Femtogram Resolution of Iron Content on a Per Cell Basis: Ex Vivo Storage of Human Red Blood Cells Leads to Loss of Hemoglobin.

The magnetic characteristics of hemoglobin (Hb) changes with the binding of dioxygen (O2) to the heme prosthetic groups of the globin chains: from paramagnetic ferrous Hb to diamagnetic ferrous oxyhemoglobin (oxyHb) with reversibly bound O2, or paramagnetic ferric methemoglobin (metHb). When multiplied over the number of Hb molecules in a red blood cell (RBC), the effect is detectable through motion analysis of RBCs in a high magnetic field and gradient. This motion is referred to as magnetophoretic mobility, which can be conveniently expressed as a fraction of the cell sedimentation velocity. In this Article, using a previously developed and reported instrument, cell tracking velocimetry (CTV), we are able to detect difference in Hb concentration in two RBC populations to a resolution of 1 × 107 Hb molecules per cell (4 × 107 atoms of Fe per cell or 4-5 femtograms of Fe). Similar resolution achieved with inductively coupled plasma-mass spectrometry requires on the order of 105-106 cells and provides an average, whereas CTV provides a measurement for each cell. CTV analysis revealed that RBCs lose, on average, 17% of their Hb after 42 days of storage, the maximum FDA-approved length of time for the cold storage of RBCs in additive solution. This difference in Hb concentration was the result of routine RBC storage; clinical implications are discussed.

Analysis of exosome release as a cellular response to MAPK pathway inhibition.

Exosome size distributions and numbers of exosomes released per cell are measured by asymmetric flow-field flow fractionation/multi-angle light scattering (A4F/MALS) for three thyroid cancer cell lines as a function of a treatment that inhibits MAPK signaling pathways in the cells. We show that these cell lines release exosomes with well-defined morphological features and size distributions that reflect a common biological process for their formation and release into the extracellular environment. We find that those cell lines with constitutive activation of the MAPK signaling pathway display MEK-dependent exosome release characterized by increased numbers of exosomes released per cell. Analysis of the measured exosome size distributions based on a generalized extreme value distribution model for exosome formation in intracellular multivesicular bodies highlights the importance of this experimental observable for delineating different mechanisms of vesicle formation and predicting how changes in exosome release can be modified by pathway inhibitors in a cell context-dependent manner.

Hemodynamics and tissue oxygenation after hemodilution with ultrahigh molecular weight polymerized albumin.

BACKGROUND: Compared to blood transfusion, plasma expanders (PEs) are more cost effective, have a longer shelf-life, and elicit a milder immune response. High molecular weight (MW) dextrans preserve microvascular function during extreme hemodilution. Dextrans, however, evokes negative hemostatic effects, including red blood cell (RBC) aggregation and reduce platelet adhesion, that limit their clinical use. Therefore, polymerization of human serum albumin (HSA) presents a simple strategy to increase HSA's molecular size. METHODS: This study was designed to test the hypothesis that polymerized HSA (PolyHSA) biophysical properties improves systemic and microvascular hemodynamics when used as a PE under anemic conditions. The study was implemented using the hamster window chamber model. Animals were first hemodiluted to 18% hematocrit (Hct) using 6% dextran 70 kDa and then to 11% Hct using either 10% PolyHSA, 10% unpolymerized HSA, or 6% dextran 70 kDa. Systemic and microvascular hemodynamics, including cardiac output (CO), mean arterial blood pressure (MAP), functional capillary density (FCD), microvascular perfusion, and oxygen tension were measured. RESULTS: Posthemodilution, PolyHSA improved MAP, CO, and oxygen delivery compared to HSA and dextran. Additionally, PolyHSA improved microvascular function in terms of blood flow and FCD. Although oxygen carrying capacity is limited at 11% Hct, tissue pO2 and oxygen delivery were higher for PolyHSA compared to HSA and dextran. CONCLUSION: PolyHSA during extreme anemia supported systemic and microvascular hemodynamics by increasing plasma viscosity without increasing vascular resistance. These findings can aid to design of studies to understand the role of the PE biophysical properties in clinical scenarios.

Ascorbic acid improves membrane fragility and decreases haemolysis during red blood cell storage.

BACKGROUND: Changes that occur to red blood cells (RBCs) during routine blood bank storage include decreased deformability, increased haemolysis and oxidative damage. Oxidative injury to the RBC membrane and haemoglobin can affect changes in shape and deformability. Ascorbic acid (AA) is an antioxidant that maintains haemoglobin in a reduced state and minimises RBC oxidative injury. We hypothesised that AA would improve membrane fragility and decrease haemolysis during storage. METHODS: Whole blood derived, AS-5 preserved, pre-storage leucoreduced RBC units were exposed to either AA or saline control solutions. Several rheological and biochemical parameters were measured serially during storage, including RBC membrane mechanical fragility, percent haemolysis and methaemoglobin levels. RESULTS: AA exposure significantly reduced mechanical fragility and haemolysis over the entire storage period. The highest two concentrations of AA affected the greatest reductions in mechanical fragility and percent haemolysis. Addition of AA to the RBCs did not significantly alter their biochemical parameters compared to control RBCs incubated with saline. CONCLUSION: AA reduced RBC membrane fragility and decreased haemolysis during storage without adversely affecting other RBC biochemical parameters. The clinical significance of these findings needs to be determined.