A rapid sample preparation technique for flow cytometric analysis of immunofluorescence allowing absolute enumeration...

A simple and rapid method was developed for immunofluorescence measurements of cells by flow cytometry which does not require washing procedures, permitting absolute enumeration of cell subpopulations. Peripheral blood cells were labeled with fluorescein and phycoerythrin conjugated monoclonal antibodies and the nucleic acid stain LDS-751. Distilled water was added following incubation to induce erythrocyte lysis by hypotonic shock. After lysis for 30 s the tonicity of the sample was increased followed by measurement on the flow cytometer. The leukocyte populations were clearly resolved in the correlation of forward and orthogonal light scattering. The immunofluorescence resolution of the labeled leukocytes was equivalent to NH4Cl and a commercial lysing preparation. Absolute number of leukocytes and percentage of leukocyte subpopulations determined with this procedure correlated well with the results obtained with a clinical hematology analyzer. Cell recovery and preservation of cellular characteristics of three different procedures for lysing the human erythrocytes were compared. The LDS-751 permitted the discrimination of intact cells from residual erythrocyte ghosts, platelets and damaged nucleated cells. A considerable loss of cells was found for both NH4Cl and commercial lysing solution; the samples prepared by NH4Cl lysing had a selective loss of lymphocyte subpopulations as compared with the other two techniques. In contrast to the two procedures in which multiple washing steps are involved, the no wash, hypotonic lysis procedure provided a means of obtaining absolute numbers of leukocyte subpopulations identified by combining light scattering and immunofluorescence characteristics with no centrifugation steps required.