FlowSpot Enables Decentralized Phenotypic and Functional Cellular Immune Profiling from Dried Blood Spots.

Abstract

Expanding access to cellular immune analysis is essential for decentralized clinical care, clinical trials, and population-based research. However, current flow cytometry workflows require rapid processing of fresh blood, proximity to a centralized laboratory, and cold chain logistics. Although dried blood spots (DBS) have transformed decentralized molecular diagnostics, no comparable approach has enabled robust flow cytometric analysis of immune cells. Here, we present FlowSpot, a novel platform that enables recovery of leukocytes from DBS and preserves their immunophenotypic characteristics, allowing downstream flow cytometric analysis following ambient-temperature storage and shipment. FlowSpot recovers intact leukocytes while preserving immune cell subset frequencies with strong concordance to fresh whole blood. We demonstrate its clinical utility by enabling remote CD4⁺ T cell immunophenotyping in people living with HIV, showing high agreement with routine clinical measurements across a broad range of CD4⁺ T cell frequencies. Beyond cellular phenotyping, FlowSpot extends immune monitoring to functional profiling by enabling detection of intracellular cytokine responses, including IFNγ, IL-2, and TNFα production by CD4⁺ and CD8⁺ T cells following ex vivo PMA/ionomycin stimulation. By overcoming a longstanding barrier to leukocyte recovery from DBS, FlowSpot extends flow cytometry beyond specialized laboratories, expanding access to cellular immune analysis for clinical care, decentralized clinical trials, and population-scale immunology.