Droplet Digital PCR: A New Technology for Detection and Quantification of Chimerism After Allogenic Hematopoietic Stem Cell Transplantation
Journal Title: Biomedical Journal of Scientific & Technical Research (BJSTR) - Year 2019, Vol 13, Issue 4
Abstract
Allogeneic hematopoietic stem cell transplantation (allo-SCT) is today an established cure for many malignant and non-malignant hematological disorders. The post-allo-SCT monitoring is very important in the engraftment phase to detect graft failure, but also in the following of post-SCT to detect the risk of disease relapse in malignant disease. The quantification of chimerism is the determination of the proportion of donor cells in recipient blood after allo-SCT. Currently, two complementary molecular biology techniques, STR PCR and qPCR methods, are routinely performed in laboratories. The practical principle is, after to have obtained informative marker between recipient and donor, the chimerism of donor/recipient is quantified by the informative markers. The STR method is based on amplification of 16-18 STR loci, which are short base sequences on chromosomes distributed throughout the genome. Each STR marker is a set (or system) of many alleles, differing in the number of tandem repeats of this sequence. After their amplification, the separation of STR products and fluorescence detection are performed on a capillary electrophoresis instrument. The calculation of amount of donor or recipient chimerism is based on ratio of informative donor and recipient signals [1]. The real-time PCR (qPCR) using Taqman technology has been developed for the chimerism analysis using insertion/deletion polymorphisms [2]. 30 to 50 probes are identified by the manufacturers. The chimerism assay qPCR protocol includes amplifications of donor -specific allele, recipient specific alleles and active reference on DNA samples of post-SCT and donor and recipient pre-SCT. The relative quantification of recipient chimerism is calculated in function of donor and recipient pre-SCT values. These two techniques have been showed accurate and reproducible. However, their use is mainly limited by their range of analysis, ie 80% - 5% for STR, 30% -0.1% for RQ-PCR [3-5], which requires managing two techniques within the laboratories. Indeed, STR assay is better method than qPCR to detect mixed chimerism after SCT for hemoglobinopathy whereas qPCR is better to detect the minimal residual disease after SCT for leukemia, because STR method lacks sensitivity. Furthermore, the recent development of micro-transplantations (chimerism < 1%) requires a chimerism technology with a very low sensitivity. Thus, a chimerism technique that combines both a large range of analysis and very low sensitivity would be very interesting. So, the digital droplet PCR (ddPCR) could be a good alternative to these issues.
Authors and Affiliations
Pascal Pedini, Noura Kouba, Matthieu Riquier, Sophie Simon, Agnès Basire, Frédéric Fina, Claire Galambrun, Jacques Chiaroni, Christophe Picard
Keywords
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- EP ID EP585807
- DOI 10.26717/BJSTR.2019.13.002420
- Views 134
- Downloads 0
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