Extracellular Vesicle Subsets and Sampling Strategies for Nucleic Acid Profiling
Johns Hopkins University School of Medicine, United States of America
Enthusiasm continues unabated for the clinical potential of extracellular RNA biomarkers, especially those associated with extracellular vesicles (EVs) that may be traced back to a cell of origin. At the same time, it has become apparent that a diversity of EVs are produced by cells under normal and pathological conditions. In this presentation, data from three “challenging biofluid studies” are shared to demonstrate the influence of sampling strategy on EV subsetting and nucleic acid profiling results. Specifically, sampling site (for blood) is shown to affect recovery of large EVs; blood additives substantially influence small RNA profiling results; and different sampling strategies for cervicovaginal secretions affect interpretation of small RNA content by qPCR and qPCR array.
Cellular and Extracellular miRNAs are Blood Compartment-Specific Diagnostic Targets for Detection and Survival Prediction in Sepsis
1Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University Munich, Germany; 2Division of Animal Physiology and Immunology, TUM School of Life Sciences Weihenstephan, Technical University Munich; 3Department of Anesthesiology, University Hospital, Ludwig-Maximilians-University, Munich; 4Department of Anaesthesiology, Neuperlach Hospital, City Hospitals of Munich, Munich
Background: Septic shock is a common medical condition with a mortality approaching 50% where early diagnosis and treatment is of particular importance for patient survival. Novel biomarkers that serve as prompt indicators of sepsis are urgently needed. High-throughput technologies assessing circulating
microRNAs represent an important tool for biomarker identification but the blood-compartment specificity of these miRNAs has not yet been investigated.
Methods: We characterized miRNA profiles from serum exosomes, total serum and blood cells (leukocytes, erythrocytes, platelets) of sepsis patients (n=22) by next-generation sequencing and RT-qPCR and established differences in miRNA expression between blood compartments. In-silico analysis was used to identify compartment-specific signaling functions of differentially regulated miRNAs in sepsis-relevant pathways.
Results: In septic shock, atotal of 77 and 103 miRNAs were down- and upregulated, respectively. A majority of these regulated miRNAs (14 in serum, 32 in exosomes, 73 in blood cells) had not been previously associated with sepsis. We found a distinctly compartment-specific regulation of miRNAs between sepsis patients and healthy volunteers. Blood cellular miR-199b-5p was identified as a potential early indicator for sepsis and septic shock. miR-125b-5p and miR-26b-5p were uniquely regulated in exosomes and serum, respectively, while one miRNA (miR-27b-3p) was present in all three compartments. In-silico analysis identified crucial signaling pathways such as LPS-stimulated toll-like receptor and HIF-1a-signaling regulated by exosome derived miRNAs.
Conclusions: The expression of sepsis-associated miRNAs is compartment-specific. Exosome-derived miRNAs contribute significant information regarding sepsis diagnosis and survival prediction and could serve as newly-identified targets for the development of novel sepsis biomarkers.
Diagnostic Performance of Plasma DNA Methylation Profiles in Lung Cancer, Pulmonary Fibrosis and COPD
1AIT Austrian Institute of Technology GmbH, Molekulare Diagnostik, Austria; 2Medical University Vienna, Austria
Disease-specific alterations of the cell-free DNA methylation status are frequently found in serum samples and are currently considered to be suitable biomarkers. Candidate markers were identified by bisulfite conversion-based genome-wide methylation screening of lung tissue from lung cancer, fibrotic ILD, and COPD. cfDNA from 400 Î¼l serum(n=204) served to test the diagnostic performance of these markers. Following methylation-sensitive restriction enzyme digestion and enrichment of methylated DNA via targeted amplification (multiplexed MSRE enrichment), a total of 96 markers were addressed by highly parallel qPCR.
Lung cancer was efficiently separated from non-cancer and controls with a sensitivity of 87.8%, (95%CI: 0.67–0.97) and specificity 90.2%, (95%CI: 0.65–0.98). Cancer was distinguished from ILD with a specificity of 88%, (95%CI: 0.57–1), and COPD from cancer with a specificity of 88% (95%CI: 0.64–0.97). Separation of ILD from COPD and controls was possible with a sensitivity of 63.1% (95%CI: 0.4–0.78) and a specificity of 70% (95%CI: 0.54–0.81). The results were confirmed using an independent sample set (n = 46) by use of the four top markers discovered in the study (HOXD10, PAX9, PTPRN2, and STAG3) yielding an AUC of 0.85 (95%CI: 0.72–0.95). This technique was capable of distinguishing interrelated complex pulmonary diseases suggesting that multiplexed MSRE enrichment might be useful for simple and reliable diagnosis of diverse multifactorial disease states.
HP’s Advanced Microfluidic Technology
HP Incorporated, United States of America
Recently, there is a lot of interest in microfluidic lab-on-a chip application for life science, forensic, point-of-care, molecular-diagnostic, other in-vitro-diagnostic, organs-on-a-chip, environmental and multiple others applications. Different scientific and commercial organizations explore a multitude of material sets and operational principles to forge microfluidic devices. Simultaneously, inkjet industry utilizes well established materials and principles of operation for complicated microfluidic systems developed for precision dispensing and manipulation of droplets with pico-liter accuracy on a massively parallel scale. The presentation describes our recent progress of low cost microfluidic platform development utilizing materials and processes developed for low-cost thermal inkjet business. The concept repurposes well established inkjet processes, microfluidic components and jetting elements for pumping, mixing, valving, fluid transport, sensing and other critical functions of complex integrated microfluidic systems. This presentation describes operating principles of microfluidic elements, examples of their integration in functional devices and discusses inkjet technology potential for broad range of microfluidic applications.
NGS analysis of the Liquid biopsy in metastatic breast cancer patients: a pilot study.
University of Florence, Italy
Circulating tumor cells (CTCs) and cell-free DNA (cfDNA), released in the bloodstream from primary tumor and metastases, are considered a “liquid biopsy” of the tumor reflecting disease complexity and evolution at any stage.
In a previous study we demonstrated the feasibility of a protocol for the mutational analysis at the single cell level in patients affected by metastatic breast cancer.
The aim of the present study is the optimization of a protocol for the detection of somatic mutations in cfDNA by Next Generation Sequencing (NGS) and the comparison with data obtained from single circulating tumor cells isolated from the same blood draw. Notwithstanding that the two components of the liquid biopsy, CTCs and cfDNA, have been extensively studied separately, few studies compared the information obtained from these two different compartments for a personalized therapeutic approach.
Cell-free DNA was extracted from 2 ml plasma by using the QIAamp circulating nucleic acid kit (QIAgen). DNA samples were sequenced by Ion s5 Sequencer by using the ion AmpliSeq Cancer Hotspot Panel v2 (Thermofisher Scientific) targeting hotspot regions in 50 oncogenes and tumor suppressor genes which are frequently mutated in cancer patients.
Results obtained from cfDNA of 5 metastatic breast cancer patients have been compared with those of 3-5 CTCs isolated from the same blood draw and the primary tumor.
From the comparison we evidenced the presence of some concordant mutations between tissue and liquid biopsy, but also discrepancies.
In at least one patient no common somatic mutation was found between tissue and liquid biopsy. In one subject two hotspot mutations in TP53 were evidenced in CTCs and primary tumor but not in cfDNA while another mutation in the same gene was common to the tissue and cfDNA but could not be found in the CTCs.
This preliminary study suggests that CTCs and cfDNA represent two complementary aspects of the liquid biopsy. Our results support the feasibility of the use of the liquid biopsy as an useful tool in personalized medicine for the management of metastatic breast cancer patients.
Validation Of Clinical Diagnostic Real-Time PCR Kits On AriaMX-DX
AB ANALITICA, Italy
Molecular diagnostic laboratories are requesting more frequently fully automated solutions to obtain the maximum level of standardization, reliability, traceability and efficiency. Recently, AB ANALITICA’s bioengineering team has developed the GENEQUALITY X120, a fully-integrated platform, for extraction of nucleic acids from a wide range of clinical samples and automated Real-Time PCR plate setup. A key point of the analytic process remains the choice of the Real-Time PCR instrument. In this study, we have started the evaluation of Agilent AriaMX-DX instrument with AB ANALITICA REALQUALITY kits and clinical validation of a new HPV screening kit.
Going to the limits of Multiplex qPCR.
TIB MOLBIOL, Berlin, Germany
Disease with the same symptoms can be caused by up to two dozens different pathogens. Successive single pathogen testing is too slow; parallel testing costs too many wells and is too expensive. Multiplex PCR testing increases the throughput and reduces the costs per assay and target while providing at least a semiquantitative result, helping to rate the significance of the results and to identify putative contaminations.
Some pathogens can cause different diseases. We combine single target assay modules in one multiplex reaction allowing customized combinations for multiplex testing using Roche 480 instruments.
Multiplex realtime qPCR is limited by the number of instrument dye channels but also by molecular interferences. Highly multiplexed PCR assays are therefore limited in their sensitivity, in particular if run as 1-step RT PCR on RNA. Stategies to weaken primer binding during the reverse transcription step increases the PCR perfomance.
For testing on bacterial resitance we combine routinely 10 targets in one well, reaching a sensitivity of significant less than 10 copies per reaction. In the Respiratory Viral Panel we combine routinely 11 targets in one well, achieving for the most targets still 10 copies per reaction.