The FE's measured for the fraction-2a are still good with the average value of 92%. When two GSF channels were connected so that the flow stream emerging from the outlet-b of the channel-1 is fed directly into the channel-2, all three FE's measured for the fraction-1a were high with the average value of 99%, indicating it contains almost purely the beads smaller than dc. Also no clear trends were observed between the FE and the sample-feeding flow rate, indicating higher sample-feeding rate can be used to increase the samplethroughput without losing resolution. No particular trends were found between FE and dc, indicating the performance of FFD-GSF does not change with dc in the range where tested. The FE's of the fraction-a are higher than 84% with the average of about 91%, while those of the fraction-b are lower than 60% with the average of about 43%. The measured FE's of the fraction-b are much lower than those of the fraction-a in all cases. In a single channel FFD-GSF operation, the fraction-a contained mostly the beads smaller than the cutoff diameter (d_c), while the fraction-b contained beads smaller than dc as well as those larger than d_c, as expected. In this study, two Gravitation SF (GSF) channels were connected in a series (Tandem GSF) to obtain a separation into three subpopulations and to improve the fractionation efficiency (FE) of the fraction-b in the full-feed depletion (FFD) mode. Separation into more than two subpopulations requires repeated SF operations. © 2016 Elsevier B.V.SPLITT Fractionation (SF) provides separation of sample into two subpopulations. Measurement of membrane potential and cell diameter could provide a new, reliable and convenient basis for evaluation of hydrodynamic effects on different cell models, allowing identification of optimal operating conditions on different scales. Some changes in SPLITT channel design are suggested to minimize cell damage. Our data do not support the assumption that SPLITT fractionation induces very low shear stress and is innocuous to cell function. Wall shear stress and maximum energy dissipation rate showed significant correlation with lethal and sublethal damage. Under the operating conditions employed, both SPLITT and centrifugation maintained cell viability above 98%, but resulted in significant sublethal damage, including echinocyte formation, decreased cell diameter, decreased mean corpuscular hemoglobin, and membrane hyperpolarization which was inhibited by EGTA. Cell viability, shape, diameter, mean corpuscular hemoglobin, and membrane potential were measured. Samples were diluted in a buffered saline solution, and were exposed to SPLITT fractionation (flow rates 1-10 ml/min) or centrifugation (100-1500 g) for 10 min. Peripheral whole blood samples were collected from healthy volunteers. The aim of this study was to investigate the hydrodynamic damage of SPLITT fractionation to human red blood cells, and to compare these effects with those induced by centrifugation. However, the hydrodynamic stress and possible consequent damaging effects of SPLITT fractionation have not been yet examined. Split-flow fractionation (SPLITT) is a family of techniques that separates in absence of labelling and uses very low flow rates and force fields, and is therefore expected to minimize cell damage. Though blood bank processing traditionally employs centrifugation, new separation techniques may be appealing for large scale processes.
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