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- Cossarizza, A., et al.
(författare)
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Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)
- 2019
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Ingår i: European Journal of Immunology. - : Wiley. - 0014-2980 .- 1521-4141. ; 49:10, s. 1457-1973
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Tidskriftsartikel (refereegranskat)abstract
- These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
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- Rassner, M., et al.
(författare)
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Development of Highly Sensitive Digital Droplet PCR for Detection of cKIT Mutations in Circulating Free DNA That Mediate Resistance to TKI Treatment for Gastrointestinal Stromal Tumor (GIST)
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067. ; 24:6
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Tidskriftsartikel (refereegranskat)abstract
- Background: Mutations in cKIT or PDGFRA are found in up to 90% of patients with gastrointestinal stromal tumors (GISTs). Previously, we described the design, validation, and clinical performance of a digital droplet (dd)PCR assay panel for the detection of imatinib-sensitive cKIT and PDFGRA mutations in circulating tumor (ct)DNA. In this study, we developed and validated a set of ddPCR assays for the detection of cKIT mutations mediating resistance to cKIT kinase inhibitors in ctDNA. In addition, we cross-validated these assays using next generation sequencing (NGS). Methods: We designed and validated five new ddPCR assays to cover the most frequent cKIT mutations mediating imatinib resistance in GISTs. For the most abundant imatinib-resistance-mediating mutations in exon 17, a drop-off, probe-based assay was designed. Dilution series (of decreasing mutant (MUT) allele frequency spiked into wildtype DNA) were conducted to determine the limit of detection (LoD). Empty controls, single wildtype controls, and samples from healthy individuals were tested to assess specificity and limit of blank (LoB). For clinical validation, we measured cKIT mutations in three patients and validated results using NGS. Results: Technical validation demonstrated good analytical sensitivity, with a LoD ranging between 0.006% and 0.16% and a LoB ranging from 2.5 to 6.7 MUT fragments/mL. When the ddPCR assays were applied to three patients, the abundance of ctDNA in serial plasma samples reflected the individual disease course, detected disease activity, and indicated resistance mutations before imaging indicated progression. Digital droplet PCR showed good correlation to NGS for individual mutations, with a higher sensitivity of detection. Conclusions: This set of ddPCR assays, together with our previous set of cKIT and PDGFRA mutations assays, allows for dynamic monitoring of cKIT and PDGFRA mutations during treatment. Together with NGS, the GIST ddPCR panel will complement imaging of GISTs for early response evaluation and early detection of relapse, and thus it might facilitate personalized decision-making.
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