Assays of cellular immunity are key to understanding the pathogenesis and mechanisms of control of viral and other infectious diseases. But such assays are difficult to perform as part of clinical studies because they are: Labile: They must either be performed on fresh blood, or on PBMC that are cryopreserved within a relatively short time after blood collection. Laborious: They require a lot of manual effort, as well as skills ad equipment not commonly found at clinical sites. Sample-intensive: They tend to require large volumes of blood, particularly if performed on cryopreserved PBMC. These challenges have limited the implementation of cellular immune function assays, particularly in children (where blood draw volumes are most limited) and in remote settings. Yet, children in remote settings are often the most affected by important infectious diseases, and stand the most to gain from advances in vaccines and other methods of control. Therefore, we propose to develop a sample-sparing and fully automated system for stimulation and stabilization of whole blood for functional cellular assays. This system will be based on the concepts pioneered by Smart Tube, Inc. in their existing whole blood stimulation system, but will use 80% less blood, and provide full automation, so that all pipetting steps are eliminated. It will draw blood directly from a collection tube, using as little as 2 cc, distribute the blood into multiple incubation chambers where it will be stimulated with lyophilized, pre-configured reagents (antigens, mitogens, etc.) to assay cellular function, then stabilized with a proteomic stabilizer for later analysis. In Specifi Aim 1, we will develop this prototype system (through a subcontract with Smart Tube, Inc.) and test it on control samples in the Stanford Human Immune Monitoring Center. Two beta units will be built out for further testing.
In Specific Aim 2, we will test these beta units at two clinical ites in Kenya through a subcontract with the Technical University of Mombasa. These clinical sites will collect blood samples from children infected with chikungunya and/or dengue virus. They will use the beta systems to stimulate and stabilize these blood samples, and ship them to Stanford for analysis by state-of-the art CyTOF mass cytometry. The results of this study will not only validate the blood collection system's performance, but will provide valuable biological data on the cellular response to these viruses in children. The blood collection system, in turn, will b useful for many different kinds of studies of cellular immunity, particularly in remote settings an situations where samples are limiting. The end result should be much more rapid advancement of our understanding of disease pathogenesis and of vaccine development for important infectious agents.
Measurements of cellular immune responses are key to understanding the pathogenesis and control of viral and other diseases, but methods to measure these responses are laborious and not suited to field settings. In this project, we will build and test an automated system for stimulating and stabilizing very small volumes of blood for such cellular assays. This system will greatly aid the conduct of field studies of viral and other infectious diseases, especially those involving children and/or remote settings.
| Subrahmanyam, Priyanka B; Maecker, Holden T (2017) CyTOF Measurement of Immunocompetence Across Major Immune Cell Types. Curr Protoc Cytom 82:9.54.1-9.54.12 |
