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Background: Red cell (RBC) transfusions are beneficial for patients with sickle cell disease (SCD), but ex vivo studies suggest that inflamed plasma from patients with SCD during crises may damage these RBCs, diminishing their potential efficacy. The hypoxic storage of RBCs may improve transfusion efficacy by minimizing the storage lesion. We tested the hypotheses that (1) The donor RBCs exposed to the plasma of patients in crisis would have lower deformability and higher hemolysis than those exposed to non-crisis plasma, and (2) hypoxic storage, compared to standard storage, of donor RBCs could preserve deformability and reduce hemolysis., Study Design and Methods: 18 SCD plasma samples from patients who had severe acute-phase symptoms (A-plasma; n = 9) or were at a steady-state (S = plasma; n = 9) were incubated with 16 RBC samples from eight units that were stored either under conventional(CRBC) or hypoxic(HRBC) conditions. Hemolysis and microcapillary deformability assays of these RBCs were analyzed using linear mixed-effect models after each sample was incubated in patient plasma overnight at 37°C RESULTS: Relative deformability was 0.036 higher (p < 0.0001) in HRBC pairs compared to CRBC pairs regardless of plasma type. Mean donor RBC hemolysis was 0.33% higher after incubation with A-plasma compared to S-plasma either with HRBC or CRBC (p = 0.04). HRBCs incubated with steady-state patient plasma demonstrated the highest deformability and lowest hemolysis., Conclusion: Hypoxic storage significantly influenced RBC deformability. Patient condition significantly influenced post-incubation hemolysis. Together, HRBCs in steady-state plasma maximized donor red cell ex vivo function and survival., (© 2022 AABB.)

Background: Hypothermic storage transforms red blood cells (RBC) from smooth biconcave discocytes into increasingly spherical spiculated echinocytes and, ultimately, fragile spherocytes (S). Individual cells undergo this transformation at different rates, producing a heterogeneous mixture of RBCs at all stages of echinocytosis in each unit of stored blood. Here we investigated how washing (known to positively affect RBC properties) changes morphology of individual RBCs at the single-cell level., Study Design and Methods: We tracked the change in shape of individual RBCs (n = 2870; drawn from six 4- to 6-week-old RBC units) that were confined in an array of microfluidic wells during washing in saline (n = 1095), 1% human serum albumin (1% HSA) solution (n = 999), and the autologous storage supernatant (control, n = 776)., Results: Shape recovery proceeded through the disappearance of spicules followed by the progressive smoothening of the RBC contour, with the majority of changes occurring within the initial 10 minutes of being exposed to the washing solution. Approximately 57% of all echinocytes recovered by at least one morphologic class when washed in 1% HSA (36% for normal saline), with 3% of cells in late-stage echinocytosis restoring their discoid shape completely. Approximately one-third of all spherocytic cells were lysed in either washing solution. Cells washed in their autologous storage supernatant continued to deteriorate during washing., Conclusion: Our findings suggest that the replacement of storage supernatant with a washing solution during washing induces actual shape recovery for RBCs in all stages of echinocytosis, except for S that undergo lysis instead., (© 2020 AABB.)

Background: Great deformability allows red blood cells (RBCs) to flow through narrow capillaries in tissues. A number of microfluidic devices with capillary-like microchannels have been developed to monitor storage-related impairment of RBC deformability during blood banking operations. This proof-of-concept study describes a new method to standardize and improve reproducibility of the RBC deformability measurements using one of these devices., Study Design and Methods: The rate of RBC flow through the microfluidic capillary network of the microvascular analyzer (MVA) device made of polydimethylsiloxane was measured to assess RBC deformability. A suspension of microbeads in a solution of glycerol in phosphate-buffered saline was developed to be used as an internal flow rate reference alongside RBC samples in the same device. RBC deformability and other in vitro quality markers were assessed weekly in six leukoreduced RBC concentrates (RCCs) dispersed in saline-adenine-glucose-mannitol additive solution and stored over 42 days at 4°C., Results: The use of flow reference reduced device-to-device measurement variability from 10% to 2%. Repeated-measure analysis using the generalized estimating equation (GEE) method showed a significant monotonic decrease in relative RBC flow rate with storage from Week 0. By the end of storage, relative RBC flow rate decreased by 22 ± 6% on average., Conclusions: The suspension of microbeads was successfully used as a flow reference to increase reproducibility of RBC deformability measurements using the MVA. Deformability results suggest an early and late aging phase for stored RCCs, with significant decreases between successive weeks suggesting a highly sensitive measurement method., (© 2020 AABB.)

Background: Higher hematocrit increases the oxygen-carrying capacity of blood but also increases blood viscosity, thus decreasing blood flow through the microvasculature and reducing the oxygen delivery to tissues. Therefore, an optimal value of hematocrit that maximizes tissue oxygenation must exist., Study Design and Methods: We used viscometry and an artificial microvascular network device to determine the optimal hematocrit in vitro. Suspensions of fresh red blood cells (RBCs) in plasma, normal saline, or a protein-containing buffer and suspensions of stored red blood cells (at Week 6 of standard hypothermic storage) in plasma with hematocrits ranging from 10 to 80% were evaluated., Results: For viscometry, optimal hematocrits were 10, 25.2, 31.9, 37.1, and 37.5% for fresh RBCs in plasma at shear rates of 3.2 or less, 11.0, 27.7, 69.5, and 128.5 inverse seconds. For the artificial microvascular network, optimal hematocrits were 51.1, 55.6, 59.2, 60.9, 62.3, and 64.6% for fresh RBCs in plasma and 46.4, 48.1, 54.8, 61.4, 65.7, and 66.5% for stored RBCs in plasma at pressures of 2.5, 5, 10, 20, 40, and 60 cm H 2 O., Conclusion: Although exact optimal hematocrit values may depend on specific microvascular architecture, our results suggest that the optimal hematocrit for oxygen delivery in the microvasculature depends on perfusion pressure. Therefore, anemia in chronic disorders may represent a beneficial physiological response to reduced perfusion pressure resulting from decreased heart function and/or vascular stenosis. Our results may help explain why a therapeutically increasing hematocrit in such conditions with RBC transfusion frequently leads to worse clinical outcomes., (© 2017 AABB.)

Background: The shape of human red blood cells (RBCs) deteriorates progressively throughout hypothermic storage, with echinocytosis being the most prevalent pathway of this morphologic lesion. As a result, each unit of stored blood contains a heterogeneous mixture of cells in various stages of echinocytosis and normal discocytes. Here we studied how the change in shape of RBCs following along the path of the echinocytic transformation affects perfusion of an artificial microvascular network (AMVN)., Study Design and Methods: Blood samples were obtained from healthy consenting volunteers. RBCs were leukoreduced, resuspended in saline, and treated with various concentrations of sodium salicylate to induce shape changes approximating the stages of echinocytosis experienced by RBCs during hypothermic storage (e.g., discocyte, echinocyte I, echinocyte II, echinocyte III, spheroechinocyte, and spherocyte). The AMVN perfusion rate was measured for 40% hematocrit suspensions of RBCs with different shapes., Results: The AMVN perfusion rates for RBCs with discocyte and echinocyte I shapes were similar, but there was a significant decline in the AMVN perfusion rate between RBCs with shapes approximating each subsequent stage of echinocytosis. The difference in AMVN perfusion between discocytes and spherocytes (the last stage of the echinocytic transformation) was 34%., Conclusion: The change in shape of RBCs from normal discocytes progressively through various stages of echinocytosis to spherocytes produced a substantial decline in the ability of these cells to perfuse an AMVN. Echinocytosis induced by hypothermic storage could therefore be responsible for a similarly substantial impairment of deformability previously observed for stored RBCs., (© 2015 AABB.)

Background: Prolonged storage of red blood cells (RBCs) leads to storage lesions, which may impair clinical outcomes after transfusion. A hallmark of storage lesions is progressive echinocytic shape transformation, which can be partially reversed by washing in albumin solutions. Here we have investigated the impact of this shape recovery on biorheologic variables., Study Design and Methods: RBCs stored hypothermically for 6 to 7 weeks were washed in a 1% human serum albumin (HSA) solution. RBC deformability was measured with osmotic gradient ektacytometry. The viscosity of RBC suspensions was measured with a Couette-type viscometer. The flow behavior of RBCs suspended at 40% hematocrit was tested with an artificial microvascular network (AMVN)., Results: Washing in 1% albumin reduced higher degrees of echinocytes and increased the frequency of discocytes, thereby shifting the morphologic index toward discocytosis. Washing also reduced RBC swelling. This shape recovery was not seen after washing in saline, buffer, or plasma. RBC shape normalization did not improve cell deformability measured by ektacytometry, but it tended to decrease suspension viscosities at low shear rates and improved the perfusion of an AMVN., Conclusions: Washing of stored RBCs in a 1% HSA solution specifically reduces echinocytosis, and this shape recovery has a beneficial effect on microvascular perfusion in vitro. Washing in 1% albumin may represent a new approach to improving the quality of stored RBCs and thus potentially reducing the likelihood of adverse clinical outcomes associated with transfusion of blood stored for longer periods of time., (© 2015 AABB.)

Background: The progressive deterioration of red blood cell (RBC) rheologic properties during refrigerated storage may reduce the clinical efficacy of transfusion of older units., Study Design and Methods: This article describes the development of a microfluidic device designed to test the rheologic properties of stored RBCs by measuring their ability to perfuse an artificial microvascular network (AMVN) comprised of capillary-size microchannels arranged in a pattern inspired by the real microvasculature. In the AMVN device, the properties of RBCs are evaluated by passing a 40% hematocrit suspension of RBCs through the network and measuring the overall perfusion rate., Results: The sensitivity of the AMVN device to the storage-induced change in rheologic properties of RBCs was tested using five prestorage leukoreduced RBC units stored in AS-1 for 41 days. The AMVN perfusion rate for stored RBCs was 26 ± 4% (19%-30%) lower than for fresh RBCs. Washing these stored RBCs in saline improved their performance by 41 ± 6% (the AMVN perfusion rate for washed stored RBCs was still 15 ± 2% lower than for fresh RBCs)., Conclusions: The measurements performed using the AMVN device confirm a significant decline in the rheologic properties of RBCs in units nearing expiration and demonstrate the sensitivity of the device to these storage-induced changes. The AMVN device may be useful for testing the effect of new storage conditions, additive solutions, and rejuvenation strategies on the rheologic properties of stored RBCs in vitro., (© 2011 American Association of Blood Banks.)

Background: Red blood cell (RBC) senescence is a process that has received considerable study, yet remains poorly understood. This has been primarily due to the difficulty in isolating a RBC cohort of narrowly distributed, well-defined age. Biotin labeling has previously been used to produce an identifiable cell cohort of known mean age; however, the variability of RBC age within the cohort is relatively large for most of its existence. Treatments typically employed on animal subjects to reduce this variability can perturb erythropoiesis and result in abnormal RBC aging., Study Design and Methods: The objective of this study was to improve on the traditional in vivo biotinylation method by introducing a chemically distinct, second labeling step. In this case, digoxigenin was used to label cells 1 to 2 days before the injection of biotin., Results: It was shown, in the rat, that two identifiable subpopulations of labeled RBCs can be followed over time: a broad, double-labeled cohort and a narrow, single-labeled cohort, the latter consisting of only those cells created between the first and second labeling steps. The utility of this technique was demonstrated by observing the age-dependent exposure of phosphatidylserine in the single-labeled RBCs., Conclusion: Its capacity to generate a cohort of narrowly distributed age, without the adverse effects associated with animal treatment, should make this a useful method for the study of RBC senescence.