prevalence and sociodemographic distributions of diabetes and the associations of diabetes with obesity over time in adult Pennsylvanians from 1995 through 2010. METHODS: We used Behavioral Risk Factor Surveillance Survey data collected from 1995 through 2010. Diabetes prevalence was assessed by self-report of physician diagnosis. Obesity was assessed by body mass index computed from self-report of height and weight. State-level data for diabetes and associated obesity prevalence from 1995 through 2010 were collected for each year. Data on sociodemographic factors (age, sex, race, income, education) and 1 known disease risk factor (obesity) were also collected. Logistic regression modeling was used to examine associations between diabetes, sociodemographic factors, and obesity. RESULTS: Diabetes prevalence in Pennsylvania, which increased from 5.6% in 1995 to 10.5% in 2010, followed national trends but exceeded the national prevalence each year by approximately 0.6 percentage points for 12 of the 16 years. The increase in prevalence was not equal across all socioeconomic groups. Obesity became a more dominant risk factor for diabetes during these 16 years. CONCLUSION: The burden of diabetes and obesity in Pennsylvania is substantial and increasing. Program managers and policy makers in Pennsylvania should consider these trends when allocating limited resources and designing programs for reducing diabetes-related illness. Other states may consider similar studies to monitor the prevalence of diabetes and determine whether disparities are changing and whether programs and resources should also shift.

A flexible microfluidic processor for molecular biology: application to microarray sample preparation.

We describe a programmable microfluidic system with onboard pumps and valves that has the ability to process reaction volumes in the sub-microlitre to hundred microlitre range. The flexibility of the architecture is demonstrated with a commercial molecular biology protocol for mRNA amplification, implemented without significant modification. The performance of the microchip system is compared to conventional bench processing at each stage of the multistep protocol, and DNA microarrays are used to assess the quality and performance of bench- and microchip-amplified RNA. The results show that the microchip system reactions are similar to bench control reactions at each step, and that the microchip- and bench-derived amplified RNAs are virtually indistinguishable in differential microarray analyses.

Factors associated with difficulty achieving initial control with crotalidae polyvalent immune fab antivenom in snakebite patients.

BACKGROUND: The prescribing information for Crotalidae Fab antivenom (FabAV) instructs clinicians to administer FabAV until initial control of the envenomation syndrome is achieved. Risk factors for difficulty achieving initial control are not known. OBJECTIVES: The study aim was to identify factors present before administration of antivenom associated with difficulty achieving initial control. METHODS: The authors conducted a retrospective study of all patients presenting to any one of 17 centers and receiving FabAV from 2002 to 2004. Demographic and historical information, as well as data about nine specific venom effects, were collected prior to the administration of antivenom. An expert panel used standard criteria to determine if initial control was achieved. The patient group that had difficulty achieving initial control was compared to the group that achieved initial control, and adjusted odds ratios were calculated using stepwise logistic regression. RESULTS: A total of 247 patients were included in the final analysis. The majority of patients were envenomated on the upper extremity and were young males. A total of 203 patients (82.2%) achieved initial control. In univariate analysis, thrombocytopenia, bleeding, neurologic effects, and a severe bite were significantly associated with difficulty achieving initial control. After logistic regression, the presence of neurologic effects and thrombocytopenia remained significantly associated with difficulty achieving initial control. When both factors were present, the patient was 13.8 times more likely to have difficulty achieving initial control. CONCLUSIONS: A number of factors were present before the administration of FabAV that were independently associated with difficulty achieving initial control of the envenomation syndrome. Predicting which patients will have difficulty achieving initial control has important ramifications for patient disposition and may provide insight into the mechanisms for lack of antivenom efficacy.

A multicenter comparison of the safety of oral versus intravenous acetylcysteine for treatment of acetaminophen overdose.

UNLABELLED: Oral and intravenous (IV) N-acetylcysteine (NAC) are used for the treatment of acetaminophen poisoning. The objective of this multicenter study was to compare the safety of these two routes of administration. METHODS: We conducted a multicenter chart review of all patients treated with NAC for acetaminophen poisoning. The primary safety outcome was the percentage of patients with NAC-related adverse events. RESULTS: A total of 503 subjects were included in the safety analysis (306 IV-only, 145 oral-only, and 52 both routes). There were no serious adverse events related to NAC for either route. Nausea and vomiting were the most common related adverse events and were more common with oral treatment (23 vs. 9%). Anaphylactoid reactions were more common with IV administration (6 vs. 2%). CONCLUSIONS: IV and oral NAC are generally mild adverse drug reactions.

Health and safety issues relating to Maine's fishing industry.

The Maine Agricultural Safety and Health Program performed a needs assessment to determine the health and safety concerns of Maine's fishing community. Information for the assessment was obtained from clinicians, focus groups of wives of fishers, and government agencies. Reports from the United States Coast Guard showed that during 1993-1994, sixty-five people were injured on commercial fishing vessels and eight people died. Decompression illness was the most frequently reported non-fatal injury (n = 15). Chronic injuries reported by clinicians included back pain, tendinitis, carpal tunnel syndrome, dysbaric osteonecrosis. Clinicians and family members were also concerned about hand and arm infections from the bait and sun exposure. Family health issues and access to care are addressed in the report. Recommendations include, in part, increasing surveillance, reducing barriers to access, increasing clinicians' knowledge about the fishing industry, and fostering collaboration between agencies providing health and safety information to fishers.