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7.1 What are the main methods available to isolate CTCs?
1- Enrichment based on expression of cell-surface markers such as EpCAm+ (positive selection) or CD45+ depletion (negative selection)
2- Enrichment based on cell size. Use of membranes with pores to retain large tumour cells (size selection)
3- Use of microfluidics platforms. Combination of cell surface markers and cell size for sorting cells
4- Electric charges combined with fluorescence tag cells Dieclectrophoresis (DEPArray)
7.2 How ctDNA can be used?
CtDNA can be used to provide a genetic landscape for a cancerous lesion (primary and metastases). This can be used for diagnostic but also for prognostic (detection of early recurrence(s)) and treatment purposes (predict the response to treatment but also detecting the development of acquired resistance). ctDNA can also offer an opportunity to track genomic evolution.
7.3 How many types of partitions are available for dPCR and how they differ from each other?
There are effectively two main methods for generating the partitions for a dPCR: prefabricated reaction wells (in a chip or plate) or droplets (water-in-oil emulsions).
7.4 What are the main differences between the commercially available dPCR platforms?’
Number of partitions, real-time PCR amplification of single molecules vs endpoint system, throughput, hands on time, turn-around time and multiplexing capability
7.5 What are the main applications for dPCR?
Applications to which Dpcr is particularly suited include; gene expression, rare allele detection, Copy number variation, NGS library quality control and single cell analysis.
7.6 What are the main advantages of dPCR in GMO testing?
1) dPCR enables the determination of absolute target copy numbers present in a reaction without the need for a reference material. This eliminates bias of amplification efficiency between the taxon and target. 2) dPCR allows the quantification of low target quantities which increases the reliability GMC detection. 3) Droplet dPCR allows multiplexing of 2-8 targets in one reaction allowing multiple GMO detection in one test. 4) Assays that work on qPCR platforms are easily transferred to dPCR platforms.
7.7 What are the main advantages of dPCR in viral load testing?
In this context dPCR is valuable for the development of certified reference materials which will further contribute to the understanding of analytical performance characteristics and promote clinical data comparability. dPCR provides precise results for low concentrations of viral materials and has demonstrated good accuracy in discriminating different genotypes of the same virus. Furthermore, it has been successfully applied in a one-step format for the quantification of RNA viruses showing tolerance to inhibitors and providing results of higher accuracy than qPCR
7.8 What is the current method used for molecular monitoring of MRD in haematological malignancies? What are the drawbacks of this? List the main advantages of dPCR in the investigation of these malignancies?
RT-qPCR is the gold-standard for monitoring MRD and is routinely applied for this purpose. However, it has inherent limitations related to the reduced precision at the lower end of the calibration curve in addition to significant variation in assay performance between different laboratories. The use of conversion factors and the introduction of international reference materials to act as calibration standards have helped mitigate this, but integration into protocols is not straightforward. RT-dPCR has unique advantages for MRD monitoring as it simplifies the standardization procedure and improves on both the sensitivity and precision of measurement. Another application for RT-dPCR is to value assign reference materials which can be used either for the calibration of secondary ‘in-house’ control materials or for directly quantifying transcript copy numbers.
7.9 What are the main advantages of dPCR in solid tumours?’
dPCR is a quick, cheap and sensitive method to quantify biomarkers used to monitor the effectiveness of treatment and identify disease progression.
Discussion question: