Multicolor FlowRNA protocol to identify the phenotype and function of the EBV-infected lymphocytes. (A) Mononuclear cells were isolated from peripheral blood, stained for lymphocyte lineage markers, and then fixed and permeabilized. Intracellular markers were then stained, and the cells subjected to fluorescence ISH (FlowRNA) for the virally encoded EBERs and housekeeping cellular B2M. (B) EBV⁺/GFP⁺ Akata cells were added to EBV⁻/GFP⁻ Akata cells at decreasing percentages (100%, 3%, 1%) and examined by FlowRNA to determine the sensitivity and specificity of the assay. (C) Lymphocytes were identified by forward vs side scatter, and single cells were gated, dead, and myeloid cells were excluded from further analysis. (D) The stained cells were examined by flow cytometry and plotted as CD19, CD3, CD4, CD8, and CD56 vs EBER to determine the subset of cells infected with EBV (dual positive). The upper panel shows a representative assay performed on blood of a healthy EBV carrier where no EBV-infected cells were detected. The lower panel shows a representative assay performed on the blood of a T/NK LPD patient and clearly shows CD3⁺, CD8⁺, and CD19⁺ lymphocyte populations expressing EBERs. The percentage of total CD19⁺ B cells and total CD3⁺, CD8⁺ T cells is given in the upper righthand quadrant. FSC, forward scatter; PMN, polymorphonuclear leukocytes; RBC, red blood cell; SSC, side scatter.