Onsite fixation preserves phosphoprotein profile in human whole blood
- 1Teiko
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.
Related
- Simvastatin Preserves Human Microvascular Endothelial Barrier Function Through Mevalonate-Dependent Regulation of RhoB Localization and Activation
- Extended normothermic machine perfusion preserves viability and tissue integrity of ex vivo human intestine segments
- Diffusion tensor imaging of whole human brains during long-term formaldehyde fixation: Temporal evolution of diffusion parameters, post-mortem conditions, and dependence on tissue structure
- Farm exposure is associated with human milk immune profile and microbiota
- Identification and characterization of a human antibody profile inversely associated with adverse cardiovascular events
BibTeX
@misc{shang2026onsite,
title = {{Onsite fixation preserves phosphoprotein profile in human whole blood}},
author = {L. Shang and M. Coughlin and C. Lancaster and A. Huang and J. A. Jarrell and L.-C. Cheng},
howpublished = {bioRxiv},
year = {2026},
month = nov,
doi = {10.64898/2026.11.05.763716},
url = {https://www.biorxiv.org/content/10.64898/2026.11.05.763716v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Onsite fixation preserves phosphoprotein profile in human whole blood
AU - L. Shang
AU - M. Coughlin
AU - C. Lancaster
AU - A. Huang
AU - J. A. Jarrell
AU - L.-C. Cheng
PY - 2026
DA - 2026/11/05
PB - bioRxiv
DO - 10.64898/2026.11.05.763716
UR - https://www.biorxiv.org/content/10.64898/2026.11.05.763716v1
AB - 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.
ER -