Tangential flow filtration facilitated washing of human red blood cells: A proof‐of‐concept study

Abstract

Background and Objectives: Red blood cell (RBC) units in hypothermic storage degrade over time, commonly known as the RBC storage lesion. These older RBC units can cause adverse clinical effects when transfused, as older RBCs in the unit lyse and release cell-free haemoglobin (Hb), a potent vasodilator that can elicit vasoconstriction, systemic hypertension and oxidative tissue injury after transfusion. In this study, we examined a novel method of washing ex vivo stored single RBC units to remove accumulated cellular waste, specifically cell-free Hb, using tangential flow filtration (TFF) driven by a centrifugal pump. Materials and Methods:The TFF RBC washing system was run under hypothermic conditions at 4 C, at a constant system volume with 0.9 wt% saline as the wash solution. The RBC washing process was conducted on 10 separate RBC units. For this proof-of-concept study, RBC units were expired at the time of washing (60-70 days old). Cell-free Hb was quantified by UV-visible absorbance spectroscopy and analysed via the Winterbourn equations. Pre-and post-wash RBC samples were analysed by Hemox Analyser, Coulter counter and Brookfield rheometer. The RBC volume fraction in solution was measured throughout the wash process by standard haematocrit (HCT) analysis.Results: No substantial decrease in the HCT was observed during the TFF RBC washing process. However, there was a significant decrease in RBC concentration in the first half of the TFF RBC wash process, with no significant change in RBC concentration during the second half of the TFF cell wash process with an 87% overall cell recovery compared with the total number of cells before initiation of cell washing. Utilization of the extinction coefficients and characteristic peaks of each Hb species potentially present in solution was quantified by Winterbourn analysis on retentate and permeate samples for each diacycle to quantify Hb concentration during the washing process. Significant cell-free Hb reduction was observed within the first four diacycles with a starting cell-free Hb concentration in the RBC unit of 0.105 mM, which plateaus to a constant Hb concentration of 0.01 mM or a total extracellular Hb mass of 0.2 g in the resultant washed unit. The oxygen equilibrium curve showed a significant decrease in P 50 between the initial and final RBC sample Shuwei Lu and Megan Allyn contributed equally to the work.