Point-of-collection whole blood fixation enables robust phosphoprotein profiling by mass cytometry for longitudinal clinical trials

Phosphoprotein profiling by cytometry is increasingly used as a pharmacodynamic readout in clinical trials, yet sample handling remains a critical and under addressed challenge. Annual JAK inhibitor trial counts increased from 61 in 2015 to 206 in 2024. Phosphoprotein signaling states are highly labile ex vivo, and without immediate stabilization, phosphorylation can shift from its in vivo state within minutes of collection. Conventional PBMC isolation and cryopreservation compound this problem. Ficoll centrifugation introduces handling-induced phosphosignaling artifacts, and freeze-thaw stress depletes monocytes and other fragile populations. Together, these effects suppress basal signals and often make ex vivo stimulation necessary to detect any signal at all. These limitations are particularly acute in longitudinal clinical trials, where samples must be shipped from remote clinical sites to central labs before processing. Here we describe a workflow that integrates point-of-collection whole blood fixation using TokuKit with phospho-mass cytometry, enabling phosphoprotein profiling across peripheral blood immune cell subsets.