Percent RBC recovery was calculated as: %RBC recovery = [(volume of washed sample * RBC count in washed sample) / (volume of initial sample * RBC count in initial sample) * 100]. RBC phosphatidylserine (PS) exposure was measured via flow cytometry: each sample was diluted in annexin binding buffer (10 mM HEPES, 0.14 M NaCl, 2.5 mM CaCl2, pH Ginsenoside Rg3 7.4) to yield a concentration of ~1 million RBC/100 L. of 300 mL/hr, producing a suspension of washed RBCs with 43 Gata6 3% hematocrit and 86 7% cell recovery. Overall, the two washing methods performed similarly for most measured parameters, lowering the concentration of free hemoglobin by 4-fold and total free protein by 10-fold. Importantly, the new washing system reduced the IgA level to 0.02 0.01 mg/mL, a concentration 5-fold lower than that produced by conventional centrifugation. This proof-of-concept study showed that centrifugation may be unnecessary for washing stored RBCs. A simple, disposable, centrifugation-free washing system could be particularly useful in smaller Ginsenoside Rg3 medical facilities and resource limited settings that may lack access to centrifugation-based cell processors. metrics of RBC quality. Materials and Methods Fabrication of the prototype washing system The centrifugation-free washing system consisted of plastic tubing 22 m in length with internal diameter (i.d.) of 1 1.02 mm (Tygon microbore, Cole Parmer, Vernon Hills, IL) wound in a spiral on a vertical column (i.d. 40 mm), three y-shaped bifurcations located at 7, 14 and 21 m along the tubing, and the membrane-hydrogel composite. Each y-shaped bifurcation was fabricated by placing a piece of tubing with outer diameter of 1 1.02 mm and a piece of tubing with outer diameter of 0.25 mm (both medical grade micro-urethane, Scientific Commodities, Lake Havasu City, AZ) flat on top of a layer (~2.5 cm in height) of cured (solid) polydimethylsiloxane (PDMS) to form the branches of the bifurcation. This assembly was then covered with uncured (liquid) PDMS to form a second layer (~2 cm in height). After curing, the tubing was pulled out, yielding a PDMS mold of the bifurcation. The dimensions of the bifurcations branches were calculated to keep an approximately 5:1 ratio of the volumetric flow rates in the larger main branch and the smaller side branch. The membrane-hydrogel composite consisted of a poly-(acrylic acid) polymer-based hydrogel (Sigma-Aldrich, Saint Lois, MO) enclosed in a pouch fabricated from asymmetric, hydrophilic polyethersulfone (PES) membrane filter sheet (Capillary Filtration Membrane, pore size 1.2 m, 3M Deutschland GmbH, Germany). The pouch was fabricated by placing 0.5 C 2.0 g of cross-linked, ready to use hydrogel between two 4.0 cm 6.0 cm rectangular pieces of the membrane, and then sealing the edges of the rectangles using a handheld heat sealer (Metronic International, Diamond Bar, CA). Blood samples RBC units (CPD AS-1, leukoreduced, storage duration 39C42 days) were purchased from the Gulf Coast Regional Blood Ginsenoside Rg3 Center (Houston, TX) and stored in a blood bank refrigerator (Helmer iB111, Helmer Scientific, Noblesville, IN) at 2C6C until use. Prior to sampling, RBC units were inverted gently by hand to ensure samples were well-mixed. All experiments were carried out at room temperature unless stated otherwise. Washing procedures Ginsenoside Rg3 For each experiment, a 30 mL sample was withdrawn from an RBC unit and re-suspended in normal saline at 1:5 ratio to dilute the samples to a hematocrit between 10C15%. (Dilution to a hematocrit of 10% corresponds to the dilution used in the conventional, centrifugation-based washing procedure performed with 2000 mL of saline per 350 mL RBC unit with a 60C65% initial hematocrit).24 50 mL of the diluted RBC sample was then passed through the prototype blood washing system using a programmable syringe pump (NE-1000, New Era Pump Systems, Farmingdale, NY) operating at various volumetric flow rates (60 C 600 mL/hour). Washed RBCs were collected continuously into a collection tube at the end of the tubing, and then the collected blood was placed from the collection tube into the membrane-module where the excess washing solution was removed by the membrane-hydrogel composite. 50 mL of the 10% hematocrit solution was conventionally washed by centrifugation at 1245g for 15 minutes (mimicking the Validated Protocol for 2-L Wash Packed or Resuspended RBCs for Cobe? 2991? cell processor). The supernatant was then removed for analysis, and the pellet of washed RBCs was reconstituted in saline to yield an output hematocrit of 40C45%, to match the output hematocrit of our prototype washing system. Measurements of washing quality The quality of washing was assessed by measuring several key parameters for the untreated, diluted, waste (washing solution), and washed RBCs, for both the prototype washing system and the conventional centrifugation-based procedure. A hematology analyzer (Medonic M-Series, Boule Diagnostics, Stockholm, Sweden) was used to measure hematocrit (HCT), RBC count, MCV, platelet count and white blood cell count. Percent RBC recovery was calculated as: %RBC recovery = [(volume of.