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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

Microfluidic lab-on-a-chip technologies enable the analysis and manipulation of small fluid volumes and particles at small scales and the control of fluid flow and transport processes at the microscale, leading to the development of new methods to address a broad range of scientific and medical challenges. Microfluidic and lab-on-a-chip technologies have made a noteworthy impact in basic, preclinical, and clinical research, especially in hematology and vascular biology due to the inherent ability of microfluidics to mimic physiologic flow conditions in blood vessels and capillaries. With the potential to significantly impact translational research and clinical diagnostics, technical issues and incentive mismatches have stymied microfluidics from fulfilling this promise. We describe how accessibility, usability, and manufacturability of microfluidic technologies should be improved and how a shift in mindset and incentives within the field is also needed to address these issues. In this report, we discuss the state of the microfluidic field regarding current limitations and propose future directions and new approaches for the field to advance microfluidic technologies closer to translation and clinical use. While our report focuses on using blood as the prototypical biofluid sample, the proposed ideas and research directions can be extrapolated to other areas of hematology, oncology, biology, and medicine.

The significant biological and functional differences between small and large platelets suggested by recent studies could have profound implications for transfusion medicine. However, investigating the relationship between platelet size and function is challenging because separating platelets by size without affecting their properties is difficult. A standard approach is centrifugation, but it inevitably leads to premature activation and aggregation of separated platelets. This paper describes the development and validation of a microfluidic device based on controlled incremental filtration (CIF) for separating platelets by size without the cell damage and usability limitations associated with centrifugation. Platelet samples derived from whole blood were used to evaluate the dependence of the CIF device separation performance on design parameters and flow rate, and to compare the properties of PLT fractions generated by the CIF device with those produced using a centrifugation protocol in a split-sample study. This was accomplished by quantifying the platelet size distribution, mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet activation before and after processing for all input and output samples. The 'large platelet' fractions produced by the CIF device and the centrifugation protocol were essentially equivalent (no significant difference in MPV and P-LCR). Platelets in the 'small platelet' fraction produced by the CIF device were significantly smaller than those produced by centrifugation (lower MPV and P-LCR). This was because the CIF 'small platelet' fraction was contaminated by much fewer large platelets (∼2-times lower recovery of >12 fL platelets) and retained the smallest platelets that were discarded by the centrifugation protocol. There was no significant difference in platelet activation between the two methods. However, centrifugation required a substantial amount of additional anticoagulant to prevent platelet aggregation during pelleting. Unlike centrifugation, the CIF device offered continuous, flow-through, single-step processing that did not cause platelet aggregation. Such a capability has the potential to accelerate the basic studies of the relationship between platelet size and function, and ultimately improve transfusion practice, particularly in the pediatric setting, where the need for low-volume, high-quality platelet transfusions is most urgent.

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.)

Regulatory T cells (Tregs) are a unique subset of lymphocytes that play a vital role in regulating the immune system by suppressing unwanted immune responses and thus preventing autoimmune diseases and inappropriate inflammatory reactions. In preclinical and clinical trials, these cells have demonstrated the ability to prevent and treat graft vs. host disease, alleviate autoimmune symptoms, and promote transplant tolerance. In this review, we provide a background on Treg cells with a focus on important Treg cell markers and Treg subsets, and outline the methodology currently used for manufacturing adoptive regulatory T cell therapies (TRACT). Finally, we discuss the approaches and outcomes of several clinical trials in which Tregs have been adoptively transferred to patients., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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).)

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.)

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: 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: 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 Disease (SCD) is among the most common single-gene diseases in the world but evidence-based comprehensive health care has not been implemented where the highest prevalence of SCD occurs, in sub-Saharan Africa (SSA). It represents an urgent health burden, both in terms of mortality and morbidity with an estimated mortality of 8-16% in children under 5 years in SSA. Addressing the high mortality of SCD in SSA and for effective management of SCD, newborn screening (NBS) should be incorporated with prevention of infections (including pneumococcal septicaemia and malaria), parental education and support at all levels of healthcare provision to enable timely recognition. The NBS working group of the Africa Sickle Cell Research Network (AfroSickleNet) collaboration surveyed current projects in NBS in SSA, and current conditions that hinder more widespread implementation of NBS for SCD. Solutions based on new point-of-care testing technology to disseminate education, and implementation science approaches that leverage existing resources are proposed.

Background and Objectives: Infusion of by-products of red blood cell (RBC) storage-induced degradation as well as of the residual plasma proteins and the anticoagulant-preservative solution contained in units of stored blood serve no therapeutic purpose and may be harmful to some patients. Here, we describe a prototype of a gravity-driven system for bedside washing of stored RBCs., Materials and Methods: Stored RBCs were diluted to 10% haematocrit (Hct) with normal saline, matching the conventional washing procedure. The dilute RBC suspensions were passed through a column of coiled tubing to allow RBC sedimentation in normal gravity, thus separating them from the washing solution. Washed RBCs were collected using bifurcations located along the tubing. Washing efficiency was quantified by measuring Hct, morphology, deformability, free haemoglobin and total-free protein., Results: The gravity-driven washing system operating at 0·5 ml/min produced washed RBCs with final Hct of 36·7 ± 3·4% (32·3-41·2%, n = 10) and waste Hct of 3·4 ± 0·7% (2·4-4·3%, n = 10), while removing 80% of free haemoglobin and 90% of total-free protein. Washing improved the ability of stored RBCs to perfuse an artificial microvascular network by 20%. The efficiency of washing performed using the gravity-driven system was not significantly different than that of conventional centrifugation., Conclusions: This proof-of-concept study demonstrates the feasibility of washing stored RBCs using a simple, disposable system with efficiency comparable to that of conventional centrifugation, and thus represents a significant first step towards enabling low-cost washing of stored blood at bedside., (© 2017 International Society of Blood Transfusion.)

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.)

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: 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: 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.

Objective: RBCs suspended in plasma form multicellular aggregates under low-flow conditions, increasing apparent blood viscosity at low shear rates. It has previously been unclear, however, if RBC aggregation affects microvascular perfusion. Here, we analyzed the impact of RBC aggregation on perfusion and 'capillary' hematocrit in an AMVN at driving pressures ranging from 5 to 60 cm H 2 O to determine if aggregation could improve tissue oxygenation., Methods: RBCs were suspended at 30% hematocrit in either 46.5 g/L dextran 40 (D40, non-aggregating medium) or 35 g/L dextran 70 (D70, aggregating medium) solutions with equal viscosity., Results: Aggregation was readily observed in the AMVN for RBCs suspended in D70 at driving pressures ≤40 cm H 2 O. The AMVN perfusion rate was the same for RBCs suspended in aggregating and non-aggregating medium, at both 'venular' and 'capillary' level. Estimated 'capillary' hematocrit was higher for D70 suspensions than for D40 suspensions at intermediate driving pressures (5-40 cm H 2 O)., Conclusions: We conclude that although RBC aggregation did not affect the AMVN perfusion rate independently of the driving pressure, a higher hematocrit in the 'capillaries' of the network for D70 suspensions suggested a better oxygen transport capacity in the presence of RBC aggregation., (© 2016 John Wiley & Sons Ltd.)

Every year, hundreds of thousands of children worldwide are born with sickle cell disease, a genetic disorder that impacts the hemoglobin molecules in blood. If left undiagnosed and untreated, most affected children will die before reaching the age of five. However, highly accurate diagnostic methods and effective treatment regimens for sickle cell disease have been known for many years, and children who receive early diagnosis and subsequent comprehensive care survive well into adulthood-as evidenced by the tremendous success of universal newborn screening programs in North America and Europe.

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 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: 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.

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.)

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.)

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.)

The number of microfluidic strategies aimed at separating particles or cells of a specific size within a continuous flow system continues to grow. The wide array of biomedical and other applications that would benefit from successful development of such technology has motivated the extensive research in this area over the past 15 years. However, despite promising advancements in microfabrication capabilities, a versatile approach that is suitable for a large range of particle sizes and high levels of enrichment, with a volumetric throughput sufficient for large-scale applications, has yet to emerge. Here we describe a straightforward method that enables the rapid design of microfluidic devices that are capable of enriching/removing particles within a complex aqueous mixture, with an unprecedented range of potential cutoff diameter (below 1 μm to above 100 μm) and an easily scalable degree of enrichment/filtration (up to 10-fold and well beyond). A simplified model of a new approach to crossflow filtration - controlled incremental filtration - was developed and validated for its ability to generate microfluidic devices that efficiently separate particles on the order of 1-10 μm, with throughputs of tens of μL min(-1), without the use of a pump. Precise control of the amount of fluid incrementally diverted at each filtration "gap" of the device allows for the gap size (~20 μm) to be much larger than the particles of interest, while the simplicity of the model allows for many thousands of these filtration points to be readily incorporated into a desired device design. This new approach should enable truly high-throughput microfluidic particle-separation devices to be generated, even by users only minimally experienced in fluid mechanics and microfabrication techniques.

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.

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.

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.

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 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.

Multicellular tumor spheroids are widely used as in vitro models for testing of anticancer drugs. The advantage of this approach is that it can predict the outcome of a drug treatment on human cancer cells in their natural three-dimensional environment without putting actual patients at risk. Several methods were utilized in the past to grow submillimeter-size tumor spheroids. However, these small models are not very useful for preclinical studies of tumor ablation where the goal is the complete destruction of tumors that can reach several centimeters in diameter in the human body. Here, we propose a PDMS well method for large tumor spheroid culture. Our experiments with HepG2 hepatic cancer cells show that three-dimensional aggregates of tumor cells with a volume as large as 44 mm(3) can be grown in cylindrical PDMS wells after the initial culture of tumor cells by the hanging drop method. This is a 350 times more than the maximum volume of tumor spheroids formed inside hanging drops (0.125 mm(3))., (© 2013 American Institute of Chemical Engineers.)

This communication describes a very simple, rapid and inexpensive point-of-care diagnostic test for sickle cell disease (SCD) that can conclusively differentiate between blood samples from normal healthy individuals, sickle cell trait carriers and SCD patients using the characteristic blood stain patterns produced by each sample on paper.

Histamine and tumor necrosis factor-α (TNF-α) are critical mediators of acute and chronic inflammation that are generated by mast cells and macrophages in atherosclerotic lesions or systemically during allergic attacks. Both of them induce activation of vascular endothelium and thus may play a role in thrombosis. Here we studied the interplay between histamine and TNF-α in glycoprotein (GP) Ibα-mediated platelet adhesion to cultured human vascular endothelial cells under static and shear flow conditions. The stimulation of endothelial cells with histamine or TNF-α increased the number of adherent or slow rolling GP Ibα-coated microbeads or washed human platelets. However, the application of histamine to endothelium pre-activated by TNF-α inhibited GP Ibα-mediated platelet adhesion. These effects were found to be associated with changes in the concentration of ultra large von Willebrand factor (ULVWF) strings anchored to endothelium. The results of this study indicate that histamine released during mast cell degranulation may cause or inhibit thrombosis, depending on whether it acts on resting endothelial cells or on cells pre-activated by other inflammatory stimuli., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Previous computational studies have suggested that the capillary blood flow oscillations frequently observed in vivo can originate spontaneously from the non-linear rheological properties of blood, without any regulatory input. Testing this hypothesis definitively in experiments involving real microvasculature has been difficult because in vivo the blood flow in capillaries is always actively controlled by the host. The objective of this study was to test the hypothesis experimentally and to investigate the relative contribution of different blood cells to the capillary blood flow dynamics under static boundary conditions and in complete isolation from the active regulatory mechanisms mediated by the blood vessels in vivo. To accomplish this objective, we passed whole blood and re-constituted blood samples (purified red blood cells suspended in buffer or in autologous plasma) through an artificial microvascular network (AMVN) comprising completely inert, microfabricated vessels with the architecture inspired by the real microvasculature. We found that the flow of blood in capillaries of the AMVN indeed oscillates with characteristic frequencies in the range of 0-0.6 Hz, which is in a very good agreement with previous computational studies and in vivo observations. We also found that the traffic of leukocytes through the network (typically neglected in computational modeling) plays an important role in generating the oscillations. This study represents the key piece of experimental evidence in support of the hypothesis that spontaneous, self-sustained oscillations of capillary blood flow can be generated solely by the non-linear rheological properties of blood flowing through microvascular networks, and provides an insight into the mechanism of this fundamentally important microcirculatory phenomenon., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.)

This paper describes the use of magnetic levitation (MagLev) to measure the association of proteins and ligands. The method starts with diamagnetic gel beads that are functionalized covalently with small molecules (putative ligands). Binding of protein to the ligands within the bead causes a change in the density of the bead. When these beads are suspended in a paramagnetic aqueous buffer and placed between the poles of two NbFeB magnets with like poles facing, the changes in the density of the bead on binding of protein result in changes in the levitation height of the bead that can be used to quantify the amount of protein bound. This paper uses a reaction-diffusion model to examine the physical principles that determine the values of rate and equilibrium constants measured by this system, using the well-defined model system of carbonic anhydrase and aryl sulfonamides. By tuning the experimental protocol, the method is capable of quantifying either the concentration of protein in a solution, or the binding affinities of a protein to several resin-bound small molecules simultaneously. Since this method requires no electricity and only a single piece of inexpensive equipment, it may find use in situations where portability and low cost are important, such as in bioanalysis in resource-limited settings, point-of-care diagnosis, veterinary medicine, and plant pathology. It still has several practical disadvantages. Most notably, the method requires relatively long assay times and cannot be applied to large proteins (>70 kDa), including antibodies. The design and synthesis of beads with improved characteristics (e.g., larger pore size) has the potential to resolve these problems.

Many diagnostic tests in a conventional clinical laboratory are performed on blood plasma because changes in its composition often reflect the current status of pathological processes throughout the body. Recently, a significant research effort has been invested into the development of microfluidic paper-based analytical devices (μPADs) implementing these conventional laboratory tests for point-of-care diagnostics in resource-limited settings. This paper describes the use of red blood cell (RBC) agglutination for separating plasma from finger-prick volumes of whole blood directly in paper, and demonstrates the utility of this approach by integrating plasma separation and a colorimetric assay in a single μPAD. The μPAD was fabricated by printing its pattern onto chromatography paper with a solid ink (wax) printer and melting the ink to create hydrophobic barriers spanning through the entire thickness of the paper substrate. The μPAD was functionalized by spotting agglutinating antibodies onto the plasma separation zone in the center and the reagents of the colorimetric assay onto the test readout zones on the periphery of the device. To operate the μPAD, a drop of whole blood was placed directly onto the plasma separation zone of the device. RBCs in the whole blood sample agglutinated and remained in the central zone, while separated plasma wicked through the paper substrate into the test readout zones where analyte in plasma reacted with the reagents of the colorimetric assay to produce a visible color change. The color change was digitized with a portable scanner and converted to concentration values using a calibration curve. The purity and yield of separated plasma was sufficient for successful operation of the μPAD. This approach to plasma separation based on RBC agglutination will be particularly useful for designing fully integrated μPADs operating directly on small samples of whole blood.