In mass cytometry, the isolation of pure lymphocytes is very important to obtain reproducible results and to shorten the time spent on data acquisition. To prepare highly purified cell suspensions of peripheral blood lymphocytes for further analysis on mass cytometer, we used the new CD81+ immune affinity chromatography cell isolation approach. Using 21 metal conjugated antibodies in a single tube we were able to identify all basic cell subsets and compare their relative abundance in final products obtained by density gradient (Ficoll-Paque) and immune affinity chromatography (CD81+ T-catch™) isolation approach. We show that T-catch isolation approach results in purer final product than Ficoll-Paque (P values 0.0156), with fewer platelets bound to target cells. As a result acquisition time of 105 nucleated cells was 3.5 shorter. We then applied unsupervised high dimensional analysis viSNE algorithm to compare the two isolation protocols, which allowed us to evaluate the contribution of unsupervised analysis over supervised manual gating. ViSNE algorithm effectively characterized almost all supervised cell subsets. Moreover, viSNE uncovered previously overseen cell subsets and showed inaccuracies in Maxpar™ Human peripheral blood phenotyping panel kit recommended gating strategy. These findings emphasize the use of unsupervised analysis tools in parallel with conventional gating strategy to mine the complete information from a set of samples. They also stress the importance of the impurity removal to sensitively detect rare cell populations in unsupervised analysis. © 2016 International Society for Advancement of Cytometry.
- MeSH
- antigeny CD81 chemie metabolismus MeSH
- Ficoll chemie MeSH
- leukocyty mononukleární cytologie MeSH
- lidé MeSH
- lymfocyty cytologie imunologie MeSH
- obrazová cytometrie metody MeSH
- protilátky chemie imunologie MeSH
- separace buněk metody MeSH
- viabilita buněk imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Cytomegalovirus (CMV) specific T-cells are known to provide long-term control of CMV reactivation, which is a frequent complication of hematopoietic stem cell transplantation. We have studied 58 pediatric patients after hematopoietic stem cell transplantation who suffered from CMV reactivation to reveal which functional T cell subset is best correlating with successful reactivation resolution and which protects from reactivation episode. METHODS: Detection of 30 combinatorial subsets of four types of response to ex vivo CMV stimulation (IFNγ secretion, IL-2 secretion, CD40L upregulation and degranulation) that were detectable on either CD8+ or CD4+ T cells through flow cytometry intracellular cytokine staining was used. RESULTS: We found that the presence of CD8+ dual positive (IFNγ+ and IL-2+) cells is the most accurate functional parameter that can predict fast resolution of CMV reactivation. Next, we show that the presence of CD8+ dual positive (IFNγ+ and IL-2+) and CD8+ IFNγ+ cells provides a protective effect (a hazard risk of 0.28 (confidence interval 0.18 - 0.43) and 0.45 (CI 0.27 - 0.75), respectively) and the presence of corticotherapy increases the risk of reactivation (HR 2.47 (CI 1.82-3.36)). Thus, a patient without corticotherapy and with both of the critical T cell subsets present has a cumulative 19.6 times lower risk of developing CMV reactivation than a patient on corticotherapy and without CD8+ dual positive (IFNγ+ and IL-2+) or CD8+ IFNγ+ cells. CONCLUSIONS: We have established parameters of CMV specific functional response ex vivo that can be used in assisting clinical management of patients with CMV reactivation. © 2015 International Clinical Cytometry Society.
- MeSH
- CD4-pozitivní T-lymfocyty metabolismus MeSH
- CD8-pozitivní T-lymfocyty metabolismus MeSH
- cytomegalovirové infekce virologie MeSH
- Cytomegalovirus metabolismus MeSH
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- předškolní dítě MeSH
- průtoková cytometrie metody MeSH
- T-lymfocyty - podskupiny imunologie MeSH
- transplantace hematopoetických kmenových buněk * metody MeSH
- viremie metabolismus MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
