title: Laser-based microdissection of single cells from tissue sections and PCR analysis of rearranged immunoglobulin genes from isolated normal and malignant human B cells.
abstract: Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the isolated cells rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of V gene family-specific primers recognizing nearly all V gene segments together with primers for the J gene segments. By sequence analysis of V genes from distinct cells, the clonal relationship of the B lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D, and J gene usage. Moreover, the presence and pattern of somatic Ig V gene mutations give valuable insight into the stage of differentiation of the B cells.
authors.affiliation: Institute of Cell Biology (Cancer Research), Medical School, University of Duisburg-Essen, Essen, Germany. ralf.kueppers@uk-essen.de
authors.forename: Ralf
authors.initials: R
authors.lastname: Küppers
authors.forename: Markus
authors.initials: M
authors.lastname: Schneider
authors.forename: Martin-Leo
authors.initials: ML
authors.lastname: Hansmann
mesh_terms: D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D019286:Endopeptidase K / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D007834:Lasers; D042282:Microdissection / Q000295:instrumentation*; D009369:Neoplasms / Q000473:pathology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D059748:Proteolysis; D017421:Sequence Analysis; D059010:Single-Cell Analysis; D013194:Staining and Labeling
chemical_list: D007135:Immunoglobulin Variable Region; D019286:Endopeptidase K