Modular microsystem for the isolation, enumeration, and phenotyping of circulating tumor cells in patients with pancreatic cancer.

In this manuscript, we discuss the development and clinical use of a thermoplastic modular microsystem for the high-throughput analysis of CTCs directly from whole blood. The modular system offers some innovative features that address challenges currently associated with many CTC platforms; it can exhaustively process 7.5 mL of blood in less than 45 min with recoveries >90%. In addition, the system automates the postselection CTC processing steps and thus, significantly reduces assay turnaround time (from selection to enumeration <1.5 h as compared to >8 h for many reported CTC platforms). The system is composed of 3 functional modules including (i) a thermoplastic CTC selection module composed of high aspect ratio (30 µm × 150 µm) channels containing anti-EpCAM antibodies that is scalable in terms of throughput by employing channel numbers ranging from 50 to 320; the channel number is user selected to accommodate the volume of blood that must be processed; (ii) an impedance sensor module for label-less CTC counting; and (iii) a staining and imaging module for the placement of released cells into a 2D array within a common imaging plane for phenotypic identification. To demonstrate the utility of this system, blood samples from patients with local resectable and metastatic pancreatic ductal adenocarcinoma (PDAC) were analyzed. We demonstrate the ability to select EpCAM positive CTCs from PDAC patients in high purity (>86%) and with excellent yields (mean = 53 CTCs per mL for metastatic PDAC patients) using our modular system. In addition, we demonstrate the ability to detect CTCs in PDAC patients with local resectable disease (mean = 11 CTCs per mL).

A titer plate-based polymer microfluidic platform for high throughput nucleic acid purification.

A 96-well solid-phase reversible immobilization (SPRI) reactor plate was designed to demonstrate functional titer plate-based microfluidic platforms. Nickel, large area mold inserts were fabricated using an SU-8 based, UV-LIGA technique on 150 mm diameter silicon substrates. Prior to UV exposure, the prebaked SU-8 resist was flycut to reduce the total thickness variation to less than 5 mum. Excellent UV lithography results, with highly vertical sidewalls, were obtained in the SU-8 by using an UV filter to remove high absorbance wavelengths below 350 nm. Overplating of nickel in the SU-8 patterns produced high quality, high precision, metal mold inserts, which were used to replicate titer plate-based SPRI reactors using hot embossing of polycarbonate (PC). Optimized molding conditions yielded good feature replication fidelity and feature location integrity over the entire surface area. Thermal fusion bonding of the molded PC chips at 150 degrees C resulted in leak-free sealing, which was verified in leakage tests using a fluorescent dye. The assembled SPRI reactor was used for simple, fast purification of genomic DNA from whole cell lysates of several bacterial species, which was verified by PCR amplification of the purified genomic DNA.

Standard electrochemical behavior of high-quality, boron-doped polycrystalline diamond thin-film electrodes

Standard electrochemical data for high-quality, boron-doped diamond thin-film electrodes are presented. Films from two different sources were compared (NRL and USU) and both were highly conductive, hydrogen-terminated, and polycrystalline. The films are acid washed and hydrogen plasma treated prior to use to remove nondiamond carbon impurity phases and to hydrogen terminate the surface. The boron-doping level of the NRL film was estimated to be in the mid 1019 B/cm3 range, and the boron-doping level of the USU films was approximately 5 x 10(20) B/cm(-3) based on boron nuclear reaction analysis. The electrochemical response was evaluated using Fe-(CN)6(3-/4-), Ru(NH3)6(3+/2+), IrCl6(2-/3-), methyl viologen, dopamine, ascorbic acid, Fe(3+/2+), and chlorpromazine. Comparisons are made between the apparent heterogeneous electron-transfer rate constants, k0(app), observed at these high-quality diamond films and the rate constants reported in the literature for freshly activated glassy carbon. Ru(NH3)6(3+/2+), IrCl6(2-/3-), methyl viologen, and chlorpromazine all involve electron transfer that is insensitive to the diamond surface microstructure and chemistry with k0(app) in the 10(-2)-10(-1) cm/s range. The rate constants are mainly influenced by the electronic properites of the films. Fe(CN)6(3-/4-) undergoes electron transfer that is extremely sensitive to the surface chemistry with k0(app) in the range of 10(-2)-10(-1) cm/s at the hydrogen-terminated surface. An oxygen surface termination severely inhibits the rate of electron transfer. Fe(3+/2+) undergoes slow electron transfer at the hydrogen-terminated surface with k0(app) near 10(-5) cm/s. The rate of electron transfer at sp2 carbon electrodes is known to be mediated by surface carbonyl functionalities; however, this inner-sphere, catalytic pathway is absent on diamond due to the hydrogen termination. Dopamine, like other catechol and catecholamines, undergoes sluggish electron transfer with k0(app) between 10(-4) and 10(-5) cm/s. Converting the surface to an oxygen termination has little effect on k0(app). The slow kinetics may be related to weak adsorption of these analytes on the diamond surface. Ascorbic acid oxidation is very sensitive to the surface termination with the most negative Ep(ox) observed at the hydrogen-terminated surface. An oxygen surface termination shifts Ep(ox) positive by some 250 mV or more. An interfacial energy diagram is proposed to explain the electron transfer whereby the midgap density of states results primarily from the boron doping level and the lattice hydrogen. The films were additionally characterized by scanning electron microscopy and micro-Raman imaging spectroscopy. The cyclic voltammetric and kinetic data presented can serve as a benchmark for research groups evaluating the electrochemical properties of semimetallic (i.e., conductive), hydrogen-terminated, polycrystalline diamond.

Outbreak of sterile peritonitis among continuous cycling peritoneal dialysis patients.

BACKGROUND: Approximately 30,000 patients receive peritoneal dialysis in the United States. In August 1996, several dialysis centers from different states reported sterile peritonitis among CCPD patients using sterile peritoneal dialysis solution (PDS) from a single manufacturer. The manufacturer recalled 53 lots of PDS that had passed established industry guidelines and Food and Drug Administration (FDA) approved quality control tests [including endotoxin levels <0.5 endotoxin units (EU)/ml], but had pre-sterilization bacterial colony counts >1 cfu/ml. METHODS: At one outpatient dialysis center, Hospital of the University of Pennsylvania (HUP), we conducted a retrospective cohort study of all CCPD patients treated during July 15 to August 30, 1996. A case-patient was defined as any HUP patient with culture-negative peritoneal fluid with a white blood cell count >100/mm3, cloudy peritoneal fluid, and/or abdominal pain. PDS and tubing were cultured for bacteria and assayed for endotoxin. RESULTS: Overall, 14 of 28 patients had sterile peritonitis. The only risk factor identified was exposure to > or =1 lot of recalled PDS (14 of 22 vs. 0/6, P = 0.02); the more recalled lots received, the higher the attack rate (P = 0.0001). Five of 47 PDS bags had detectable endotoxin; recalled lots were more likely to have measurable endotoxin than nonrecalled lots (5/19 vs. 0/17, P = 0.05). When case-patients resumed CCPD using PDS from non-recalled lots, no further cases were reported. CONCLUSIONS: Our results suggest that this outbreak was caused by intrinsic PDS contamination with endotoxin. Pre-sterilization colony counts may be an important quality control indicator for CCPD fluids in conjunction with endotoxin levels.