Immunophenotyping of cerebrospinal fluid cells by Chipcytometry

Abstract Background The gold standard in cerebrospinal fluid (CSF) cell immunophenotyping is flow cytometry. Nevertheless, the small amount of CSF cells and the invasive character of lumbar puncture limit the spectrum of possible investigation. Chipcytometry, a modified approach to slide-based cytometry, might be a useful tool for CSF analysis due to the possibility of iterative staining, imaging, and bleaching cycles. The aim of this study was to compare flow cytometric leukocyte subset analysis with Chipcytometry comparing the percentage distribution of distinct cell populations and the T-cell CD4:CD8 ratio. Moreover, this study investigated the interpretability of chips loaded with CSF cells and examined the applicability of Chipcytometry in clinical practice. Methods 375 CSF samples from 364 patients were analyzed by Chipcytometry using an automated upright microscope. Cell surface molecules were stained using fluorescence-labeled monoclonal antibodies. For cross-validation experiments, flow cytometry data of six patients were analyzed and matched with Chipcytometry data. Results Our experiments showed a better agreement examined by Bland-Altman analysis for samples with CSF pleocytosis than for normocellular CSF samples. Data were more consistent for B cells and CD4:CD8 ratio than for T cells and monocytes. Advantages of Chipcytometry compared to flow cytometry are that cells once fixated can be analyzed for up to 20 months with additional markers at any time. The clinical application of Chipcytometry is demonstrated by two illustrative case reports. However, the low amount of CSF cells limits the analysis of normocellular CSF samples, as in our cohort only 11.7% of respectively loaded chips had sufficient cell density for further investigation compared to 59.8% of all chips loaded with samples with elevated cell counts (≥ 5/μl). Varying centrifuge settings, tube materials and resuspension technique were not able to increase the cell yield. Conclusion In summary, the results demonstrate the great potential of Chipcytometry of CSF cells for both scientific questions and routine diagnostic. A new chip design optimized to meet the requirements of CSF would greatly enhance the value of this method. Cross-validation results need to be confirmed in a larger cohort

A nano-enabled cancer-specific ITCH RNAi chemotherapy booster for pancreatic cancer

AbstractGemcitabine is currently the standard therapy for pancreatic cancer. However, growing concerns over gemcitabine resistance mean that new combinatory therapies are required to prevent loss of efficacy with prolonged treatment. Here, we suggest that this could be achieved through co-administration of RNA interference agents targeting the ubiquitin ligase ITCH. Stable anti-ITCH siRNA and shRNA dendriplexes with a desirable safety profile were prepared using generation 3 poly(propylenimine) dendrimers (DAB-Am16). The complexes were efficiently taken up by human pancreatic cancer cells and produced a 40-60% decrease in ITCH RNA and protein expression in vitro (si/shRNA) and in a xenograft model of pancreatic cancer (shRNA). When co-administered with gemcitabine (100mg/kg/week) at a subtherapeutic dose, treatment with ITCH-shRNA (3x 50mg/week) was able to fully suppress tumour growth for 17days, suggesting that downregulation of ITCH mediated by DAB-Am16/shRNA sensitizes pancreatic cancer to gemcitabine in an efficient and specific manner.From the Clinical EditorGemcitabine delivery to pancreatic cancer often results in the common problem of drug resistance. This team overcame the problem through co-administration of siRNA and shRNA dendriplexes targeting the ubiquitin ligase ITCH