Faculty Bibliography

OBJECTIVE: Interobserver agreement in the context of oral epithelial dysplasia (OED) grading has been notoriously unreliable and can impose barriers for developing new molecular markers and diagnostic technologies. This paper aimed to report the details of a 3-stage histopathology review and adjudication process with the goal of achieving a consensus histopathologic diagnosis of each biopsy. STUDY DESIGN: Two adjacent serial histologic sections of oral lesions from 846 patients were independently scored by 2 different pathologists from a pool of 4. In instances where the original 2 pathologists disagreed, a third, independent adjudicating pathologist conducted a review of both sections. If a majority agreement was not achieved, the third stage involved a face-to-face consensus review. RESULTS: Individual pathologist pair kappa values ranged from 0.251 to 0.706 (fair-good) before the 3-stage review process. During the initial review phase, the 2 pathologists agreed on a diagnosis for 69.9% of the cases. After the adjudication review by a third pathologist, an additional 22.8% of cases were given a consensus diagnosis (agreement of 2 out of 3 pathologists). After the face-to-face review, the remaining 7.3% of cases had a consensus diagnosis. CONCLUSIONS: The use of the defined protocol resulted in a substantial increase (30%) in diagnostic agreement and has the potential to improve the level of agreement for establishing gold standards for studies based on histopathologic diagnosis.

Measuring low concentrations of clinically-important biomarkers using porous bead-based lab-on-a-chip (LOC) platforms is critical for the successful implementation of point-of-care (POC) devices. One way to meet this objective is to optimize the geometry of the bead holder, referred to here as a micro-container. In this work, two geometric micro-containers were explored, the inverted pyramid frustum (PF) and the inverted clipped pyramid frustum (CPF). Finite element models of this bead array assay system were developed to optimize the micro-container and bead geometries for increased pressure, to increase analyte capture in porous bead-based fluorescence immunoassays. Custom micro-milled micro-container structures containing an inverted CPF geometry resulted in a 28% reduction in flow-through regions from traditional anisotropically-etched pyramidal geometry derived from Si-111 termination layers. This novel "reduced flow-through" design resulted in a 33% increase in analyte penetration into the bead and twofold increase in fluorescence signal intensity as demonstrated with C-Reactive Protein (CRP) antigen, an important biomarker of inflammation. A consequent twofold decrease in the limit of detection (LOD) and the limit of quantification (LOQ) of a proof-of-concept assay for the free isoform of Prostate-Specific Antigen (free PSA), an important biomarker for prostate cancer detection, is also presented. Furthermore, a 53% decrease in the bead diameter is shown to result in a 160% increase in pressure and 2.5-fold increase in signal, as estimated by COMSOL models and confirmed experimentally by epi-fluorescence microscopy. Such optimizations of the bead micro-container and bead geometries have the potential to significantly reduce the LODs and reagent costs for spatially programmed bead-based assay systems of this type.

OBJECTIVE: There is currently a gap in on-site drug of abuse monitoring. Current detection methods involve invasive sampling of blood and urine specimens, or collection of oral fluid, followed by qualitative screening tests using immunochromatographic cartridges. While remote laboratories then may provide confirmation and quantitative assessment of a presumptive positive, this instrumentation is expensive and decoupled from the initial sampling making the current drug-screening program inefficient and costly. The authors applied a noninvasive oral fluid sampling approach integrated with the in-development chip-based Programmable bio-nano-chip (p-BNC) platform for the detection of drugs of abuse. METHOD: The p-BNC assay methodology was applied for the detection of tetrahydrocannabinol, morphine, amphetamine, methamphetamine, cocaine, methadone and benzodiazepines, initially using spiked buffered samples and, ultimately, using oral fluid specimen collected from consented volunteers. RESULTS: Rapid ( approximately 10min), sensitive detection ( approximately ng/mL) and quantitation of 12 drugs of abuse was demonstrated on the p-BNC platform. Furthermore, the system provided visibility to time-course of select drug and metabolite profiles in oral fluids; for the drug cocaine, three regions of slope were observed that, when combined with concentration measurements from this and prior impairment studies, information about cocaine-induced impairment may be revealed. CONCLUSIONS: This chip-based p-BNC detection modality has significant potential to be used in the future by law enforcement officers for roadside drug testing and to serve a variety of other settings, including outpatient and inpatient drug rehabilitation centers, emergency rooms, prisons, schools, and in the workplace.

Point-of-care (POC) diagnostic platforms have the potential to enable low-cost, large-scale screening. As no single biomarker is shed by all ovarian cancers, multiplexed biomarker panels promise improved sensitivity and specificity to address the unmet need for early detection of ovarian cancer. We have configured the programmable bio-nano-chip (p-BNC) - a multiplexable, microfluidic, modular platform - to quantify a novel multimarker panel comprising CA125, HE4, MMP-7 and CA72-4. The p-BNC is a bead-based immunoanalyzer system with a credit-card-sized footprint that integrates automated sample metering, bubble and debris removal, reagent storage and waste disposal, permitting POC analysis. Multiplexed p-BNC immunoassays demonstrated high specificity, low cross-reactivity, low limits of detection suitable for early detection, and a short analysis time of 43 minutes. Day-to-day variability, a critical factor for longitudinally monitoring biomarkers, ranged between 5.4-10.5%, well below the biological variation for all four markers. Biomarker concentrations for 31 late-stage sera correlated well (R2 = 0.71 to 0.93 for various biomarkers) with values obtained on the Luminex(R) platform. In a 31 patient cohort encompassing early- and late-stage ovarian cancers along with benign and healthy controls, the multiplexed p-BNC panel was able to distinguish cases from controls with 68.7% sensitivity at 80% specificity. Utility for longitudinal biomarker monitoring was demonstrated with pre-diagnostic sera from 2 cases and 4 controls. Taken together, the p-BNC shows strong promise as a diagnostic tool for large-scale screening that takes advantage of faster results and lower costs while leveraging possible improvement in sensitivity and specificity from biomarker panels.

This manuscript describes programmable Bio-Nano-Chip (p-BNC) approach that serves as miniaturized assay platform designed for the rapid detection and quantitation of multiple analytes in biological fluids along with the specific applications in salivary diagnostics intended for the point of need (PON). Included here are oral fluid-based tests for local periodontal disease, systemic cardiac disease and multiplexed tests for drugs of abuse.

With the advent of an increased emphasis on the potential to utilize biomarkers in saliva for systemic diseases, the issue of existing oral disease is an important consideration that could adversely affect the interpretation of diagnostic results obtained from saliva. We addressed the question does a patient's oral inflammation status confound biomarker levels used in diagnosis of acute myocardial infarction (AMI). The results demonstrated that multiple serum biomarkers and a few salivary biomarkers reflected the cardiac event. Importantly, oral health of the individual had minimal impact on the validity of the serum or salivary biomarker effectiveness.

The comparative utility of serum and saliva as diagnostic fluids for identifying biomarkers of acute myocardial infarction (AMI) was investigated. The goal was to determine if salivary biomarkers could facilitate a screening diagnosis of AMI, especially in cases of non-ST elevation MI (NSTEMI), since these cases are not readily identified by electrocardiogram (ECG). Serum and unstimulated whole saliva (UWS) collected from 92 AMI patients within 48 hours of chest pain onset and 105 asymptomatic healthy control individuals were assayed for 13 proteins relevant to cardiovascular disease, by Beadlyte technology (Luminex(R)) and enzyme immunoassays. Data were analyzed with concentration cut-points, ECG findings, logistic regression (LR) (adjusted for matching for age, gender, race, smoking, number of teeth, and oral health status), and classification and regression tree (CART) analysis. A sensitivity analysis was conducted by repetition of the CART analysis in 58 cases and 58 controls, each matched by age and gender. Serum biomarkers demonstrated AMI sensitivity and specificity superior to that of saliva, as determined by LR and CART. The predominant discriminators in serum by LR were troponin I (TnI), B-type natriuretic peptide (BNP), and creatine kinase-MB (CK-MB), and TnI and BNP by CART. In saliva, LR identified C-reactive protein (CRP) as the biomarker most predictive of AMI. A combination of smoking tobacco, UWS CRP, CK-MB, sCD40 ligand, gender, and number of teeth identified AMI in the CART decision trees. When ECG findings, salivary biomarkers, and confounders were included, AMI was predicted with 80.0% sensitivity and 100% specificity. These analyses support the potential utility of salivary biomarker measurements used with ECG for the identification of AMI. Thus, saliva-based tests may provide additional diagnostic screening information in the clinical course for patients suspected of having an AMI.