Mass Cytometry Workflow to Achieve High-Dimensional Immunophenotyping in Resource-Limited or Decentralized...

Globally, regional and remote communities are burdened by both an increased prevalence and worse prognosis of many infectious and chronic diseases. However, largely owing to logistical challenges, these communities are under-represented in clinical trials and research studies. As individuals from rural communities experience unique environmental exposures and risk factors for disease, immune phenotyping data collected from metropolitan populations may not be broadly generalizable. To address this, we present a workflow that enables the inclusion of resource-limited sites in high-parameter mass cytometry studies. In this approach, whole blood (WB) or peripheral blood mononuclear cells (PBMCs) are collected, stained fresh for surface antigens, and cryopreserved at the collection site. Samples are then shipped to the central site for further processing, including neutrophil depletion, fixation, barcoding, intracellular staining, and data acquisition. Importantly, the WB staining approach does not require specialized equipment such as centrifuges and is therefore feasible to perform in a resource-limited environment. A support protocol details steps for data preprocessing and cleanup. We present example data demonstrating the application of this workflow to determine immune differences between eight patients with late-stage lung cancer and four healthy blood donors. Overall, this workflow may improve access to underserved communities and facilitate, for the first time, the scalability of immune phenotyping studies to harness geographically dispersed clinical centers. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation and staining of PBMCs for cytometry Basic Protocol 2: Preparation and staining of whole blood for cytometry Basic Protocol 3: Fixation, permeabilization, intracellular staining, and data acquisition for blood sample immunophenotyping Support Protocol: Data preprocessing and cleanup.