Comparison of analysis of bovine surface immunoglobulin bearing and peanut agglutinin binding lymphocytes by flow cytometry and fluorescence microscopy

Abstract

Bovine peripheral blood lymphocytes were examined for their binding to anti-immunoglobulin serum, peanut agglutinin, and μ, α, and ϵ heavy chain specific antisera by immunofluorescence. The percentage of total lymphocytes with positive staining was determined independently by flow cytometry and fluorescence microscopy. The correlation of data from both methods was best for analysis of total surface immunoglobulin and IgM bearing cells. The percentage of lymphocytes bearing surface immunoglobulin (B cells) was determined using both whole antiserum and a F(ab') 2 reagent. Quantitation by flow cytometry did not show a significant difference when the two reagents were used, whereas fluorescence microscopy revealed a significant difference ( p < .05). The mean percent of total surface immunoglobulin bearing cells was 30 ± 3% by either method. Flow cytometry gave significantly larger values than fluorescence microscopy for samples stained with fluorescein conjugated peanut agglutinin. Peanut agglutinin binding cells comprised 70 ± 3% by flow cytometry and 51 ± 3% by fluorescence microscopy. Similarly, there was a significant difference between both methods when IgA bearing lymphocytes were examined. Percentages of immunoglobulin E, A, and M bearing lymphocytes as well as total B and T cells in spleen and bronchial lymph node were determined by immunofluorescence using the cytofluorograph. Peanut agglutinin binding cells were less numerous in spleen and lymph node than in peripheral blood. Immunoglobulin E bearing lymphocytes increased from 0.07% in peripheral blood to 4% in spleen and 1.9% in lymph node. In this paper we demonstrate how flow cytometry can be used to examine a large number of samples in a rapid and reproducible manner. This is the first report in which bovine lymphocytes bearing surface IgE are quantitated.