1 Department of Clinical Medicine - Hæmatologisk Afdeling, Aalborg Sygehus, Department of Clinical Medicine, Health, Aarhus University2 Department of Clinical Medicine, Health, Aarhus University3 Service of Cytometry & Department of Medicine, CICancer - University of Salamanca, Spain4 unknown5 Department of Biomedicine - Forskning og uddannelse, Øst, Department of Biomedicine, Health, Aarhus University6 Department of Clinical Medicine, Health, Aarhus University7 Department of Biomedicine - Forskning og uddannelse, Øst, Department of Biomedicine, Health, Aarhus University
In order to improve insights into the B-cell biology and thereby B-cell myelomagenesis we have established a MSCNET standard for multiparametric flow cytometry (MFC) and cell sorting (FACS) for subsequent genetic analysis. The material analysed was fresh tonsils, blood and bone marrow. The method included homogenization, isolation of mononuclear cells, MFC and FACS sorting using multicolour fluorescence single tube panels.of antibodies against surface molecules as CD10/20/27/38/45, supplemented with tissue related antibodies. Isolated B-cell subpopulations were evaluated by morphological inspection and single gene expression analysis (qRT-PCR) for transcription factors as well as global gene expression profiling (GEP; GeneChip Human Exon 1.0 ST Array). For example for tonsils, based on the immunophenotypic presentation (including CD3/44/CXCR4 in the panel), B-cell subsets were identified and sorted, naïve, centroblast, centrocyte, memory, and plasmablasts. The identity of the tonsillar subpopulations was verified using qRT-PCR and exon microarray GEP based on the used discriminative phenotypic markers as well as transcriptions factors BACH2, BCL6, PAX5, IRF4, P27, PRDM1 and XBP1. Globally, the B-cell subpopulations identified have distinct gene expression profiles reflecting their functions but also revealing genes with subpopulation specific exon splicing. In conclusion a combination of surface markers expressed antigens and gene expression analysis of B cell subsets confirm a strong methodology to be used in myelomagenesis and evaluated as for prognostic information.