Fig. 1

The “Whiskey Method” provides unique access to malignant cells and facilitates detection of PCNSL. A Experimental scheme of the “Whiskey Method.” Brain biopsy was collected into a sterile sampling tube filled with 2 ml 0.9% sodium chloride. The sample container was swirled for 10 s, and the supernatant was immediately subjected to flow cytometric and scRNA-seq analyses. The biopsy tissue was sent for neuropathological evaluation. B Representative flow cytometry dot plots of CD45⁺ leukocytes and flow cytometry analysis of the percentage and number of CD45⁺ leukocytes in the samples of PCNSL (n = 8) and glioblastoma (n = 8) patients (Additional file 1: Table S1 and Additional file 2: Supplementary Methods). C Flow cytometry analysis of CD19⁺CD20⁺ B cells (Bc) within the CD45⁺ leukocyte gate in peripheral blood (n = 7) and biopsy samples (n = 8) of PCNSL samples. B cell subpopulations were further characterized by analyzing the distribution of CD27⁺CD20⁺, CD38⁺, CD38⁺CD27⁺, CD138+, CD5⁺CD10⁻, CD5⁻CD10⁺, and of Ig kappa or lambda light chain expressing cells. The gating strategy is shown in Additional file 4: Fig. S1. D Flow cytometry analysis of CD3⁺CD56^- T cells (Tc) and CD3⁻CD56⁺ NK cells in the CD45⁺ lymphocytes gate within peripheral blood (n = 7) and biopsy samples (n = 7) are shown. NK cells were further subclustered into CD56^dimCD16⁺ and CD56^brightCD16⁻ NK cells. T cells were further characterized by the percentage and ratio of CD4⁺ and CD8⁺ Tc, and CD4⁺CD25⁺IL7R⁻ Treg, as well as the distribution of naive T cells (Tn; CCR7⁺CD45RA⁺), central memory T cells (Tcm; CCR7⁺CD45RA^-), effector memory T cells (Tem; CCR7⁻CD45RA⁻), effector memory recently activated T cells (TEMRA; CCR7⁻CD45RA⁺). In addition, percentages of CD4⁺ and CD8⁺ T cells expressing HLA-DR and PD-1 were determined. Gating strategies are depicted in Additional file 4: Fig. S2. Data are depicted as mean, error bars show the SE; two-sided Mann-Whitney U test was used to calculate statistical significance between groups; * p<0.05, ** p<0.01, *** p <0.001, **** p <0.0001, ns not significant