A Mass Cytometry-Based Blood Cell Phenotyping Workflow Enabling Inclusion of Resource-Limited and Rural Sites in Immune System Studies.

Insight into disease detection and treatment through comprehensive immune phenotyping relies on the generation of high-quality data. However, the execution of robust immune monitoring in clinical trials by flow cytometry is complex due to logistical challenges in sample preparation and reagent stability. These challenges are exacerbated when considering remote and rural communities in Australia, which are burdened by both an increased prevalence and a worse prognosis of many chronic and infectious pathologies. This is not unique to the Australian context as globally remote and rural communities are underrepresented in biomedical research studies, contributing to persistent health inequities. To address these challenges, we have harnessed the unique benefits of mass cytometry to develop and test a workflow that enables inclusion of remote communities in high-parameter immune phenotyping studies. The stability of heavy-metal conjugated antibodies and the lack of any signal derived from cellular autofluorescence allows samples stained for mass cytometry to be cryopreserved and stored long-term. In this approach, whole blood (WB) samples are collected and stained fresh with dry-format antibodies specific for 38 surface targets, then cryopreserved without washing. By removing the need for centrifugation, we enable staining and preservation of fresh samples in resource-limited settings without access to specialized equipment. Samples are delivered to the central site, where neutrophils are removed and samples are barcoded with a 6-channel cocktail, pooled and stained for an additional 8 intracellular targets, achieving 52-plex coverage. In a pilot to demonstrate workflow feasibility, we characterized blood immune and myeloid cell populations from cancer patients recruited at 3 sites across Australia, including 26 patients recruited from Dubbo Base Hospital which is located approximately 400 km from the state capital city, Sydney. We find consistency in immune phenotype between WB samples prepared with our simplified WB approach and matched PBMC samples prepared in parallel. This is the first time, to our knowledge, that a multi-centre high-parameter immune phenotyping study has included cancer patients from regional Australia. In summary, we show here that the preparation of samples for high-dimensional mass cytometry can be simplified to be compatible with resource-limited environments while conserving data quality.