*Considerations for Utilizing Circulating Blood Cells for Respirometry Studies*

An important consideration for blood-based bioenergetics is selecting an appropriate cell type to use for respirometry experiments and whether mixed cell populations, such as PBMCs, are suitable samples for respirometry. Rausser et al. used a high-throughput mitochondrial phenotyping platform to compare PBMCs to leukocyte subtypes from the same individual [135]. They show large functional differences in both mitochondrial content and respiratory chain enzymatic activity between immune cell subtypes. Furthermore, PBMC cell type composition can vary between individuals and within the same individual over time. Therefore, they caution that using PBMCs for mitochondrial bioenergetics measurements can mask age- and sex-related changes. Furthermore, Darley-Usmar et al. have shown that respiration rates, the ratio of OXPHOS to glycolysis, as well as mitochondrial protein composition vary between circulating blood cell subpopulations [116]. For example, platelets have the highest basal and ATP-linked OCR compared with leukocyte cell types but lower maximal respiration and reserve capacity [116,136], which could alter bioenergetic results and interpretation if platelet contamination is present in the test samples.

Platelets are a common contaminant in human PBMC samples and vary depending on blood collection and PBMC isolation strategies [137]. Urata et al. have demonstrated that platelet contamination is common and can vary to a large extent amongst PBMC preparations [138]. They measured mtDNA copy number before and after platelet depletion and showed that the amount of platelet contamination varied between individuals and that PBMC mtDNA content is overestimated almost two-fold due to platelet contamination. This work was framed in the context of using PBMC mtDNA copy number as a potential biomarker to monitor disease but has implications for blood-based bioenergetics, as well. Additionally, cellular and mitochondrial metabolism including mitochondrial bioenergetic profiles changes in resting cells compared with activated cells [122,139]. Thrombinactivated platelets increase basal and ATP-linked oxygen consumption and glycolysis compared with control cells [140]. Therefore, it is important to consider cell activation, optimal collection, and isolation conditions when isolating platelets and leukocytes from whole blood [141,142].

For further information on respirometry in circulating blood cells, Braganza et al. have published a comprehensive review of blood-based bioenergetics outlining the research published in platelets and leukocytes as well as considerations for the use of blood-based bioenergetics as a translational and clinical tool [133]. While more research is needed to understand what cell populations are most representative of systemic mitochondrial function and specific diseases, blood-based bioenergetics offers a minimally invasive alternative to muscle biopsies and could provide a systemic biomarker of mitochondrial function and key readout for clinical trials. However, one major consideration for the utilization of this approach is that samples generally need to be collected and immediately processed to isolate circulating platelets and leukocytes from whole blood. Further isolating subpopulations of cells requires more starting material and processing. Therefore, there is still a need for a non-invasive sample that requires minimal on-site processing.
