Sensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR.

Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy. Therefore, there is an urgent need for CAR-T cell monitoring by clinicians to assess cell expansion and persistence in patients. CAR-T cell manufacturers and researchers need to evaluate transduction efficiency and vector copy number for quality control. Here, CAR expression was analyzed in peripheral blood samples from patients and healthy donors by flow cytometry with four commercially available detection reagents and on the gene level by quantitative polymerase chain reaction (qPCR). Flow cytometric analysis of CAR expression showed higher mean CAR expression values for CD19 CAR detection reagent and the F(ab')2 antibody than Protein L and CD19 Protein. In addition, the CD19 CAR detection reagent showed a significantly lower median background staining of 0.02% (range 0.007-0.06%) when compared to the F(ab')2 antibody, CD19 protein and Protein L with 0.80% (range 0.47-1.58%), 0.65% (range 0.25-1.35%) and 0.73% (range 0.44-1.23%). Furthermore, flow cytometry-based CAR-T cell frequencies by CD19 CAR detection reagent showed a good correlation with qPCR results. In conclusion, quality control of CAR-T cell products can be performed by FACS and qPCR. For the monitoring of CAR-T cell frequencies by FACS in patients, CAR detection reagents with a low background staining are preferable.

Improvement of in vitro potency assays by a resting step for clinical-grade chimeric antigen receptor engineered T cells.

BACKGROUND: Chimeric antigen receptor engineered T (CAR-T) cell therapy is a promising approach currently revolutionizing the field of cancer immunotherapy. However, data concerning clinical-grade CAR-T cell stability and functionality after months of cryopreservation have not been released by companies so far. To investigate the effect of cryopreservation on CAR-T cells and to further optimize the potency assays, we performed this study. METHODS: A third generation of CD19 CAR-T cells was manufactured according to Good Manufacturing Practice (GMP) requirements, which is applied to patients in an ongoing clinical phase 1 study. Quality control tests for sterility, endotoxin and mycoplasma were performed for each batch. Stability in terms of viability, recovery, transduction efficiency and functional capacity was determined using microscopy, multiparametric flow cytometry as well as chromium-51 release tests. RESULTS: Up to 90days of cryopreservation had no influence on viability, recovery and transduction efficiency of CAR-T cells. However, higher cell concentration for cryopreservation could alter the cell viability and recovery but not the transduction efficiency. Moreover, directly after thawing, both the quantity and quality of the functionality of CAR-T cells were transiently hampered by the negative effects of cryopreservation. Notably, the impaired functionality could be fully restored and even strengthened after an overnight resting process. DISCUSSION: Cryopreservation is a challenge for the functional activity of CAR-T cells. However, CAR-T cells regain their potency by overnight incubation at 37°C, which mimics the clinical application setting. Therefore, an overnight resting step should be included in in vitro potency assays.

Injury severity and seating position in accidents with German EMS helicopters.

INTRODUCTION: Accident rates and fatality rates for Helicopter Emergency Medical Service (HEMS) missions have been investigated recently, but none of these studies considered the influence of the seating position in the helicopter. The aim of the present descriptive and observational study was to analyze injury severity depending on the seating position during HEMS accidents in Germany. MATERIAL AND METHODS: Data from the German Federal Agency for Flight Accident Investigation was gathered for a period of 40 years (from 1970 to 2009). The seating position in the aircraft during the accident and the resulting injury severity (i.e., 1=no; 2=slight; 3=severe; and 4=fatal) were recorded. Injury severity was compared using the Fisher's exact test. P values <0.05 were considered statistically significant. RESULTS: A total of n=99 accidents were investigated (n=61 accidents did not lead to any injuries in the occupants, n=7 accidents resulted in minor, and n=6 in severe injuries, and lethal injuries resulted from n=15 accidents). The occupant in the "patient" position was most likely to suffer from deadly injuries (44.9%), followed by the HEMS crew member rear seat (25.0%), compared to lower lethality rates in the other seating positions (9.4-11.2%). Sitting on the HEMS crew member rear seat also was associated with the highest percentage of severe and minor injuries (12.5% each). CONCLUSIONS: In HEMS accidents, the patients' position and the HEMS crew member rear seat were found to be at the highest risk for fatal or severe injuries. These results support the urgent requirement of a large international data base for HEMS accidents as a basis for further studies to improve the safety in HEMS missions.