Person:

Khormaee, Sariah

Monitoring for drug-induced liver injury (DILI) via serial transaminase measurements in patients on potentially hepatotoxic medications (e.g., for HIV and tuberculosis) is routine in resource-rich nations, but often unavailable in resource-limited settings. Towards enabling universal access to affordable point-of-care (POC) screening for DILI, we have performed the first field evaluation of a paper-based, microfluidic fingerstick test for rapid, semi-quantitative, visual measurement of blood alanine aminotransferase (ALT). Our objectives were to assess operational feasibility, inter-operator variability, lot variability, device failure rate, and accuracy, to inform device modification for further field testing. The paper-based ALT test was performed at POC on fingerstick samples from 600 outpatients receiving HIV treatment in Vietnam. Results, read independently by two clinic nurses, were compared with gold-standard automated (Roche Cobas) results from venipuncture samples obtained in parallel. Two device lots were used sequentially. We demonstrated high inter-operator agreement, with 96.3% (95% C.I., 94.3–97.7%) agreement in placing visual results into clinically-defined “bins” (<3x, 3–5x, and >5x upper limit of normal), >90% agreement in validity determination, and intraclass correlation coefficient of 0.89 (95% C.I., 0.87–0.91). Lot variability was observed in % invalids due to hemolysis (21.1% for Lot 1, 1.6% for Lot 2) and correlated with lots of incorporated plasma separation membranes. Invalid rates <1% were observed for all other device controls. Overall bin placement accuracy for the two readers was 84% (84.3%/83.6%). Our findings of extremely high inter-operator agreement for visual reading–obtained in a target clinical environment, as performed by local practitioners–indicate that the device operation and reading process is feasible and reproducible. Bin placement accuracy and lot-to-lot variability data identified specific targets for device optimization and material quality control. This is the first field study performed with a patterned paper-based microfluidic device and opens the door to development of similar assays for other important analytes.

Short interfering RNA (siRNA) is a class of nucleotide drugs with a profound potential to improve patient health through its ability to silence the expression of specific genes at the post-transcriptional level. However, the clinical application of siRNA therapeutics remains hindered by a lack of efficient delivery systems that deposit siRNA into the cytoplasm of cells, a step necessary for siRNA’s silencing effect. Much research has focused on the development of siRNA delivery agents to overcome this challenge. There are no standard pre-clinical models for testing of siRNA delivery agents, and investigators have chosen to evaluate efficacy in a variety of systems including in vitro tissue culture and animal models. These systems have vastly different cellular microenvironments which may modulate cellular behavior and affect the response of cells to siRNA, thus altering the apparent efficacy of siRNA delivery agents. The substrate on which cells adhere is one aspect of the microenvironment that has been previously shown to alter cellular behavior. In this work, we tested the hypothesis that changing the properties of cellular adhesion substrates can change the apparent efficacy of a siRNA delivery agent. Specifically, we used a commonly employed in vitro cationic lipid siRNA delivery vector and evaluated siRNA silencing efficacy in U251 cells seeded on alginate hydrogel surfaces. These surfaces were synthesized to have systematic variation in integrin ligand arginine-glycine-aspartate (RGD) density and elastic modulus. We found that an eightfold increase in RGD content of the alginate grown substrate increased siRNA knockdown efficacy from 25 ± 12% to 52 ± 10%, with constant concentrations of siRNA and delivery agent. We found no difference in siRNA mediated knockdown efficacy over the elastic modulus range tested (53-133 kPa). These results indicate that the cell-adhesion substrate interaction can modulate siRNA protein silencing efficacy, a finding important for evaluation of siRNA therapeutics in the in vitro setting.