Rouleaux Formation of Live Human RBCs Affected by Oxygen and Heat

다국어 초록 (Multilingual Abstract)

RBCs are known to aggregate, or coalesce, forming rouleaux, which are closely packed stacks of cells arranged face‐to‐face; both linear and branching structures can form (1,2,3). Healthy blood flow can disaggregate the loosely bound RBC, however, in pathological conditions, rouleaux formation remains and contributes to occlusion of microvessels (1,2,3). One of these pathologies is thermal burn injury where rouleaux are known to form at the margin between the burn and healthy tissue. Our purpose was to examine the process of early aggregation to determine how rouleaux form, which RBC shapes can form rouleaux and whether oxygen or temperature affected rouleaux formation. RBCs are normally biconcave disks, but other shapes readily form, including echinocytes (ECH): ECH‐I (short broad spicules, flat cell); ECH‐II (formed spicules, elliptical cell); ECH‐III (well formed spicules, round cell). Washed human RBCs (BioIVT, Westbury, NY) were suspended in platelet poor plasma (PPP), equilibrated with high oxygen (to ensure fully saturated hemoglobin) or 0% oxygen were loaded into washed microwells (at 37°C) and allowed to settle into a monolayer. RBCs were visualized using a Nikon Diaphot 200 Microscope and 60X water immersion objective; images were acquired using an Iphone XS Max attached to the objective. Baseline images were obtained for 1 minute at 37°C, then the stage temperature was increased to 45°C for 5 minutes. Sequential images were analyzed using ImageJ to follow movement of individual RBC and rouleaux (distance) and velocity. RBC were scored by their morphology per clinical standards as discocyte (biconcave disk), or ECH‐I, II or III. Single RBC joined existing rouleaux or formed new rouleaux. Only discocytes or ECH‐I joined/formed rouleaux, not ECH‐II/III. At 37°C with 0% oxygen, there were 33% more rouleaux (36% more cells in rouleaux) already formed (compared to high oxygen). After reaching 45°C, there were 52% more rouleaux (2‐X more cells in rouleaux) were present in 0% oxygen compared to high oxygen. Baseline RBC velocity was close to that expected for simple diffusion of RBC in solution (0.08um/s). Baseline at 37°C, with 0% oxygen, RBC velocity (mean+/‐SD) was 0.07+/‐0.04um/s, increasing to 0.10+/‐0.05um/s with high oxygen. Increasing stage temperature to 45°C fully heated the microchannels within 2 minutes. During this transition time, velocity with high oxygen increased 10‐fold to 1.1+/‐0.7um/s. At 45°C, with 0% oxygen, RBC velocity was now 0.22+/‐0.14um/s (3‐fold increase from 37°C), and with high oxygen, RBC velocity was 0.36+/‐0.3um/s (also 3‐fold increase from 37°C). RBC velocity did not differ by scored morphology. There was a tendency for the final ‘step’ in joining a rouleaux to have a slower velocity. Thus, both increasing temperature and high oxygen promoted faster RBC movement, yet the oxygen depleted environment clearly promoted more rouleaux. [1. Levin, G.Y., and Egorihina, M.N., 2011, Int J Burns Trauma 1, 34‐41; 2. Sebastian, J.L., et al., 2005, Phys Rev E Stat Nonlin Soft Matter Phys 72, 031913; 3. Wagner, C., et al., 2013, Comptes Rendus Physique 14, 459‐469]