The need to activate thermoplastic surfaces using robust and efficient methods has been driven by the fact that replication techniques can be used to produce microfluidic devices in a high production mode and at low cost, making polymer microfluidics invaluable for in vitro diagnostics, such as circulating tumor cell (CTC) analysis, where device disposability is critical to mitigate artifacts associated with sample carryover. Modifying the surface chemistry of thermoplastic devices through activation techniques can be used to increase the wettability of the surface or to produce functional scaffolds to allow for the covalent attachment of biologics, such as antibodies for CTC recognition. Extensive surface characterization tools were used to investigate UV activation of various surfaces to produce uniform and high surface coverage of functional groups, such as carboxylic acids in microchannels of different aspect ratios. We found that the efficiency of the UV activation process is highly dependent on the microchannel aspect ratio and the identity of the thermoplastic substrate. Colorimetric assays and fluorescence imaging of UV-activated microchannels following EDC/NHS coupling of Cy3-labeled oligonucleotides indicated that UV-activation of a PMMA microchannel with an aspect ratio of ∼3 was significantly less efficient toward the bottom of the channel compared to the upper sections. This effect was a consequence of the bulk polymer's damping of the modifying UV radiation due to absorption artifacts. In contrast, this effect was less pronounced for COC. Moreover, we observed that after thermal fusion bonding of the device's cover plate to the substrate, many of the generated functional groups buried into the bulk rendering them inaccessible. The propensity of this surface reorganization was found to be higher for PMMA compared to COC. As an example of the effects of material and microchannel aspect ratios on device functionality, thermoplastic devices for the selection of CTCs from whole blood were evaluated, which required the immobilization of monoclonal antibodies to channel walls. From our results, we concluded the CTC yield and purity of isolated CTCs were dependent on the substrate material with COC producing the highest clinical yields for CTCs as well as better purities compared to PMMA.

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 post-selection 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 comprised 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).

Background Pediatric pheochromocytomas and paragangliomas (PC/PGLs) are rare with limited data as to what the optimal management approach is. The aim of this study was to determine the role of genetic testing and imaging to detect extra-adrenal and/or metastatic tumors in pediatric PC/PGLs. Methods A retrospective study of 55 patients diagnosed at ≤ 21 years of age with PC/PGLs was performed with analysis of data on genetic testing and multimodal imaging. Results Eighty percent of patients (n=44/55) had a germline mutation. The majority were found to have either VHL (38%) or SDHB (25.4%) mutation. PC was present in 67% (n=37/55) of patients and was bilateral in 51.3% (n=19/37). The majority of patients with bilateral PC had VHL (79%). Abdominal PGL was present in 21.8% (n=12/55), head and neck PGL in 11% (n=6/55) and thoracic PGL in 3.6% (n=2/55) of patients. For PGL, SDHx accounted for 72% (n=13/18) of mutations. The rate of malignancy was 16.4% (n=9/55), 55.5% had SDHB mutations. In two-thirds of patients, functional imaging identified either extra-adrenal PGL and/or metastatic disease. Conclusions The majority of pediatric patients with PC/PGL have germline mutations. Therefore, all pediatric patients with PC/PGLs require genetic testing and imaging to detect extra-adrenal PGL and metastatic disease to guide treatment and follow up.