Bioanalytical Methods for Characterization of CAR-T Cellular Kinetics: Comparison of PCR Assays and Matrices.

Recently, multiple chimeric antigen receptor T-cell (CAR-T)-based therapies have been approved for treating hematological malignancies, targeting CD19 and B-cell maturation antigen. Unlike protein or antibody therapies, CAR-T therapies are "living cell" therapies whose pharmacokinetics are characterized by expansion, distribution, contraction, and persistence. Therefore, this unique modality requires a different approach for quantitation compared with conventional ligand binding assays implemented for most biologics. Cellular (flow cytometry) or molecular assays (polymerase chain reaction (PCR)) can be deployed with each having unique advantages and disadvantages. In this article, we describe the molecular assays utilized: quantitative PCR (qPCR), which was the initial platform used to estimate transgene copy numbers and more recently droplet digital PCR (ddPCR) which quantitates the absolute copy numbers of CAR transgene. The comparability of the two methods in patient samples and of each method across different matrices (isolated CD3+ T-cells or whole blood) was also performed. The results show a good correlation between qPCR and ddPCR for the amplification of same gene in clinical samples from a CAR-T therapy trial. In addition, our studies show that the qPCR-based amplification of transgene levels was well-correlated, independent of DNA sources (either CD3+ T-cells or whole blood). Our results also highlight that ddPCR can be a better platform for monitoring samples at the early phase of CAR-T dosing prior to expansion and during long-term monitoring as they can detect samples with very low copy numbers with high sensitivity, in addition to easier implementation and sample logistics.

Systematic use of the RAM nasal cannula in the Yale-New Haven Children's Hospital Neonatal Intensive Care Unit: a quality improvement project.

OBJECTIVE: To evaluate the clinical efficacy of the RAM nasal cannula (NC) with different modes of non-invasive ventilation (NIV) in the neonatal intensive care unit (NICU). METHODS: A single center prospective, observational study of infants placed on RAM NC. A small trial (Study 1) was completed in 16 infants on NIV via the RAM NC over a 48-h period to create Summary Statement recommendations. Next, over a 10-month period (Study 2), data were prospectively collected for the outcome of all infants receiving respiratory support with the RAM NC. Outcomes were compared between different modes of NIV and whether the recommendations were followed. RESULTS: The Study 2 population consisted of 88 infants of whom 67 infants received nasal continuous positive airway pressure ventilation (NCPAP) and 21 received nasal intermittent positive pressure ventilation (NIPPV) via the RAM NC. The NIPPV group tended to be younger, smaller and stayed on the RAM NC longer. The overall success rate in weaning off the RAM NC, if our guidelines were followed, was 63%. CONCLUSION: RAM NC use with NIV was well tolerated in the neonatal population with the use of our guidelines. We speculate that use of our guidelines will lead to a more systematic use of the RAM NC in the NICU.