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The isolation of a specific lymphocyte subset from blood is the required first step in the manufacturing of many novel cellular immunotherapies. Microfluidic size-based separation methods are poised to significantly simplify this process because they require neither centrifugation nor magnetic or fluorescent labeling to operate. Lymphocytes can be separated from red blood cells (RBCs) and platelets as well as monocytes and granulocytes because their size differs from each of these cell types. However, further separation of a specific lymphocyte subset from other unwanted lymphocytes using size-based methods is impossible because all lymphocytes have approximately the same size and can only be distinguished by surface markers. This paper describes a new approach that made it possible for a size-based separation method to isolate a desired subset of lymphocytes by making unwanted lymphocytes as well as other blood cells artificially larger. The separation was enabled by selectively binding multiple RBCs to each unwanted cell to create 'rosettes' with an effective size significantly larger than the diameter of a typical lymphocyte. The desired lymphocytes remained unaffected by rosetting and were separated from the rosettes by passing the mixture through a microfluidic size-based separation device based on controlled incremental filtration (CIF). This new rosette-enabled size-based (RESIZE) separation approach demonstrated recovery of 80-90% for all lymphocyte subsets tested (CD3 + , CD4 + , CD56 + ) which was ∼2.5-fold higher than that for the standard immunodensity method (RBC rosetting followed by density gradient centrifugation). The purity of separation was >90% for CD3 + cells but declined with increasing cell rarity. Unlike the immunodensity approach, RESIZE required neither centrifugation nor cell washing after the separation and was ∼2.5-fold faster when processing the same sample volume. The results of this study suggest that integration of the RESIZE approach for high-yield isolation of lymphocyte subsets from blood could significantly streamline the manufacturing workflow and thus have a potentially transformative impact on the cost and availability of novel cellular immunotherapies.

Changes in red blood cell (RBC) morphology distribution have emerged as a quantitative biomarker for the degradation of RBC functional properties during hypothermic storage. Previously published automated methods for classifying the morphology of stored RBCs often had insufficient accuracy and relied on proprietary code and datasets, making them difficult to use in many research and clinical applications. Here we describe the development and validation of a highly accurate open-source RBC morphology classification pipeline based on ensemble deep learning (DL). The DL-enabled pipeline utilized adaptive thresholding or semantic segmentation for RBC identification, a deep ensemble of four convolutional neural networks (CNNs) to classify RBC morphology, and Kalman filtering with Hungarian assignment for tracking changes in the morphology of individual RBCs over time. The ensembled CNNs were trained and evaluated on thousands of individual RBCs from two open-access datasets previously collected to quantify the morphological heterogeneity and washing-induced shape recovery of stored RBCs. Confusion matrices and reliability diagrams demonstrated under-confidence of the constituent models and an accuracy of about 98% for the deep ensemble. Such a high accuracy allowed the CNN ensemble to uncover new insights over our previously published studies. Re-analysis of the datasets yielded much more accurate distributions of the effective diameters of stored RBCs at each stage of morphological degradation (discocyte: 7.821 ± 0.429 µm, echinocyte 1: 7.800 ± 0.581 µm, echinocyte 2: 7.304 ± 0.567 µm, echinocyte 3: 6.433 ± 0.490 µm, sphero-echinocyte: 5.963 ± 0.348 µm, spherocyte: 5.904 ± 0.292 µm, stomatocyte: 7.080 ± 0.522 µm). The effective diameter distributions were significantly different across all morphologies, with considerable effect sizes for non-neighboring classes. A combination of morphology classification with cell tracking enabled the discovery of a relatively rare and previously overlooked shape recovery of some sphero-echinocytes to early-stage echinocytes after washing with 1% human serum albumin solution. Finally, the datasets and code have been made freely available online to enable replication, further improvement, and adaptation of our work for other applications., (© 2023. The Author(s).)

Ektacytometry has been the primary method for evaluating deformability of red blood cells (RBCs) in both research and clinical settings. This study was designed to test the hypothesis that the flow of RBCs through a network of microfluidic capillaries could provide a more sensitive assessment of the progressive impairment of RBC deformability during hypothermic storage than ektacytometry. RBC units (n = 9) were split in half, with one half stored under standard (normoxic) conditions and the other half stored hypoxically, for up to 6 weeks. RBC deformability was measured weekly using two microfluidic devices, an artificial microvascular network (AMVN) and a multiplexed microcapillary network (MMCN), and two commercially available ektacytometers (RheoScan-D and LORRCA). By week 6, the elongation indexes measured with RheoScan-D and LORRCA decreased by 5.8-7.1% (5.4-6.9% for hypoxic storage). Over the same storage duration, the AMVN perfusion rate declined by 27.5% (24.5% for hypoxic) and the MMCN perfusion rate declined by 49.0% (42.4% for hypoxic). Unlike ektacytometry, both AMVN and MMCN measurements showed statistically significant differences between the two conditions after 1 week of storage. RBC morphology deteriorated continuously with the fraction of irreversibly-damaged (spherical) cells increasing significantly faster for normoxic than for hypoxic storage. Consequently, the number of MMCN capillary plugging events and the time MMCN capillaries spent plugged was consistently lower for hypoxic than for normoxic storage. These data suggest that capillary networks are significantly more sensitive to both the overall storage-induced decline of RBC deformability, and to the differences between the two storage conditions, than ektacytometry.

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.)

Washed red blood cells (RBCs) are indicated for immunoglobulin A (IgA) deficient recipients. Centrifugation-based cell processors commonly used by hospital blood banks cannot consistently reduce IgA below the recommended levels, hence double washing is frequently required. Here, we describe a prototype of a simple, portable, disposable system capable of washing stored RBCs without centrifugation, while reducing IgA below 0.05 mg/dL in a single run. Samples from RBC units (n = 8, leukoreduced, 4-6 weeks storage duration) were diluted with normal saline to a hematocrit of 10%, and then washed using either the prototype washing system, or via conventional centrifugation. The efficiency of the two washing methods was quantified and compared by measuring several key in vitro quality metrics. The prototype of the washing system was able to process stored RBCs at a rate of 300 mL/hour, producing a suspension of washed RBCs with 43 ± 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 medical facilities and resource limited settings that may lack access to centrifugation-based cell processors., (© 2017 Wiley Periodicals, Inc.)

Objective: Hct in narrow vessels is reduced due to concentration of fast-flowing RBCs in the center, and of slower flowing plasma along the wall of the vessel, which in combination with plasma skimming at bifurcations leads to the striking heterogeneity of local Hct in branching capillary networks known as the network Fåhraeus effect. We analyzed the influence of feeding Hct and perfusion pressure on the Fåhraeus effect in an AMVN., Methods: RBC suspensions in plasma with Hcts between 20% and 70% were perfused at pressures of 5-60 cm H 2 O through the AMVN. A microscope and high-speed camera were used to measure RBC velocity and Hct in microchannels of height of 5 μm and widths of 5-19 μm., Results: Channel Hcts were reduced compared with Hct feeding in 5 and 7 μm microchannels, but not in larger microchannels. The magnitude of Hct reduction increased with decreasing Hct feeding and decreasing ΔP (flow velocity), showing an about sevenfold higher effect for 40% Hct feeding and low pressure/flow velocity than for 60% Hct feeding and high pressure/flow velocity., Conclusions: The magnitude of the network Fåhraeus effect in an AMVN is inversely related to Hct feeding and ΔP., (© 2017 John Wiley & Sons Ltd.)

Background: During hypothermic storage, a substantial fraction of red blood cells (RBCs) transforms from flexible discocytes to rigid sphero-echinocytes and spherocytes. Infusion of these irreversibly-damaged cells into the recipient during transfusion serves no therapeutic purpose and may contribute to adverse outcomes in some patients. In this proof-of-concept study we describe the use of hypotonic washing for selective removal of the irreversibly-damaged cells from stored blood., Materials and Methods: Stored RBCs were mixed with saline of various concentrations to identify optimal concentration for inducing osmotic swelling and selective bursting of spherical cells (sphero-echinocytes, spherocytes), while minimising indiscriminate lysis of other RBCs. Effectiveness of optimal treatment was assessed by measuring morphology, rheological properties, and surface phosphatidylserine (PS) exposure for cells from several RBCs units (n=5, CPD>AS-1, leucoreduced, 6 weeks storage duration) washed in hypotonic vs isotonic saline., Results: Washing in mildly hypotonic saline (0.585 g/dL, osmolality: 221.7±2.3 mmol/kg) reduced the fraction of spherical cells 3-fold from 9.5±3.4% to 3.2±2.8%, while cutting PS exposure in half from 1.48±0.86% to 0.59±0.29%. Isotonic washing had no effect on PS exposure or the fraction of spherical cells. Both isotonic and hypotonic washing increased the fraction of well-preserved cells (discocytes, echinocytes 1) substantially, and improved the ability of stored RBCs to perfuse an artificial microvascular network by approximately 25%, as compared with the initial sample., Discussion: This study demonstrated that washing in hypotonic saline could selectively remove a significant fraction of the spherical and PS-exposing cells from stored blood, while significantly improving the rheological properties of remaining well-preserved RBCs. Further studies are needed to access the potential effect from hypotonic washing on transfusion outcomes.

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: Hypothermic storage of red blood cells (RBCs) results in progressive deterioration of the rheological properties of the cells, which may reduce the efficacy of RBC transfusions. Recent studies have suggested that storing RBC units under anaerobic conditions may reduce this storage-induced deterioration., Materials and Methods: The aim of this study was to compare the rheological properties of conventionally and anaerobically stored RBC and provide a measure of the relationship between oxidative damage to stored RBC and their ability to perfuse microvascular networks. Three different microfluidic devices were used to measure the ability of both types of stored RBC to perfuse artificial microvascular networks. Flow rates of the RBC passing through the entire network (bulk perfusion) and the individual capillaries (capillary perfusion) of the devices were measured on days 2, 21, 42, and 63 of storage., Results: The bulk perfusion rates for anaerobically stored RBC were significantly higher than for conventionally stored RBCs over the entire duration of storage for all devices (up to 10% on day 42; up to 14% on day 63). Capillary perfusion rates suggested that anaerobically stored RBC units contained significantly fewer non-deformable RBC capable of transiently plugging microfluidic device capillaries. The number of plugging events caused by these non-deformable RBC increased over the 63 days of hypothermic storage by nearly 16- to 21-fold for conventionally stored units, and by only about 3- to 6-fold for anaerobically stored units., Discussion: The perfusion measurements suggest that anaerobically stored RBC retain a greater ability to perfuse networks of artificial capillaries compared to conventionally (aerobically) stored RBC. It is likely that anaerobic storage confers this positive effect on the bulk mechanical properties of stored RBC by significantly reducing the number of non-deformable cells present in the overall population of relatively well-preserved RBC.

Background and Objectives: The morphology of red blood cells (RBCs) deteriorates progressively during hypothermic storage. The degree of deterioration varies between individual cells, resulting in a highly heterogeneous population of cells contained within each RBC unit. Current techniques capable of categorizing the morphology of individual stored RBCs are manual, laborious and error-prone procedures that limit the number of cells that can be studied. Our objective was to create a simple, automated system for high-throughput RBC morphology classification., Materials and Methods: A simple microfluidic device, designed to enable rapid, consistent acquisition of images of optimally oriented RBCs, was fabricated using soft lithography. A custom image analysis algorithm was developed to categorize the morphology of each individual RBC in the acquired images. The system was used to determine morphology of individual RBCs in several RBC units stored hypothermically for 6-8 weeks., Results: The system was used to automatically determine the distribution of cell diameter within each morphological class for >1 000 000 individual stored RBCs (speed: >10 000 cells/h; accuracy: 91·9% low resolution, 75·3% high resolution). Diameter mean and standard deviation by morphology class were as follows: discocyte 7·80 ± 0·49 μm, echinocyte 1 7·61 ± 0·63 μm, echinocyte 2 7·02 ± 0·61 μm, echinocyte 3 6·47 ± 0·42 μm, sphero-echinocyte 6·01 ± 0·26 μm, spherocyte 6·02 ± 0·27 μm, stomatocyte 1 6·95 ± 0·61 μm and stomatocyte 2 7·32 ± 0·47 μm., Conclusions: The automated morphology classification procedure described in this study is significantly simpler, faster and less subjective than conventional manual procedures. The ability to evaluate the morphology of individual RBCs automatically, rapidly and in statistically significant numbers enabled us to perform the most extensive study of stored RBC morphology to date., (© 2015 International Society of Blood Transfusion.)

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.)

Plasma sodium concentration is normally held within a narrow range. It may however vary greatly under pathophysiological conditions. Changes in osmolality lead to either swelling or shrinkage of red blood cells (RBCs). Here we investigated the influence of suspension osmolality on biophysical properties of RBCs and their ability to perfuse an artificial microvascular network (AMVN). Blood was drawn from healthy volunteers. RBC deformability was measured by osmotic gradient ektacytometry over a continuous range of osmolalities. Packed RBCs were suspended in NaCl solutions (0.45, 0.6, 0.9, 1.2, and 1.5 g/dL), resulting in supernatant osmolalities of 179 ± 4, 213 ± 1, 283 ± 2, 354 ± 3, and 423 ± 5 mOsm/kg H2O. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) were determined using centrifuged microhematocrit. RBC suspensions at constant cell numbers were used to measure viscosity at shear rates ranging from 0.11 to 69.5s(-1) and the perfusion rate of the AMVN. MCV was inversely and MCHC directly proportional to osmolality. RBC deformability was maximized at isosmotic conditions (290 mOsm/kg H2O) and markedly decreased by either hypo- or hyperosmolality. The optimum osmolality for RBC suspension viscosity was shifted toward hyperosmolality, while lower osmolalities increased suspension viscosity exponentially. However, the AMVN perfusion rate was maximized at 290 mOsm/kg H2O and changed by less than 10% over a wide range of osmolalities. These findings contribute to the basic understanding of blood flow in health and disease and may have significant implications for the management of osmotic homeostasis in clinical practice., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: Complement system is activated in patients with trauma. Although complement activation is presumed to contribute to organ damage and constitutional symptoms, little is known about the involved mechanisms. Because complement components may deposit on RBCs, we asked whether complement deposits on the surface of RBC in trauma and whether such deposition alters RBC function., Design: A prospective experimental study., Setting: Research laboratory., Subjects: Blood samples collected from 42 trauma patients and 21 healthy donors., Intervention: None., Measurements and Main Results: RBC and sera were collected from trauma patients and control donors. RBCs from trauma patients (n = 40) were found to display significantly higher amounts of C4d on their surface by flow cytometry compared with RBCs from control (n = 17) (p < 0.01). Increased amounts of iC3b were found in trauma sera (n = 27) (vs 12 controls, p < 0.01) by enzyme-linked immunosorbent assay. Incubation of RBC from universal donors (type O, Rh negative) with trauma sera (n = 10) promoted C4d deposition on their surface (vs six controls, p< 0.05). Complement-decorated RBC (n = 6) displayed limited their deformability (vs six controls, p < 0.05) in two-dimensional microchannel arrays. Incubation of RBC with trauma sera (n = 10) promoted the phosphorylation of band 3, a cytoskeletal protein important for the function of the RBC membrane (vs eight controls, p < 0.05), and also accelerated calcium influx (n = 9) and enhanced nitric oxide production (n = 12) (vs four and eight controls respectively, p < 0.05) in flow cytometry., Conclusions: Our study found the presence of extensive complement activation in trauma patients and presents new evidence in support of the hypothesis that complement activation products deposit on the surface of RBC. Such deposition could limit RBC deformability and promote the production of nitric oxide. Our findings suggest that RBC in trauma patients malfunctions, which may explain organ damage and constitutional symptoms that is not accounted for otherwise by previously known pathophysiologic mechanisms.

The ability of red blood cells (RBC) to undergo a wide range of deformations while traversing the microvasculature is crucial for adequate perfusion. Interpretation of RBC deformability measurements performed in vitro in the context of microvascular perfusion has been notoriously difficult. This study compares the measurements of RBC deformability performed using micropore filtration and ektacytometry with the RBC ability to perfuse an artificial microvascular network (AMVN). Human RBCs were collected from healthy consenting volunteers, leukoreduced, washed and exposed to graded concentrations (0-0.08%) of glutaraldehyde (a non-specific protein cross-linker) and diamide (a spectrin-specific protein cross-linker) to impair the deformability of RBCs. Samples comprising cells with two different levels of deformability were created by adding non-deformable RBCs (hardened by exposure to 0.08% glutaraldehyde) to the sample of normal healthy RBCs. Ektacytometry indicated a nearly linear decline in RBC deformability with increasing glutaraldehyde concentration. Micropore filtration showed a significant reduction only for concentrations of glutaraldehyde higher than 0.04%. Neither micropore filtration nor ektacytometry measurements could accurately predict the AMVN perfusion. Treatment with diamide reduced RBC deformability as indicated by ektacytometry, but had no significant effect on either micropore filtration or the AMVN perfusion. Both micropore filtration and ektacytometry showed a linear decline in effective RBC deformability with increasing fraction of non-deformable RBCs in the sample. The corresponding decline in the AMVN perfusion plateaued above 50%, reflecting the innate ability of blood flow in the microvasculature to bypass occluded capillaries. Our results suggest that in vitro measurements of RBC deformability performed using either micropore filtration or ektacytometry may not represent the ability of same RBCs to perfuse microvascular networks. Further development of biomimetic tools for measuring RBC deformability (e.g. the AMVN) could enable a more functionally relevant testing of RBC mechanical properties.

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.)

The use of microfabrication technology in the study of biological systems continues to grow rapidly in both prevalence and ascendancy. Customised microdevices that provide superior results than traditional macroscopic methods can be designed in order to investigate specific cell types and cellular processes. This study showed the benefit of this approach in precisely characterising the progressive losses of surface area and haemoglobin (Hb) content by the human red blood cell (RBC), from newborn reticulocyte to senescent erythrocyte. The high-throughput, multiparametric measurements made on individual cells with a specialised microdevice enabled, for the first time, delineation and quantification of the losses that occur during the two stages of the human RBC lifespan. Data acquired on tens of thousands of red cells showed that nearly as much membrane area is lost during the 1-2 d of reticulocyte maturation (c. 10-14%) as in the subsequent 4 months of erythrocyte ageing (c. 16-17%). The total decrease in Hb over the red cell lifespan is also estimated (c. 15%) and a model describing the complete time-course of diminishing mean RBC area and Hb is proposed. The relationship between the losses of Hb and area, and their possible influence on red cell lifespan, are discussed.

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.