Onsite fixation preserves phosphoprotein profile in human whole blood

Abstract

Phospho-flow cytometry is increasingly used as a pharmacodynamic readout of kinase-pathway activity, including in JAK inhibitor trials. Phosphoprotein states are intrinsically labile ex vivo and are further altered by conventional workflows, which typically involve PBMC isolation, cryopreservation, and transit times of hours to days before measurement. Here, we fix whole blood at collection and quantify six phosphoproteins by mass cytometry, benchmarked against matched cryopreserved PBMCs. Unlike PBMCs, whole blood retained neutrophils, ~75-80% of which were pSTAT3-positive at baseline. Basal pSTAT3 was also markedly higher in whole blood monocytes (~41% positive vs. ~9% in PBMCs), whereas T, B, and NK cells differed by less than 1 percentage point. Stimulation produced subset-specific phosphorylation patterns that were retained after 6 weeks of storage. Because fixation does not require centrifugation and retains neutrophils, samples can be fixed at collection sites without on-site processing laboratories and analyzed centrally in batches.