title: Lymphocyte enrichment using CD81-targeted immunoaffinity matrix.
abstract: 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 105nucleated 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.
authors.affiliation: CLIP - Childhood Leukemia Investigation Prague, Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University Prague and University Hospital Motol, Prague, Czech Republic.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Pelák
authors.affiliation: CLIP - Childhood Leukemia Investigation Prague, Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University Prague and University Hospital Motol, Prague, Czech Republic.
authors.forename: Daniela
authors.initials: D
authors.lastname: Kužílková
authors.affiliation: CLIP - Childhood Leukemia Investigation Prague, Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University Prague and University Hospital Motol, Prague, Czech Republic.
authors.forename: Daniel
authors.initials: D
authors.lastname: Thürner
authors.affiliation: IBA GmbH, Göttingen, Germany.
authors.forename: Marie-Luise
authors.initials: ML
authors.lastname: Kiene
authors.affiliation: IBA GmbH, Göttingen, Germany.
authors.forename: Kristian
authors.initials: K
authors.lastname: Stanar
authors.affiliation: CLIP - Childhood Leukemia Investigation Prague, Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University Prague and University Hospital Motol, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Stuchlý
authors.affiliation: CLIP - Childhood Leukemia Investigation Prague, Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University Prague and University Hospital Motol, Prague, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Vášková
authors.affiliation: CLIP - Childhood Leukemia Investigation Prague, Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University Prague and University Hospital Motol, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Starý
authors.affiliation: CLIP - Childhood Leukemia Investigation Prague, Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University Prague and University Hospital Motol, Prague, Czech Republic.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Hrušák
authors.affiliation: IBA GmbH, Göttingen, Germany.
authors.forename: Herbert
authors.initials: H
authors.lastname: Stadler
authors.affiliation: CLIP - Childhood Leukemia Investigation Prague, Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University Prague and University Hospital Motol, Prague, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Kalina
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival / Q000276:immunology; D005362:Ficoll / Q000737:chemistry; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D008214:Lymphocytes / Q000166:cytology* / Q000276:immunology; D060225:Tetraspanin 28 / Q000737:chemistry / Q000378:metabolism
chemical_list: D000906:Antibodies; D060225:Tetraspanin 28; D005362:Ficoll
keywords: CD81; CyTOF; Ficoll-Paque; T-catch™; human PBMC; mass cytometry; platelets; viSNE