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Madeleine Lu, Dalia L Lezzar, Eszter Vörös, Sergey S Shevkoplyas Department of Biomedical Engineering, University of Houston, Houston, TX 77204-5060, USA Abstract: Millions of blood components including red blood cells, platelets, and granulocytes are transfused each year in the United States. The transfusion of these blood products may be associated with adverse clinical outcomes in some patients due to residual proteins and other contaminants that accumulate in blood units during processing and storage. Blood products are, therefore, often washed in normal saline or other media to remove the contaminants and improve the quality of blood cells before transfusion. While there are numerous methods for washing and volume reducing blood components, a vast majority utilize centrifugation-based processing, such as manual centrifugation, open and closed cell processing systems, and cell salvage/autotransfusion devices. Although these technologies are widely employed with a relatively low risk to the average patient, there is evidence that centrifugation-based processing may be inadequate when transfusing to immunocompromised patients, neonatal and infant patients, or patients susceptible to transfusion-related allergic reactions. Cell separation and volume reduction techniques that employ centrifugation have been shown to damage blood cells, contributing to these adverse outcomes. The limitations and disadvantages of centrifugation-based processing have spurred the development of novel centrifugation-free methods for washing and volume reducing blood components, thereby causing significantly less damage to the cells. Some of these emerging technologies are already transforming niche applications, poised to enter mainstream blood cell processing in the not too distant future. Keywords: washing, volume reduction, red blood cells, platelets, granulocytes, transfusion

Leukapheresis is a common extracorporeal procedure for leukodepletion and cellular collection. During the procedure, a patient's blood is passed through an apheresis machine to separate white blood cells (WBCs) from red blood cells (RBCs) and platelets (PLTs), which are then returned to the patient. Although it is well-tolerated by adults and older children, leukapheresis poses a significant risk to neonates and low-weight infants because the extracorporeal volume (ECV) of a typical leukapheresis circuit represents a particularly large fraction of their total blood volume. The reliance of existing apheresis technology on centrifugation for separating blood cells limits the degree to which the circuit ECV could be miniaturized. The rapidly advancing field of microfluidic cell separation holds excellent promise for devices with competitive separation performance and void volumes that are orders of magnitude smaller than their centrifugation-based counterparts. This review discusses recent advancements in the field, focusing on passive separation methods that could potentially be adapted to perform leukapheresis. We first outline the performance requirements that any separation method must meet to replace centrifugation-based methods successfully. We then provide an overview of the passive separation methods that can remove WBCs from whole blood, focusing on the technological advancements made in the last decade. We describe and compare standard performance metrics, including blood dilution requirements, WBC separation efficiency, RBC and PLT loss, and processing throughput, and discuss the potential of each separation method for future use as a high-throughput microfluidic leukapheresis platform. Finally, we outline the primary common challenges that must still be overcome for these novel microfluidic technologies to enable centrifugation-free, low-ECV leukapheresis in the pediatric setting.

The majority of adoptive cellular therapies are produced from peripheral mononuclear cells obtained via leukapheresis and further enriched for the cells of interest (e.g., T cells). Here, we present a first-of-its-kind closed system, which effectively removes ~85% of monocytes and ~88% of platelets, while recovering ~88% of concentrated T cells in a separate output stream, as the leukapheresis sample flows through a microfluidic device at 5 mL/min. The system is driven by a common peristaltic pump, enabled by a novel pressure wave dampener, and operates in a closed bag-to-bag configuration, without requiring any specialized, dedicated equipment. When compared to standard density gradient centrifugation on paired samples, the new system demonstrated a 1.5-fold increase in T cell recovery and a 2-fold reduction in inter-sample variability for this separation outcome. The T cell-to-monocyte ratio of the leukapheresis sample was increased to 20:1, whereas with density gradient processing it decreased to 2:1. As a result of superior purity and/or gentler processing, T cells enriched by the system showed a 2.7-times higher fold expansion during subsequent culture, and an overall 3.5-times higher cumulative yield. This centrifugation-free and label-free closed system for enriching lymphocytes could significantly simplify and standardize the manufacturing of life-saving cellular therapies., Competing Interests: Sean C. Gifford and Sergey S. Shevkoplyas are inventors of U.S. Patent #9,789,235 ‘Separation and concentration of particles’ describing the ‘controlled incremental filtration’ technology, and are co‐founders of Halcyon Biomedical, Incorporated, a company that would benefit from its commercialization. Riley C. Reddington and Marc Manheim are employees of Halcyon Biomedical, Incorporated. SSS has received research funding from Halcyon Biomedical, Incorporated. Anton Mukhamedshin, Mai T. P. Dinh, Kumar Abhishek, and Mubasher Iqbal declare no competing interests., (© 2023 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

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).)

Leukapheresis, the extracorporeal separation of white blood cells (WBCs) from red blood cells (RBCs) and platelets (PLTs), is a life-saving procedure used for treating patients with cancer and other conditions, and as the initial step in the manufacturing of cellular and gene-based therapies. Well-tolerated by adults, leukapheresis poses a significant risk to neonates and low-weight infants because the extracorporeal volume (ECV) of standard centrifugation-based machines represents a particularly large fraction of these patients' total blood volume. Here we describe a novel high-throughput microfluidic device (with a void volume of 0.4 mL) based on controlled incremental filtration (CIF) technology that could replace centrifugation for performing leukapheresis. The CIF device was tested extensively using whole blood from healthy volunteers at multiple hematocrits (5-30%) and flow rates (10-30 mL/min). In the flow-through regime, the CIF device separated WBCs with > 85% efficiency and 10-15% loss of RBCs and PLTs while processing whole blood diluted with saline to 10% hematocrit at a flow rate of 10 mL/min. In the recirculation regime, the CIF device demonstrated a similar level of separation performance, virtually depleting WBCs in the recirculating blood (~ 98% reduction) by the end of a 3.5-hour simulated leukapheresis procedure. Importantly, the device operated without clogging or decline in separation performance, with minimal activation of WBCs and PLTs and no measurable damage to RBCs. Compared to the typical parameters of centrifugation-based leukapheresis, the CIF device had a void volume at least 100-fold smaller, removed WBCs about twice as fast, and lost ~ 2-3-fold fewer PLTs, while operating at a flow rate compatible with the current practice. The hematocrit and flow rate at which the CIF device operated were significantly higher than previously published for other microfluidic cell separation methods. Finally, this study is the first to demonstrate a highly efficient separation of cells from recirculating blood using a microfluidic device. Overall, these findings suggest the feasibility of using high-throughput microfluidic cell separation technology to ultimately enable centrifugation-free, low-ECV leukapheresis. Such a capability would be particularly useful in young children, a vulnerable group of patients who are currently underserved., (© 2022. The Author(s).)

Biomarker development is a key clinical research need in sickle cell disease (SCD). Hemorheological parameters are excellent candidates as abnormal red blood cell (RBC) rheology plays a critical role in SCD pathophysiology. Here we describe a microfluidic device capable of evaluating RBC deformability and adhesiveness concurrently, by measuring their effect on perfusion of an artificial microvascular network (AMVN) that combines microchannels small enough to require RBC deformation, and laminin (LN) coating on channel walls to model intravascular adhesion. Each AMVN device consists of three identical capillary networks, which can be coated with LN (adhesive) or left uncoated (non-adhesive) independently. The perfusion rate for sickle RBCs in the LN-coated networks (0.18 ± 0.02 nL/s) was significantly slower than in non-adhesive networks (0.20 ± 0.02 nL/s), and both were significantly slower than the perfusion rate for normal RBCs in the LN-coated networks (0.22 ± 0.01 nL/s). Importantly, there was no overlap between the ranges of perfusion rates obtained for sickle and normal RBC samples in the LN-coated networks. Interestingly, treatment with poloxamer 188 decreased the perfusion rate for sickle RBCs in LN-coated networks in a dose-dependent manner, contrary to previous studies with conventional assays, but in agreement with the latest clinical trial which showed no clinical benefit. Overall, these findings suggest the potential utility of the adhesive AMVN device for evaluating the effect of novel curative and palliative therapies on the hemorheological status of SCD patients during clinical trials and in post-market clinical practice., Competing Interests: SS was an inventor of the artificial microvascular network technology described in this article, and has received compensation as a consultant for Hemanext (formerly New Health Sciences, Inc.) which is commercializing this technology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lu, Kanne, Reddington, Lezzar, Sheehan and Shevkoplyas.)

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: The isolation of lymphocytes - and removal of platelets (PLTs) and red blood cells (RBCs) - from an initial blood sample prior to culture is a key enabling step for effective manufacture of cellular therapies. Unfortunately, currently available methods suffer from various drawbacks, including low cell recovery, need for complex equipment, potential loss of sterility and/or high materials/labor cost., Methods: A newly developed system for selectively concentrating leukocytes within precisely designed, but readily fabricated, microchannels was compared with conventional density gradient centrifugation with respect to: (i) ability to recover lymphocytes while removing PLTs/RBCs and (ii) growth rate and overall cell yield once expanded in culture., Results: In the optimal embodiment of the new microfluidic approach, recoveries of CD3+, CD19+ and CD56+ cells (85%, 89% and 97%, respectively) were significantly higher than for paired samples processed via gradient-based separation (51%, 53% and 40%). Although the removal of residual PLTs and RBCs was lower using the new approach, its enriched T-cell fraction nevertheless grew at a significantly higher rate than the gradient-isolated cells, with approximately twice the cumulative cell yield observed after 7 days of culture., Discussion: The standardization of each step of cellular therapy manufacturing would enable an accelerated translation of research breakthroughs into widely available clinical treatments. The high-throughput approach described in this study - requiring no ancillary pumping mechanism nor expensive disposables to operate - may be a viable candidate to standardize and streamline the initial isolation of lymphocytes for culture while also potentially shortening the time required for their expansion into a therapeutic dose., (Copyright © 2018 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

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.)

Background: The use of centrifugation-based approaches for processing donated blood into components is routine in the industrialized world, as disparate storage conditions require the rapid separation of 'whole blood' into distinct red blood cell (RBC), platelet, and plasma products. However, the logistical complications and potential cellular damage associated with centrifugation/apheresis manufacturing of blood products are well documented. The objective of this study was to evaluate a proof-of-concept system for whole blood processing, which does not employ electromechanical parts, is easily portable, and can be operated immediately after donation with minimal human labor., Methods and Findings: In a split-unit study (n = 6), full (~500mL) units of freshly-donated whole blood were divided, with one half processed by conventional centrifugation techniques and the other with the new blood separation system. Each of these processes took 2-3 hours to complete and were performed in parallel. Blood products generated by the two approaches were compared using an extensive panel of cellular and plasma quality metrics. Comparison of nearly all RBC parameters showed no significant differences between the two approaches, although the portable system generated RBC units with a slight but statistically significant improvement in 2,3-diphosphoglyceric acid concentration (p < 0.05). More notably, several markers of platelet damage were significantly and meaningfully higher in products generated with conventional centrifugation: the increase in platelet activation (assessed via P-selectin expression in platelets before and after blood processing) was nearly 4-fold higher for platelet units produced via centrifugation, and the release of pro-inflammatory mediators (soluble CD40-ligand, thromboxane B2) was significantly higher for centrifuged platelets as well (p < 0.01)., Conclusion: This study demonstrated that a simple, passive system for separating donated blood into components may be a viable alternative to centrifugation-particularly for applications in remote or resource-limited settings, or for patients requiring highly functional platelet product.

Sickle cell anemia (SCA) is a genetic blood disorder that is particularly lethal in early childhood. Universal newborn screening programs and subsequent early treatment are known to drastically reduce under-five SCA mortality. However, in resource-limited settings, cost and infrastructure constraints limit the effectiveness of laboratory-based SCA screening programs. To address this limitation our laboratory previously developed a low-cost, equipment-free, point-of-care, paper-based SCA test. Here, we improved the stability and performance of the test by replacing sodium hydrosulfite (HS), a key reducing agent in the hemoglobin solubility buffer which is not stable in aqueous solutions, with sodium metabisulfite (MS). The MS formulation of the test was compared to the HS formulation in a laboratory setting by inexperienced users ( n = 3), to determine visual limit of detection (LOD), readout time, diagnostic accuracy, intra- and inter-observer agreement, and shelf life. The MS test was found to have a 10% sickle hemoglobin LOD, 21-min readout time, 97.3% sensitivity and 99.5% specificity for SCA, almost perfect intra- and inter-observer agreement, at least 24 weeks of shelf stability at room temperature, and could be packaged into a self-contained, distributable test kits comprised of off-the-shelf disposable components and food-grade reagents with a total cost of only $0.21 (USD)., Competing Interests: S.S.S. and N.Z.P. are inventors on a utility PCT application “Paper based diagnostic test” (PCT/US2012/064856, 11/13/2012) claiming priority benefit of US 61/692,994 (8/24/2012) and US 61/558,009 (11/10/2011) []. S.S.S. is a part-owner of Halcyon Biomedical Incorporated, a company that may benefit from commercialization of the paper-based SCA test(s). All other authors declare no conflict of interest.

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.

The high cost, complexity and reliance on electricity, specialized equipment and supplies associated with conventional diagnostic methods limit the scope and sustainability of newborn screening for sickle cell disease (SCD) in sub-Saharan Africa and other resource-limited areas worldwide. Here we describe the development of a simple, low-cost, rapid, equipment- and electricity-free paper-based test capable of detecting sickle hemoglobin (HbS) in newborn blood samples with a limit of detection of 2% HbS. We validated this newborn paper-based test in a cohort of 159 newborns at an obstetric hospital in Cabinda, Angola. Newborn screening results using the paper-based test were compared to conventional isoelectric focusing (IEF). The test detected the presence of HbS with 81.8% sensitivity and 83.3% specificity, and identified SCD newborns with 100.0% sensitivity and 70.7% specificity. The use of the paper-based test in a two-stage newborn screening process could have excluded about 70% of all newborns from expensive confirmatory testing by IEF, without missing any of the SCD newborns in the studied cohort. This study demonstrates the potential utility of the newborn paper-based test for reducing the overall cost of screening newborns for SCD and thus increasing the practicality of universal newborn SCD screening programs in resource-limited settings.

Leukocyte reduction of donated blood products substantially reduces the risk of a number of transfusion-related complications. Current 'leukoreduction' filters operate by trapping leukocytes within specialized filtration material, while allowing desired blood components to pass through. However, the continuous release of inflammatory cytokines from the retained leukocytes, as well as the potential for platelet activation and clogging, are significant drawbacks of conventional 'dead end' filtration. To address these limitations, here we demonstrate our newly-developed 'controlled incremental filtration' (CIF) approach to perform high-throughput microfluidic removal of leukocytes from platelet-rich plasma (PRP) in a continuous flow regime. Leukocytes are separated from platelets within the PRP by progressively syphoning clarified PRP away from the concentrated leukocyte flowstream. Filtrate PRP collected from an optimally-designed CIF device typically showed a ~1000-fold (i.e. 99.9%) reduction in leukocyte concentration, while recovering >80% of the original platelets, at volumetric throughputs of ~1 mL/min. These results suggest that the CIF approach will enable users in many fields to now apply the advantages of microfluidic devices to particle separation, even for applications requiring macroscale flowrates.

Background: The high childhood mortality and life-long complications associated with sickle cell anemia (SCA) in developing countries could be significantly reduced with effective prophylaxis and education if SCA is diagnosed early in life. However, conventional laboratory methods used for diagnosing SCA remain prohibitively expensive and impractical in this setting. This study describes the clinical validation of a low-cost paper-based test for SCA that can accurately identify sickle trait carriers (HbAS) and individuals with SCA (HbSS) among adults and children over 1 year of age., Methods and Findings: In a population of healthy volunteers and SCA patients in the United States (n = 55) the test identified individuals whose blood contained any HbS (HbAS and HbSS) with 100% sensitivity and 100% specificity for both visual evaluation and automated analysis, and detected SCA (HbSS) with 93% sensitivity and 94% specificity for visual evaluation and 100% sensitivity and 97% specificity for automated analysis. In a population of post-partum women (with a previously unknown SCA status) at a primary obstetric hospital in Cabinda, Angola (n = 226) the test identified sickle cell trait carriers with 94% sensitivity and 97% specificity using visual evaluation (none of the women had SCA). Notably, our test permits instrument- and electricity-free visual diagnostics, requires minimal training to be performed, can be completed within 30 minutes, and costs about $0.07 in test-specific consumable materials., Conclusions: Our results validate the paper-based SCA test as a useful low-cost tool for screening adults and children for sickle trait and disease and demonstrate its practicality in resource-limited clinical settings.

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.

Quantification of sickle hemoglobin (HbS) in patients with sickle cell disease (SCD) undergoing hydroxyurea or chronic transfusion therapy is essential to monitoring the effectiveness of these therapies. The clinical monitoring of %HbS using conventional laboratory methods is limited by high per-test costs and long turnaround times usually associated with these methods. Here we demonstrate a simple, rapid, inexpensive paper-based assay capable of quantifying %HbS in blood samples from patients with SCD. A 20 μL droplet of whole blood and hemoglobin solubility buffer was deposited on chromatography paper. The relative color intensities of regions of the resulting blood stain, determined by automated image analysis, are used to estimate %HbS. We compared the paper-based assay with hemoglobin electrophoresis (comparison method) using blood samples from 88 subjects. The test shows high correlation (R(2)  = 0.86) and strong agreement (standard deviation of difference = 7%HbS) with conventional Hb electrophoresis measurement of %HbS, and closely approximates clinically predicted change in %HbS with transfusion therapy (mean difference 2.6%HbS, n = 5). The paper-based assay can be completed in less than 35 min and has a per-test cost less than $0.25. The assay is accurate across a wide range of HbS levels (10-97%) and hemoglobin concentrations (5.6-12.9 g/dL) and is unaffected by high levels of HbF (up to 80.6%). This study demonstrates the feasibility of the paper-based %HbS assay. The paper-based test could improve clinical care for SCD, particularly in resource-limited settings, by enabling more rapid and less expensive %HbS monitoring., (© 2015 Wiley Periodicals, Inc.)

Although effective low-cost interventions exist, child mortality attributable to sickle cell disease (SCD) remains high in low-resource areas due, in large part, to the lack of accessible diagnostic methods. The presence of dense (ρ > 1.120 g/cm(3)) cells is characteristic of SCD. The fluid, self-assembling step-gradients in density created by aqueous multiphase systems (AMPSs) identifies SCD by detecting dense cells. AMPSs separate different forms of red blood cells by density in a microhematocrit centrifuge and provide a visual means to distinguish individuals with SCD from those with normal hemoglobin or with nondisease, sickle-cell trait in under 12 min. Visual evaluation of a simple two-phase system identified the two main subclasses of SCD [homozygous (Hb SS) and heterozygous (Hb SC)] with a sensitivity of 90% (73-98%) and a specificity of 97% (86-100%). A three-phase system identified these two types of SCD with a sensitivity of 91% (78-98%) and a specificity of 88% (74-98%). This system could also distinguish between Hb SS and Hb SC. To the authors' knowledge, this test demonstrates the first separation of cells by density with AMPSs, and the usefulness of AMPSs in point-of-care diagnostic hematology.

This study investigates the stability of a finger of air as it propagates into a liquid-filled model of a liquid-filled model of a pulmonary bifurcation. We seek to elucidate the stability characteristics of the reopening of daughter airways, an event that may be important to the treatment of acute lung disease. To do so, we investigated the symmetry of reopening under conditions of nearly constant surface tension with 1) purified H2O or 2) an anionic surfactant (sodium dodecyl sulfate). Dynamic surface tension was investigated using pulmonary surfactant (Infasurf) with and without the presence of albumin. Flow visualization was accomplished using a microparticle image velocimetry (μ-PIV)/shadowgraph system through which we measured 1) the propagation velocity of the finger of air that reopens each daughter branch, and 2) the instantaneous and averaged velocity field of liquid phase surrounding the tip of the propagating bubble. Only pulmonary surfactant demonstrated the ability of maintaining a nearly symmetric propagation in the daughter channels, which is likely to lead to homogeneous airway reopening. In contrast, when pulmonary surfactant was inactivated by albumin or when the system was held at a nearly constant surface tension, reopening occurred asymmetrically. Our analysis suggests that Infasurf's dynamic surface tension qualities are important to stabilize the removal of liquid obstructions. This demonstrates a new important function of pulmonary surfactant for airway reopening of a multibranched network.

Background: The measurement of hemoglobin concentration ([Hb]) is performed routinely as a part of a complete blood cell count to evaluate the oxygen-carrying capacity of blood. Devices currently available to physicians and clinical laboratories for measuring [Hb] are accurate, operate on small samples, and provide results rapidly, but may be prohibitively expensive for resource-limited settings. The unavailability of accurate but inexpensive diagnostic tools often precludes proper diagnosis of anemia in low-income developing countries. Therefore, we developed a simple paper-based assay for measuring [Hb]., Methods: A 20-μL droplet of a mixture of blood and Drabkin reagent was deposited onto patterned chromatography paper. The resulting blood stain was digitized with a portable scanner and analyzed. The mean color intensity of the blood stain was used to quantify [Hb]. We compared the performance of the paper-based Hb assay with a hematology analyzer (comparison method) using blood samples from 54 subjects., Results: The values of [Hb] measured by the paper-based assay and the comparison method were highly correlated (R(2) = 0.9598); the standard deviation of the difference between the two measurements was 0.62 g/dL. The assay was accurate within 1 g/dL 90.7% of the time, overestimating [Hb] by ≥1 g/dL in 1.9% and underestimating [Hb] by ≥1 g/dL in 7.4% of the subjects., Conclusions: This study demonstrates the feasibility of the paper-based Hb assay. This simple, low-cost test should be useful for diagnosing anemia in resource-limited settings, particularly in the context of care for malaria, HIV, and sickle cell disease patients in sub-Saharan Africa.

A critical accomplishment in the rapidly developing field of regenerative medicine will be the ability to foster repair of neurons severed by injury, disease, or microsurgery. In C. elegans, individual visualized axons can be laser-cut in vivo and neuronal responses to damage can be monitored to decipher genetic requirements for regeneration. With an initial interest in how local environments manage cellular debris, we performed femtosecond laser axotomies in genetic backgrounds lacking cell death gene activities. Unexpectedly, we found that the CED-3 caspase, well known as the core apoptotic cell death executioner, acts in early responses to neuronal injury to promote rapid regeneration of dissociated axons. In ced-3 mutants, initial regenerative outgrowth dynamics are impaired and axon repair through reconnection of the two dissociated ends is delayed. The CED-3 activator, CED-4/Apaf-1, similarly promotes regeneration, but the upstream regulators of apoptosis CED-9/Bcl2 and BH3-domain proteins EGL-1 and CED-13 are not essential. Thus, a novel regulatory mechanism must be utilized to activate core apoptotic proteins for neuronal repair. Since calcium plays a conserved modulatory role in regeneration, we hypothesized calcium might play a critical regulatory role in the CED-3/CED-4 repair pathway. We used the calcium reporter cameleon to track in vivo calcium fluxes in the axotomized neuron. We show that when the endoplasmic reticulum calcium-storing chaperone calreticulin, CRT-1, is deleted, both calcium dynamics and initial regenerative outgrowth are impaired. Genetic data suggest that CED-3, CED-4, and CRT-1 act in the same pathway to promote early events in regeneration and that CED-3 might act downstream of CRT-1, but upstream of the conserved DLK-1 kinase implicated in regeneration across species. This study documents reconstructive roles for proteins known to orchestrate apoptotic death and links previously unconnected observations in the vertebrate literature to suggest a similar pathway may be conserved in higher organisms., Competing Interests: The authors have declared that no competing interests exist.

Objective: Systemic lupus erythematosus (SLE) is characterized by intravascular activation of the complement system and deposition of complement fragments (C3 and C4) on plasma membranes of circulating cells, including red blood cells (RBCs). The aim of this study was to address whether this process affects the biophysical properties of RBCs., Methods: Serum and RBCs were isolated from patients with SLE and healthy controls. RBCs from healthy universal donors (type O, Rh negative) were incubated with SLE or control serum. We used flow cytometry to assess complement fragment deposition on RBCs. RBC membrane deformability was measured using 2-dimensional microchannel arrays. Protein phosphorylation levels were quantified by Western blotting., Results: Incubation of healthy universal donor RBCs with sera from patients with SLE, but not with control sera, led to deposition of C4d fragments on the RBCs. Complement-decorated RBCs exhibited significant decreases in both membrane deformability and flickering. Sera from SLE patients triggered a transitory Ca(++) influx in RBCs that was associated with decreased phosphorylation of β-spectrin and with increased phosphorylation of band 3, two key proteins of RBC cytoskeleton. Finally, incubation with SLE sera led to the production of nitric oxide by RBCs, whereas this did not occur with control sera., Conclusion: Our data suggest that complement activation in patients with SLE leads to calcium-dependent cytosketeletal changes in RBCs that render them less deformable, probably impairing their flow through capillaries. This phenomenon may negatively affect the delivery of oxygen to the tissues., (Copyright © 2011 by the American College of Rheumatology.)

Microbes as well as immune complexes and other continuously generated inflammatory particles are efficiently removed from the human circulation by red blood cells (RBCs) through a process called immune-adherence clearance. During this process, RBCs use complement receptor 1 (CR1, CD35) to bind circulating complement-opsonized particles and transfer them to resident macrophages in the liver and spleen for removal. We here show that ligation of RBC CR1 by antibody and complement-opsonized particles induces a transient Ca(++) influx that is proportional to the RBC CR1 levels and is inhibited by T1E3 pAb, a specific inhibitor of TRPC1 channels. The CR1-elicited RBC Ca(++) influx is accompanied by an increase in RBC membrane deformability that positively correlates with the number of preexisting CR1 molecules on RBC membranes. Biochemically, ligation of RBC CR1 causes a significant increase in phosphorylation levels of β-spectrin that is inhibited by preincubation of RBCs with DMAT, a specific casein kinase II inhibitor. We hypothesize that the CR1-dependent increase in membrane deformability could be relevant for facilitating the transfer of CR1-bound particles from the RBCs to the hepatic and splenic phagocytes.

We demonstrate the assembly of biohybrid materials from engineered tissues and synthetic polymer thin films. The constructs were built by culturing neonatal rat ventricular cardiomyocytes on polydimethylsiloxane thin films micropatterned with extracellular matrix proteins to promote spatially ordered, two-dimensional myogenesis. The constructs, termed muscular thin films, adopted functional, three-dimensional conformations when released from a thermally sensitive polymer substrate and were designed to perform biomimetic tasks by varying tissue architecture, thin-film shape, and electrical-pacing protocol. These centimeter-scale constructs perform functions as diverse as gripping, pumping, walking, and swimming with fine spatial and temporal control and generating specific forces as high as 4 millinewtons per square millimeter.