29023707 OR Guidelines for the use of flow cytometry and cell sorting in immunological studies Dotaz Zobrazit nápovědu
- MeSH
- DNA analýza MeSH
- falešně pozitivní reakce MeSH
- imunofenotypizace MeSH
- imunologické techniky * MeSH
- lidé MeSH
- proliferace buněk MeSH
- průtoková cytometrie MeSH
- řízení kvality MeSH
- RNA analýza MeSH
- separace buněk MeSH
- směrnice jako téma * MeSH
- software MeSH
- T-lymfocyty cytologie MeSH
- výzkumný projekt MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
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.
BACKGROUND: CD157 has been recently reported as a useful glycosylphosphatidylinositol (GPI)-linked marker for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clones in patients with suspected paroxysmal nocturnal hemoglobinuria by flow cytometry as it targets both neutrophils and monocytes. The aim of this study is to test the feasibility of a non-fluorescent aerolysin (FLAER) approach and propose an alternative for laboratories, where FLAER is not available. METHODS: We validated a non-FLAER-based single-tube, 6-color assay targeting the GPI-linked structures CD157, CD24, and CD14. We determined its performance characteristics on 20 PNH patient samples containing a variety of clone sizes and compared results with a previously validated FLAER-based approach. RESULTS: Coefficient of variation (CV) for intra-/interassay precision analyses ranged from 0.1%/0.2% to 3.02%/7.58% for neutrophils and from 0.10%/0.3% to 5.39%/6.36% for monocytes. Coefficient of determination (r2 ) for linear regression analysis of PNH clones from 20 patients ranging from 0.06% to 99.7% was 0.99 in all cases, Wilcoxon ranks test showed no statistically significant differences (P > 0.05), Bland-Altman analysis demonstrated performance agreement with mean bias ranging from 0.06 to 0.2. CONCLUSION: Our results confirm very good performance characteristics for both intra- and interassay precision analyses, favorable correlation, and agreement between the FLAER and non-FLAER-based approaches, using the CD157 GPI marker. Our experience suggests that a rapid and cost-effective simultaneous evaluation of PNH neutrophils and monocytes by flow cytometry without using FLAER is possible in areas where FLAER may not be widely available. © 2016 International Clinical Cytometry Society.
- MeSH
- ADP-ribosylcyklasa imunologie metabolismus MeSH
- antigen CD24 imunologie metabolismus MeSH
- antigeny CD14 imunologie metabolismus MeSH
- bakteriální toxiny MeSH
- biologické markery metabolismus MeSH
- CD antigeny imunologie metabolismus MeSH
- cytotoxické proteiny tvořící póry MeSH
- GPI-vázané proteiny imunologie metabolismus MeSH
- lidé MeSH
- monocyty imunologie metabolismus MeSH
- neutrofily imunologie metabolismus MeSH
- paroxysmální hemoglobinurie imunologie metabolismus MeSH
- průtoková cytometrie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.
- MeSH
- CD antigeny metabolismus MeSH
- chronická lymfatická leukemie patologie terapie MeSH
- dospělí MeSH
- imunofenotypizace MeSH
- kombinovaná terapie MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- následné studie MeSH
- prognóza MeSH
- průtoková cytometrie normy MeSH
- reziduální nádor diagnóza genetika metabolismus MeSH
- staging nádorů MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
