Fig. 2. Lymphocyte subset analysis of the peripheral blood (Case 2) by flow cytometry using Primary Immunodeficiency Orientation Tube (PIDOT) panel. In this patient, naïve CD4⁺/CD8⁺ T-cells were markedly reduced along with B-cell subsets, but NK cell population was preserved. (A) After gating lymphocytes on CD45 channel, forward scatter channel (FSC) and side scatter channel (SSC), CD3 and CD19 in combination with TCRγδ were used to define lymphocytes subsets; T-cells (upper left), B-cells (lower right) and NK cells (lower left). (B) CD16⁺CD56 marker and CD3 marker were used to define NK cells (upper left). (C) The CD3⁺ T-cells were divided into CD4⁺, CD8⁺ and double negative T-cells (DNT). In this plot, the distribution of CD3⁺ T-cells showed an overall reduction. (D) The double negative T-cells were subdivided into two subpopulations based on the expression of TCRγδ; TCRγδ^- T-cells (upper left) and TCRγδ⁺ T-cells (upper right). (E) CD4⁺ T-cell subsets were subdivided into naïve (CD27⁺CD45RA⁺; upper right), central memory/transitional memory (CM/TM; CD27⁺CD45RA^-; upper left), effector memory (EM; CD27^-CD45RA^-; lower left) and terminally differentiated (TD; CD27^-CD45RA⁺; lower right) CD4⁺ T-cells. All CD4⁺ T-cell subsets were decreased, particularly lack of naïve CD4⁺ was pronounced. (F) CD8⁺ T-cell subsets were subdivided into naïve (CD27⁺CD45RA⁺; upper right), CM/TM (CD27⁺CD45RA^-; upper left), EM (CD27^-CD45RA^-; lower left), effector CD27^dim (middle right) and TD (CD27^-CD45RA⁺; lower right) CD8⁺ T-cells. In this case also, there was a decrease in the overall CD8⁺ T-cell subsets and severe reduction in naïve CD8⁺ T-cells. (G) Subdivision of the B-cell subsets demonstrated an overall reduction in pre-GC B-cells (CD27^-CD16^-CD56^-; lower left) and post-GC B-cells (CD27⁺CD16^-CD56^-; upper left). (H) Reduced pre-GC B-cells were further classified into mature naïve B-cells (CD27^-smIgM⁺IgD⁺⁺; upper right) and transitional/immature B-cells (CD27^-smIgM⁺IgD⁺; lower right). (I) Post-GC B-cells were sub-classified into unswitched MBC/PC (CD27⁺smIgM⁺IgD⁺; upper right) and switched MBC/PC (CD27⁺smIgM^-IgD^-; lower left).

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