High-Throughput Metabolomics Platform for the Rapid Data-Driven Development of Novel Additive Solutions for Blood Storage

Abstract

Red blood cell transfusion is a life-saving intervention, and storage is a logistic necessity to make ~110 million units available for transfusion every year worldwide. However, storage in the blood bank is associated with a progressive metabolic decline, which correlates with the accumulation of morphological lesions, increased intra- and extra-vascular hemolysis upon transfusion, and altered oxygen binding/off-loading kinetics. Prior to storage, red blood cells are suspended in nutrient formulations known as additive solutions to prolong cellular viability. Despite a thorough expansion of knowledge regarding red blood cell biology over the past few decades, only a single new additive solution has been approved by the Food and Drug Administration this century, owing in part to the limited capacity for development of novel formulations. As a proof of principle, we leveraged a novel high-throughput metabolomics technology as a platform for rapid data-driven development and screening of novel additive solutions for blood storage under both normoxic and hypoxic conditions. To this end, we obtained leukocyte-filtered red blood cells (RBCs) and stored them under normoxic or hypoxic conditions in 96 well plates (containing polyvinylchloride plasticized with diethylhexylphthalate to concentrations comparable to full size storage units) in the presence of an additive solution supplemented with six different compounds. To inform this data-driven strategy, we relied on previously identified metabolic markers of the RBC storage lesion that associates with measures of hemolysis and post-transfusion recovery, which are the FDA gold standards to predict stored blood quality, as well as and metabolic predictors of oxygen binding/off-loading parameters. Direct quantitation of these predictors of RBC storage quality were used here—along with detailed pathway analysis of central energy and redox metabolism—as a decision-making tool to screen novel additive formulations in a multiplexed fashion. Candidate supplements are shown here that boost-specific pathways. These metabolic effects are only in part dependent on the SO2 storage conditions. Through this platform, we anticipate testing thousands of novel additives and combinations thereof in the upcoming months.

Supplementation with uric and ascorbic acid protects stored red blood cells through enhancement of non-enzymatic antioxidant activity and metabolic rewiring

“…Recently, through a high-throughput metabolomics platform, the addition of several metabolites was applied to test their influence upon energy and redox metabolism. For example, supplementation of stored RBCs with the antioxidant N-acetylcysteine seemed to beneficially impact the pentose phosphate pathway (PPP), while the addition of glutamine did not alter glutathione (GSH) synthesis, with both results being supported by other studies [ [10] , [11] , [12] ]. Incubation of stored RBCs in fruit and vegetable extracts showed that the bio-flavonoid naringin protected them from lysis and externalization of removal signals [ 13 ], whereas polyphenol quercetin had a minor impact [ 14 ].…”

A deep 96-well plate RBC storage platform for high-throughput screening of novel storage solutions

“…The final candidate(s) can then proceed to a full-unit storage study in the final storage configuration, followed by a clinical study to determine post-transfusion recovery of stored autologous RBCs. Recently, an accompanying paper from Nemkov et al has shown that the overall metabolic profile generated by this platform is comparable to that of RBCs stored in a standard storage bag, and successfully demonstrated the platform’s feasibility by applying it to examine the effect of spiking five different compounds into the AS-3 additive ( Nemkov et al, 2022 ).…”

“…This study was conducted under both the conventional normoxic (N) condition and hypoxic/hypocapnic (H) storage condition. The study was designed to investigate RBC samples stored under the H condition as well as the standard N storage condition because; i) H storage has been shown to significantly enhance the quality of stored RBCs (Han et al, 2010;Dumont et al, 2016;Nemkov et al, 2022); and ii) the currently approved ASs are not optimized for H storage.…”

“…The full utility of this approach is realized when the stored RBCs plates are subsequently processed using the high-throughput metabolomics/lipidomics workflow. Nemkov and colleagues recently demonstrated the feasibility of this approach by analyzing RBC plate samples generated by this platform to examine the overall metabolic and oxidative status of stored RBCs in AS-3 spiked with five supplemented compounds ( Nemkov et al, 2022 ).…”

Dried blood spot characterization of sex‐based metabolic responses to acute running exercise

“…One main roadblock to the feasibility of remote testing of at home collected blood samples for these emerging medical applications is not only tied to collection and shipment protocols, but also to the intrinsically elevated costs of metabolomics studies. To tackle this issue, we recently developed an ultra‐high‐throughput metabolomics method that enables the analysis of ∼1000 samples/day, making it economically and logistically feasible to scale metabolomics testing for large volumes of samples 74 . Coupling of dried blood field sampling and ultra‐high‐throughput omics approaches can pave the way for the dissemination of metabolomics from a laboratory science to a more widely applicable discipline; a revolution foretold by early application of metabolomics approaches in the fields of clinical biochemistry 75 and medical diagnostics and prognostics, especially when coupled to machine learning approaches 76 .…”