A multiparameter whole blood platform for assessment of anti-CMV immunity in allogeneic stem cell transplantation recipients.

Human cytomegalovirus (HCMV) infection remains a major challenge in allogeneic stem cell recipients. Current immune monitoring largely relies on IFN-γ-based readouts following ex vivo stimulation, which cannot fully capture the complex anti-HCMV immune defense. Therefore, we established a multiparameter HCMV-reactive whole blood (WB) assay that can be initiated at the bedside, requires minimal blood volume, preserves physiological cellular interactions, and enables integrated cellular and cytokine readouts within a closed-tube workflow. Using enhanced α-CD40 co-stimulation for robust detection of T-cell activation markers without widely used exocytosis inhibitors, our assay facilitates parallel flow cytometric quantification of activation-induced HCMV-reactive CD4+ and CD8+ T-cell responses and multiplexed detection of secreted cytokines. In parallel, our WB platform allows multimer-based detection of HCMV-specific CD8+ T cells and quantification of additional cell populations implicated in anti-HCMV protection, including Vδ1+ γδ T cells, "memory-like" natural killer (NK) cells, and HCMV-specific B cells. Across matched samples, readouts in WB and isolated peripheral blood mononuclear cells showed strong correlation. High intra-assay precision was achieved for key readouts across several immune cell compartments. Overall, our WB platform offers a simple, modular framework to advance post-transplant HCMV immune profiling while also being adaptable to other pathogens and immunological applications.