Microfluidic evaluation of red cells collected and stored in modified processing solutions used in blood banking

Abstract

The most recent American Association of Blood Banks survey found that 40,000 units of blood are required daily for general medicine, hematology/oncology, surgery, and for accident and trauma victims. While blood transfusions are an extremely important component of critical healthcare, complications associated with transfusion of blood components still exist. It is well-established that the red blood cell (RBC) undergoes many physical and chemical changes during storage. Increased oxidative stress, formation of advanced glycation endproducts, and microparticle formation are all known to occur during RBC storage. Furthermore, it is also known that patients who receive a transfusion have reduced levels of available nitric oxide (NO), a major determinant in blood flow. However, the origin of this reduced NO bioavailability is not completely understood. Here, we show that a simple modification to the glucose concentration in the solutions used to process whole blood for subsequent RBC storage results in a remarkable change in the ability of these cells to stimulate NO. In a controlled in vitro microflow system, we discovered that storage of RBCs in normoglycemic versions of standard storage solutions resulted in RBC-derived ATP release values 4 weeks into storage that were significantly greater than day 1 release values for those RBCs stored in conventional solutions. During the same storage duration, microfluidic technologies enabled measurements of endothelium-derived NO that were stimulated by the ATP release from the stored RBCs. In comparison to currently accepted processing solutions, the NO production increased by more than 25% in the presence of the RBCs stored in the normoglycemic storage solutions. Control experiments using inhibitors of ATP release from the RBCs, or ATP binding to the endothelium, strongly suggest that the increased NO production by the endothelium is directly related to the ability of the stored RBCs to release ATP. We anticipate these findings to represent a starting point in controlling glucose levels in solutions used for blood component storage, especially considering that current solutions contain glucose at levels that are nearly 20-fold greater than blood glucose levels of a healthy human, and even 10-fold greater than levels found in diabetic bloodstreams.

Restoration of intracellular ATP production in banked red blood cells improves inducible ATP export and suppresses RBC-endothelial adhesion

“…We demonstrate that sRBCs have blunted ATP export even when the Hb conformation is taken into account (Fig. 1C), thus substantiating previous reports for impaired ATP export in these RBCs (41,43).…”

“…Along these lines, the recent publication by Sikora et al (36) reemphasizes the need to stringently monitor hemolysis, although surprisingly, their limited study design did not specifically target approaches that safeguarded against hemolysis-induced increases in extracellular ATP, thus limiting generalization and interpretation of the investigation. Nonetheless, our present observations confirm those of numerous other investigators who have, like us, reported inducible and lysis-independent ATP export from intact, healthy human RBCs (3,7,13,15,24,38,41,43).…”

Centrifugation-induced release of ATP from red blood cells

“…In comparison, for many of the measurements listed in Table 1, the ATP yielded by centrifugation observed here is negligible. For some experiments with microbore tubing [18, 19, 23], however, the reported ATP concentrations are comparable to those yielded by centrifugation.…”

“…Later work employed continuous flow systems comprised of microbore tubing [7, 8, 14], which yielded a similar correlation between decreased internal tubing diameter and increased ATP concentration. More recent work using microbore tubing has evaluated the effects of storage methods on ATP release from RBCs [23]. The advent of microfluidics afforded the unique opportunity to combine continuous flow detection of ATP with systems on the length scale of the microvasculature.…”

A microfluidic interface for the culture and sampling of adiponectin from primary adipocytes

“…Additionally, due to the volume or number of cells needed for well-plates and downstream assays, multiple animals (typically mice or rats) are required for even simple studies 10, 11 . Offering alternatives to static sampling methodology, microfluidic systems not only allow for significant reductions in cell numbers, sample volumes, and reagents needed, but such constant-flow culture systems are arguably better representations of in vivo, biological conditions 12–14, 15, 16 .…”