Association Between Local Boards of Health Authority Over Budgets and PHAB Accreditation Standard Score.

Introduction: This study examined the relationship between local board of health authority and local health departments' budget-related activities and performance scores in the Public Health Accreditation Board standards while considering the governance structure under which the local health agencies operate. Methods: Data from 250 local health departments were obtained from the Public Health Accreditation Board and were combined with data from the 2016 National Association of County and City Officials Profile Survey. Multilevel regression analysis was used to examine the relationship between local board of health authority on local health departments' budget-related activities, using the governance structure as the group-level variable. Results: Analyses identified positive associations between local board of health authority on local health departments' budget-related activities and local health departments' aggregate average performance scores in Public Health Accreditation Board accreditation. No apparent association was found between the type of governance structure under which a local health department operates and performance scores in Public Health Accreditation Board accreditation standards, perhaps attributable to variation in the characteristics and roles of their governing bodies. Conclusions: The analyses suggest that local boards of health with authority related to local health departments' budgets appear to have an influential role in budget-related activities and may improve local health departments' performance scores in Public Health Accreditation Board accreditation standards. However, vast variations in more specific local boards of health roles and characteristics exist across local health departments and for which there are no national data. More research is thus needed to control for or examine the influences of specific local boards of health characteristics before the benefits of expanded local boards of health authority over local health departments' budgetary decision making on local health departments' performance can be fully understood.

Associations Between Local Health Department Expenditures on Foundational Capabilities and PHAB Accreditation Standards Scores.

Context: Foundational Capabilities (FC) are the public health (PH) infrastructure areas that are essential for local health departments (LHDs) to support a "minimum package" of programs and services that promote population health. Despite being a critical component of LHD programs, FC are chronically underfunded, and studies specific to the relationship between LHD FC expenditures and their performance-the LHDs' ability to provide essential PH programs and services to their community-have not been previously reported. Public Health Accreditation Board (PHAB) accreditation is a nationally recognized accreditation program for PH agencies. PHAB accreditation assesses LHDs' performance against sets of standards that are based on the 10 essential PH services. Alignment between FC and the PHAB standards presents a means for assessing LHD FC expenditures relative to their performance in PHAB accreditation standards. Objectives: We examined the association between LHD total FC expenditures, as well as FC funding allocation patterns, and performance score on selected PHAB accreditation standards. Methods: We used Bayesian regression methods to estimate the coefficients for the aggregate performance score, and performance scores on individual PHAB standards. Results: Analyses showed that a dollar increase in total FC expenditures is associated with a 0.2% increase in the aggregate performance score in selected PHAB standards as well as the performance score on most of the standards examined. LHDs that allocated FC budgets more evenly across FC programs were found to be more likely to have higher scores. Conclusions: Investment in FC could improve LHD performance scores in PHAB accreditation standards and support LHDs' capability for improving community health outcomes. Allocating available FC resources across the various FC programs could support better LHD performance, as indicated by accreditation scores. This study contributes to advancing the understanding of public health finances in relation to performance and could help guide effective LHD resource allocation.

Laser-induced fluorescence detector with a fiber-coupled micro GRIN lens for capillary electrophoresis.

Capillary electrophoresis coupled with sheath-flow laser-induced fluorescence (LIF) detection has been shown to offer outstanding sensitivity for chemical and biochemical analysis. However, a major drawback remains with the complexity of the optical configuration traditionally employed. Here we present a simplified confocal optics based on fiber optics and micro gradient-index (GRIN) lenses for modular optical design in capillary electrophoresis with laser-induced fluorescence. We demonstrate the use of the optical system with a sheath-flow cuvette as the laser-induced fluorescence detector for capillary electrophoresis. The system's performance was established with concentration detection limits of 8±2pM and mass detection limits of 57 zeptomole for a standard sodium fluorescein sample.

High-Resolution Capillary Zone Electrophoresis with Mass Spectrometry Peptide Mapping of Therapeutic Proteins: Peptide Recovery and Post-translational Modification Analysis in Monoclonal Antibodies and Antibody-Drug Conjugates.

Reversed phase liquid chromatography with mass spectrometry (RPLC-MS) peptide mapping is routinely used for interrogating molecular and structural attributes such as amino acid composition, sequence variants, and post-translational modifications (PTMs) in antibody-derived therapeutics. RPLC has some limitations that often impact the analysis of certain peptides including large hydrophobic peptides, hydrophilic di-/tripeptides and glycopeptides. Capillary zone electrophoresis with mass spectrometry (CZE-MS) has great potential for peptide mapping due to high efficiency and outstanding sensitivity. In this report we demonstrate the utility of CZE-MS as an orthogonal and complementary technique to RPLC-MS for peptide mapping analyses of antibody-drug conjugates (ADCs) and their parent antibodies. This work is based on high-resolution CZE-MS separation recently developed in our group, where a mixed aqueous-organic solvent system containing N,N-dimethylacetamide (DMA) or N,N-dimethylformamide (DMF) was used to improve the separation selectivity. The results described here show several advantages of CZE-MS for the analysis of small hydrophilic di-/tripeptides, large hydrophobic peptides, glycopeptides, and hydrophobic drug-linked peptides.

High-Resolution Capillary Zone Electrophoresis with Mass Spectrometry Peptide Mapping of Therapeutic Proteins: Improved Separation with Mixed Aqueous-Aprotic Dipolar Solvents (N,N-Dimethylacetamide and N,N-Dimethylformamide) as the Background Electrolyte.

Peptide mapping with mass spectrometry (MS) detection is a powerful technique routinely used for interrogating physicochemical properties of proteins. Peptide mapping benefits from an efficient front-end separation to increase selectivity and reduce complexity prior to MS detection. The most commonly used method for peptide mapping is based on reverse phase liquid chromatography with mass spectrometry. Capillary zone electrophoresis with mass spectrometry (CZE-MS) is an orthogonal technique with growing attention for peptide mapping of biotherapeutic proteins due to its high efficiency and sensitivity. However, that growth has been slow due to poorer peptide resolution and method robustness compared to RPLC. Here we present results from optimization of CZE-MS peptide mapping separation using mixed aqueous-aprotic dipolar solvent (N,N-dimethylacetamide (DMA) and N,N-dimethylformamide (DMF), as the background electrolyte (BGE) to improve the separation performance. Addition of DMA or DMF to the BGE impacts separation selectivity through differential change in pKa of the peptides. The CZE-MS peptide mapping method with the modified BGE produced significant improvement in resolution over the conventional CZE-MS methods. The method was evaluated with both sheathless and sheathflow CE-MS ion sources.

Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1 monoclonal antibodies.

Fragmentation of monoclonal antibodies is a critical quality attribute routinely monitored to assess the purity and integrity of the product from development to commercialization. Cleavage in the upper hinge region of IgG1 monoclonal antibodies is a common fragmentation pattern widely studied by size exclusion chromatography (SEC). Capillary electrophoresis with sodium dodecylsulfate (CE-SDS) is a well-established technique commonly used for monitoring antibody fragments as well, but its comparability to SEC in monitoring hinge fragments has not been established until now. We report a characterization strategy that establishes the correlation between hinge region fragments analyzed by SEC and CE-SDS. Monoclonal antibodies with elevated hinge fragments were generated under low pH stress conditions and analyzed by SEC and CE-SDS. The masses of the fragments generated were determined by LC-MS. Electrophoretic migration of the hinge fragmentation products in CE-SDS were determined based on their mass values. Comparative assessment of fragments by SEC, and CE-SDS showed similar correlation with incubation time. This study demonstrates that CE-SDS can be employed as a surrogate technique to SEC for monitoring hinge region fragments. Most importantly, combination of these techniques can be used to obtain comprehensive understanding of fragment related characteristics of therapeutic protein products.

Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation.

Characterization of both the acidic and basic regions of imaged capillary isoelectric focusing (icIEF) profile of an IgG1 antibody was achieved through preparative immobilized pH gradient isoelectric focusing (IPG-IEF) fractionation. Recent attempts at using this method to fractionate charge variants of monoclonal antibodies (mAbs) have shown promising results, but identification of the chemical modifications in the variants was limited to the basic species. We have optimized the method to achieve enrichment of each variant across the icIEF profile of an IgG1 mAb. The fractionation was followed by extended characterization to elucidate the composition of the acidic, main, and basic species observed in the icIEF profile. Deamidation, sialylation, glycation, and fragmentation were identified as the main modifications contributing to acidic variants of the mAb while C-terminal lysine, C-terminal proline amidation, and uncyclized N-terminal glutamine were the major species contributing to the basic variants. This characterization allows a better understanding of the modifications that contribute to the charge variants observed by icIEF, facilitating the evaluation of impacts on product safety and efficacy.

Capillary electrophoresis coupled with automated fraction collection.

A fraction collector based on a drop-on-demand ink-jet printer was developed to interface capillary zone electrophoresis with a 96 well microtiter plate. We first evaluated the performance of the collector by using capillary zone electrophoresis to analyze a 1mM solution of tetramethylrhodamine; a fluorescent microtiter plate reader was then used to detect the analyte and characterize fraction carryover between wells. Relative standard deviation in peak height was 20% and the relative standard deviation in migration time was 1%. The mean and standard deviation of the tetramethylrhodamine peak width was 5 ± 1 s and likely limited by the 4-s period between droplet deposition. We next injected a complex mixture of DNA fragments and used real-time PCR to quantify the product in a CE-SELEX experiment. The reconstructed electrophoretic peak was 27 s in duration. Finally, we repeated the experiment in the presence of a 30-µM thrombin solution under CE-SELEX conditions; fractions were collected and next-generation sequencing was used to characterize the DNA binders. Over 25,000 sequences were identified with close matches to known thrombin binding aptamers.

Ultrasensitive online SERS detection of structural isomers separated by capillary zone electrophoresis.

A mixture of structural isomers was separated and identified at nanomolar concentrations (∼100,000 molecules) by incorporating capillary zone electrophoresis (CZE) with a sheath flow surface-enhanced Raman scattering (SERS) detector. Baseline resolution was obtained from three structural isomers of rhodamine using a planar silver SERS substrate, demonstrating the utility of this approach for trace chemical analysis.

Ultrasensitive surface-enhanced Raman scattering flow detector using hydrodynamic focusing.

Label-free, chemical specific detection in flow is important for high throughput characterization of analytes in applications such as flow injection analysis, electrophoresis, and chromatography. We have developed a surface-enhanced Raman scattering (SERS) flow detector capable of ultrasensitive optical detection on the millisecond time scale. The device employs hydrodynamic focusing to improve SERS detection in a flow channel where a sheath flow confines analyte molecules eluted from a fused silica capillary over a planar SERS-active substrate. Increased analyte interactions with the SERS substrate significantly improve detection sensitivity. The performance of this flow detector was investigated using a combination of finite element simulations, fluorescence imaging, and Raman experiments. Computational fluid dynamics based on finite element analysis was used to optimize the flow conditions. The modeling indicates that a number of factors, such as the capillary dimensions and the ratio of the sheath flow to analyte flow rates, are critical for obtaining optimal results. Sample confinement resulting from the flow dynamics was confirmed using wide-field fluorescence imaging of rhodamine 6G (R6G). Raman experiments at different sheath flow rates showed increased sensitivity compared with the modeling predictions, suggesting increased adsorption. Using a 50 ms acquisition, a sheath flow rate of 180 µL/min, and a sample flow rate of 5 µL/min, a linear dynamic range from nanomolar to micromolar concentrations of R6G with a limit of detection (LOD) of 1 nM is observed. At low analyte concentrations, rapid analyte desorption is observed, enabling repeated and high-throughput SERS detection. The flow detector offers substantial advantages over conventional SERS-based assays such as minimal sample volumes and high detection efficiency.

Nicked-sleeve interface for two-dimensional capillary electrophoresis.

We report an improved interface for two-dimensional capillary electrophoresis. This interface is based on capillary tubing and a Plexiglas chip, both of which were milled using a micro-dicing saw. The interface was evaluated and compared to a traditional interface design for both pseudo one-dimensional and two-dimensional capillary electrophoresis. We observe less than 70% transfer efficiency for the traditional design and greater than 90% transfer efficiency with this new interface.

Simplified sheath flow cuvette design for ultrasensitive laser induced fluorescence detection in capillary electrophoresis.

We present a design for a sheath-flow cuvette that uses a relatively inexpensive quartz cuvette. The cuvette has a high optical quality square flow chamber that is fused to quartz tubes at each end. PEEK/TEFZEL fittings hold and seal the quartz flow chamber without putting strain on the cuvette. The performance of the cuvette is evaluated as a laser-induced fluorescence detector for capillary electrophoresis. The cuvette produces mass detection limits of 50 yoctomoles (30 copies) for 5-carboxyl tetramethylrhodamine (5 TAMRA SE) with a separation efficiency of 400,000 theoretical plates.

Thermal diffusivity imaging with the thermal lens microscope.

A coaxial thermal lens microscope was used to generate images based on both the absorbance and thermal diffusivity of histological samples. A pump beam was modulated at frequencies ranging from 50 kHz to 5 MHz using an acousto-optic modulator. The pump and a CW probe beam were combined with a dichroic mirror, directed into an inverted microscope, and focused onto the specimen. The change in the transmitted probe beam's center intensity was detected with a photodiode. The photodiode's signal and a reference signal from the modulator were sent to a high-speed lock-in amplifier. The in-phase and quadrature signals were recorded as a sample was translated through the focused beams and used to generate images based on the amplitude and phase of the lock-in amplifier's signal. The amplitude is related to the absorbance and the phase is related to the thermal diffusivity of the sample. Thin sections of stained liver and bone tissues were imaged; the contrast and signal-to-noise ratio of the phase image was highest at frequencies from 0.1-1 MHz and dropped at higher frequencies. The spatial resolution was 2.5 µm for both amplitude and phase images, limited by the pump beam spot size.

Capillary isoelectric focusing with pH 9.7 cathode for the analysis of gastric biopsies.

Capillary isoelectric focusing tends to suffer from poor reproducibility, particularly for the analysis of complex protein samples from cellular or tissue homogenates. This poor reproducibility appears to be associated with erratic variations in electroosmotic flow. One cause of electroosmotic flow variation is degradation of the capillary coating caused by the extremely basic solution commonly used during mobilization and focusing; this degradation of the capillary coating can be reduced by employing a CAPS mobilization buffer at pH 9. Another cause of variation is protein adsorption to the capillary wall, which causes an increase in electroosmotic flow. The effects of protein adsorption can be reduced by use of surfactants in the buffer and by employing an extremely low sample loading. We report the use of CAPS mobilization buffer in combination with an ultrasensitive laser-induced fluorescence detector for the reproducible analysis of ∼2 ng of protein from a Barrett's esophagus biopsy.

Nine orders of magnitude dynamic range: picomolar to millimolar concentration measurement in capillary electrophoresis with laser induced fluorescence detection employing cascaded avalanche photodiode photon counters.

The dynamic range of capillary electrophoresis analysis is ultimately limited by molecular shot noise at low concentrations and by concentration-induced band broadening at high concentrations. We report a system that approaches these fundamental limits. A laser-induced fluorescence detector is reported that employs a cascade of four fiber-optic beam splitters connected in series to generate a primary signal and four attenuated signals, each monitored by a single-photon counting avalanche photodiode. Appropriate scaling of the signals from the five photodiodes produces a linear optical calibration curve for 5-carboxyl-tetramethylrhodamine from the concentration detection limit of 1 pM to the upper limit of 1 mM. Mass detection limits are 120 yoctomoles (70 molecules) injected into the instrument. The very-wide dynamic range instrument was used to study the metabolic products of the fluorescently labeled glycosphingolipid tetramethylrhodamine labeled GM1 (GM1-TMR) produced by single cells isolated from the rat cerebellum.

Simplified capillary electrophoresis nanospray sheath-flow interface for high efficiency and sensitive peptide analysis.

We report a simple nanospray sheath-flow interface for capillary electrophoresis. This interface relies on electrokinetic flow to drive both the separation and the electrospray; no mechanical pump is used for the sheath flow. This system was interfaced with an LCQ mass spectrometer. The best results were observed with a 2-microm diameter emitter tip and a 1-mm spacing between the separation capillary tip and the emitter tip. Under these conditions, mass detection limits (3sigma) of 100 amol were obtained for insulin receptor fragment 1142-1153. The separation efficiency exceeded 200,000 plates for this compound. The relative standard deviation generated during continual infusion of a 50 microM solution of angiotensin II was 2% for the total ion count and 3% for the extracted ion count over a 40-min period. Finally, the interface was also demonstrated for negative ion mode.

Two-dimensional capillary electrophoresis: capillary isoelectric focusing and capillary zone electrophoresis with laser-induced fluorescence detection.

CIEF and CZE are coupled with LIF detection to create an ultrasensitive 2-D separation method for proteins. In this method, two capillaries are joined through a buffer-filled interface. Separate power supplies control the potential at the injection end of the first capillary and at the interface; the detector is held at ground potential. Proteins are labeled with the fluorogenic reagent Chromeo P503, which preserves the isoelectric point of the labeled protein. The labeled proteins were mixed with ampholytes and injected into the first-dimension capillary. A focusing step was performed with the injection end of the capillary at high pH and the interface at low pH. To mobilize components, the interface was filled with a high pH buffer, which was compatible with the second-dimension separation. A fraction was transferred to the second-dimension capillary for separation. The process of fraction transfer and second dimension separation was repeated two dozen times. The separation produced a spot capacity of 125.

Capillary array isoelectric focusing with laser-induced fluorescence detection: milli-pH unit resolution and yoctomole mass detection limits in a 32-channel system.

We report a multiplexed capillary electrophoresis system employing an array of 32 capillaries with a micromachined sheath-flow cuvette as the detection chamber. The sample streams were simultaneously excited with a 473-nm laser beam, and the fluorescence emission was imaged on a CCD camera with a pair of doublet achromat lens. The instrument produced mass detection limits of 380 +/- 120 yoctomoles for fluorescein in zone electrophoresis. Capillary isoelectric focusing of fluorescent standards produced peaks with an average width of 0.0029 +/- 0.0008 pH. Capillary coating stability limits the reproducibility of the analysis.