Chapple, Mary R.; (1992) Subpopulations of human bone marrow b cells: a phenotypic study. Doctoral thesis (M.D.), University College Hospital Medical School.

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Abstract

The regulatory mechanisms that monitor the size of the peripheral B-cell pool and determine cell death or survival are poorly understood. In rodents, B-lymphopoiesis is maintained at a high rate throughout adult life and under resting conditions there is little recruitment into the long-lived peripheral pool; it therefore follows that the majority of newly-formed B lymphocytes have a very short life-span. The maturation stages of B-lymphocytes in man and experimental mammals appear to be similar. We have determined the phenotype of sIgM and sIgD-expressing cells from normal adult human bone marrow, peripheral blood and tonsil, by dual immunofluorescence with an extensive panel of monoclonal antibodies representative of major B cell clusters, in order to identify antigenic differences which might play a regulatory role. All slgM+ bone marrow, peripheral blood and tonsil lymphocytes expressed HLA class n antigens and the antigens recognised by the CD19, CD20, CD37 and CD40 clusters. Antibodies of the CD21, CD22, CD9 and CD54 clusters and anti-IgD were reactive with different proportions of slgM+ cells in blood and bone marrow: 29.5% (range 5-60%) of slgM+ cells in marrow were sIgD-ve and most of these cells were also CD21-ve and CD22-ve, thus defining a unique marrow population. Almost all CD54+ bone marrow cells were IgD-ve. Newly-formed and mature recirculating cells comprising the slgM+ slgD-ve population could not however, be distinguished by the panel of antibodies. Whilst carrying out dual immunofluorescence phenotyping studies on human tonsil B cells using the fluorescence activated cell analyser, it was noted that in some experiments the fluorescence of one dye appeared to be quenched by the other. This phenomenon was investigated in detail. The dye pair fluorescein and R-phycoerythrin have been widely employed for dual fluorescence analysis using a single-laser fluorescence activated cell analyser and interaction between the two dyes has not been observed. Evidence is presented in this thesis that at high concentrations R-phycoerythrin can completely quench the fluorescein signal in dual fluorescence analysis of human tonsil lymphocytes labelled with pairs of monoclonal antibodies. Reduction of the fluorescein signal correlated with the amount of R-phycoerythrin attached and the relative intensity of emission from the two fluorochromes. This phenomenon can seriously compromise the interpretation of dual immunofluorescence carried out on a single-laser instrument.

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