Andrew W. Browne

An on-chip whole blood/plasma separator with bead-packed microchannel on COC polymer

A disposable on-chip whole blood/plasma separator, which is able to separate plasma from whole human blood by capillary force through a bead-packed microchannel, has been designed, fabricated and characterized in this work. Various sizes of silica beads were slurry-packed through a microchannel using a bump structure which held beads in a defined region. The bead-packed microchannel induces a capillary force which allows plasma to move forward through the bead-packed column more rapidly than red blood cells (RBCs). The blood/plasma separator with bead-packed microchannel has successfully separated plasma from the whole blood without haemolysis of RBCs. The separation method developed in this work can be applied to various on-chip stationary filtrations of RBC for point-of-care clinical diagnostics.

Optimizing Precision in Toric Lens Selection by Combining Keratometry Techniques

PURPOSE To increase precision in toric intraocular lens selection by reducing the frequency of outliers that arise from technology-dependent variability during the preoperative assessment for routine cataract surgery. METHODS Mean preoperative values for absolute sphere, amount of astigmatism, and steepest cylindrical axis were obtained for 87 eyes (54 patients) each with a manual keratometer and four automated keratometers. The mean sphere, amount of astigmatism, and steepest cylindrical axis across five technologies for each eye were defined as the meld sphere, meld astigmatism, and meld axis, respectively. Each technology was evaluated against the meld by Bland–Altman analysis, Student’s paired t test, and correlation coefficients. Further comparison between individual technologies and the meld quantified the number of outlier measurements each technology produced. RESULTS The number of outliers between individual keratometers and the meld differed with specific measurement of sphere, axis, or amount of astigmatism. Although statistical analysis using Bland–Altman plots, correlation coefficients, and paired t tests suggested insignificant difference from meld measurements for each parameter, precision-guided analysis presented more clinically significant outliers. The number of outliers can be reduced for sphere (range: 2%-46% to 1%-6%), astigmatism (range: 6%-23% to 0%-2%), and axis outliers (range: 15%-27% to 3%-6%) by averaging measurements from automated and manual keratometers. CONCLUSIONS Although multiple keratometry technologies produced similar, average measurements, the authors found a disturbing number of outliers that may be overlooked when employing a single technology. Measurement errors can be dramatically reduced by averaging measurements from manual keratometry with any automated technology to make toric lens selection more precise.

A PDMS pinch-valve module embedded in rigid polymer lab chips for on-chip flow regulation

A new dead-volume-free PDMS pinch microvalve has been designed, fabricated and characterized in this work for valving and flow regulation on rigid polymer lab chips as an embedded modular platform. The pinch microvalve is operated by compressing a microfabricated flexible channel with an external actuation force. The assembly of modular PDMS pinch microvalves within rigid thermoplastic microfluidic devices provides numerous benefits in realizing hybrid polymer lab chips with functionality to precisely regulate flow. Flow rates of the developed pinch microvalve were characterized with respect to the pinch valve channel dimension and the pinching displacement distance applied to the valve. Finally, a microfluidic system with a single channel that branches into four pinch-valved channels was realized and a novel scheme for simultaneously actuating all four valves with a single actuator was demonstrated. The microfluidic PDMS pinch valves developed in this work will have a variety of applications in microfluidic systems developed in rigid polymers.

Cancer Screening by Systemic Administration of a Gene Delivery Vector Encoding Tumor-Selective Secretable Biomarker Expression

Cancer biomarkers facilitate screening and early detection but are known for only a few cancer types. We demonstrated the principle of inducing tumors to secrete a serum biomarker using a systemically administered gene delivery vector that targets tumors for selective expression of an engineered cassette. We exploited tumor-selective replication of a conditionally replicative Herpes simplex virus (HSV) combined with a replication-dependent late viral promoter to achieve tumor-selective biomarker expression as an example gene delivery vector. Virus replication, cytotoxicity and biomarker production were low in quiescent normal human foreskin keratinocytes and high in cancer cells in vitro. Following intravenous injection of virus >90% of tumor-bearing mice exhibited higher levels of biomarker than non-tumor-bearing mice and upon necropsy, we detected virus exclusively in tumors. Our strategy of forcing tumors to secrete a serum biomarker could be useful for cancer screening in high-risk patients, and possibly for monitoring response to therapy. In addition, because oncolytic vectors for tumor specific gene delivery are cytotoxic, they may supplement our screening strategy as a “theragnostic” agent. The cancer screening approach presented in this work introduces a paradigm shift in the utility of gene delivery which we foresee being improved by alternative vectors targeting gene delivery and expression to tumors. Refining this approach will usher a new era for clinical cancer screening that may be implemented in the developed and undeveloped world.

Hot-Embossed Piezoelectric Polymer Micro-Diaphragm Arrays Integrated with Lab-on-a-Chip for Protein Analysis

Piezoelectric copolymer, PVDF-TrFE, diaphragm arrays with integrated microfluidic chip for high performance protein immunosensors were developed and characterized in this work. Mold-transfer and hot-embossing techniques provide a high throughput and repeatable way to fabricate piezoelectric polymer diaphragms and PVDF-TrFEs hydrophobic surface acts as a natural bioreceptor to capture proteins of interest. In addition, integrated microfluidic devices enhance reaction efficiency and reduce the assay time.

An in-line microfluidic blood sampling interface between patients and saline infusion systems.

This work seeks to extend the utility of microfluidics to conventional blood sampling aperati. Daily medical care of hospitalized patients demands repeated needle punctures or interfacing with a catheter to collect blood samples. Large, research grade systems can autonomously sample blood from laboratory animals; however, a disposable aperatus that can be used to repeatedly sample blood from hospitalized patients does not exist. We have designed, fabricated and demonstrated a 3-layered rigid polymer microfluidic blood sampling device with integrated polymer pinch valves for placement in-line between a patient and a saline infusion system. The blood sampler we designed seeks to mitigate sample cross contamination, reduce risks of microbial contamination associated with invasive blood sampling and improve technical ease of blood sampling. Clinical laboratory tests and microfluidic devices for rapid point-of-care-testing (POCT) of patient samples require human sampling procedures for collection of a patient sample at defined time points. The microfluidic sampling device is designed ultimately to be backwards compatible with existing clinical saline infusion protocols and function as a universal front-end blood sampling unit for the variety of microfluidic lab chips and POCT devices.

Intraoperative Optical Coherence Tomography–Assisted Chorioretinal Biopsy in the DISCOVER Study

TOPIC To assess the role and utility of intraoperative optical coherence tomography (iOCT) guidance during pars plana vitrectomy with chorioretinal biopsy for patients with suspicious chorioretinal lesions. CLINICAL RELEVANCE Chorioretinal lesions suspicious for malignancy sometimes require surgical biopsy for diagnosis and treatment guidance. Biopsy site selection is currently guided by presurgical imaging, and clinical assessment. We sought to determine whether iOCT provides useful information for biopsy site selection to ensure a diagnostic specimen and assessment of wound repair after biopsy. MATERIALS AND METHODS DISCOVER is a prospective study examining microscope-integrated iOCT systems in ophthalmic surgery. In this report, we examine all eyes in the DISCOVER study that underwent chorioretinal biopsy. Clinical characteristics, surgical technique, and imaging findings were reviewed. In addition, surgeon feedback data on iOCT utility and value to the procedure was also evaluated. RESULTS Six eyes were identified within the DISCOVER study that underwent chorioretinal biopsy. iOCT was used to assess retinal and choroidal lesions prior to chorioretinal incision. Following biopsy, iOCT was utilized to assess retinal attachment and wound margin integrity. Additionally, iOCT was performed in real-time during biopsy to ensure proper biopsy depth and position. In 2 cases, preoperative OCT evaluation of retinal lesions was not possible. Utilizing iOCT, comprehensive evaluation of anatomic features was successfully performed prior to biopsy. This facilitated identification of biopsy sites most likely to provide a diagnostic sample based on lesion morphology on iOCT. After completion of chorioretinal biopsy, iOCT verified completeness of biopsy site and the retinal-RPE relationship (e.g., complete apposition, partial detachment). CONCLUSION Microscope-integrated iOCT provides real-time guidance of biopsy site selection and verification of biopsy site stability following specimen collection.

Combined Phacoemulsification and Ahmed Glaucoma Drainage Implant Surgery for Patients With Refractory Glaucoma and Cataract.

Purpose:To examine the indications, safety, efficacy, and complications of combined phacoemulsification and Ahmed glaucoma drainage implant surgery. Methods:A retrospective case review of 35 eyes (31 patients) subjected to combined phacoemulsification and Ahmed glaucoma drainage implant surgery. Demographic characteristics of the study population, indications for combined surgery, and operative and postoperative complications were recorded. Visual acuity, intraocular pressure (IOP), and number of glaucoma medications were evaluated preoperatively and postoperatively. Complete success was defined as IOP⩽21 mm Hg without medication, qualified success if IOP⩽21 mm Hg with ≥1 medications, and failure if IOP>21 mm Hg or ⩽5 mm Hg on ≥2 consecutive visits. Results:Mean follow-up was 29.5 months (range, 6 to 87 mo). The most common indication for combined surgery was a history of prior failed trabeculectomy (60%). Postoperative visual acuity improved in 30 of 35 eyes (85%) (P<0.01) regardless of the indication for combined surgery. IOP was reduced from a mean of 24.7 to 15.0 mm Hg at the last follow-up visit (P<0.01). The number of IOP-lowering medications was reduced from a median of 3.1 preoperatively to 1.7 at the last follow-up (P<0.01). Overall, there were 31 eyes (89%) classified as qualified success and 4 eyes (11%) as complete success. The most common postoperative complication was a hypertensive phase in 18 eyes (51%). Conclusions:Combined phacoemulsification and Ahmed glaucoma drainage implant surgery seems to be a safe and effective surgical option, providing good visual rehabilitation and control of IOP for patients with refractory glaucoma and cataract.

The Relationship between Central Corneal Thickness (CCT) and Papilledema from Idiopathic Intracranial Hypertension (IIH).

Purpose Human pluripotent stem cell (hPSC)-derived retinal organoids are a platform for investigating retinal development, pathophysiology, and cellular therapies. In contrast to histologic analysis in which multiple specimens fixed at different times are used to reconstruct developmental processes, repeated analysis of the same living organoids provides a more direct means to characterize changes. New live imaging modalities can provide insights into retinal organoid structure and metabolic function during in vitro growth. This study employed live tissue imaging to characterize retinal organoid development, including metabolic changes accompanying photoreceptor differentiation. Methods Live hPSC-derived retinal organoids at different developmental stages were examined for microanatomic organization and metabolic function by phase contrast microscopy, optical coherence tomography (OCT), fluorescence lifetime imaging microscopy (FLIM), and hyperspectral imaging (HSpec). Features were compared to those revealed by histologic staining, immunostaining, and microcomputed tomography (micro-CT) of fixed organoid tissue. Results We used FLIM and HSpec to detect changes in metabolic activity as organoids differentiated into organized lamellae. FLIM detected increased glycolytic activity and HSpec detected retinol and retinoic acid accumulation in the organoid outer layer, coinciding with photoreceptor genesis. OCT enabled imaging of lamellae formed during organoid maturation. Micro-CT revealed three-dimensional structure, but failed to detect lamellae. Conclusions Live imaging modalities facilitate real-time and nondestructive imaging of retinal organoids as they organize into lamellar structures. FLIM and HSpec enable rapid detection of lamellar structure and photoreceptor metabolism. Live imaging techniques may aid in the continuous evaluation of retinal organoid development in diverse experimental and cell therapy settings.

A polymer cell chip with in situ monitoring capability of cell viability for cadmium cytotoxicity test of human rhabdomyosarcoma cells

This paper presents a polymer cell chip integrated with interdigitated array electrodes (IDA) and a planar cadmium (Cd) micro sensor for on-chip monitoring of cell culture and performing Cd cytotoxicity test of human rhabdomyosarcoma (HR) cells. HR cells were maintained in static culture on the cyclic olefin copolymer (COC) chip for up to seven days, and the cellular behavior was characterized using IDA by electrochemical impedance spectroscopy. A microfabricated Cd sensor with bismuth working electrode was adapted for in situ Cd sensing when cells were exposed to Cd at different concentrations, and the corresponding Cd cytotoxicity was monitored.