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’s 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 using the paper-based assay and the comparison method were highly correlated (R2 = 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.

BACKGROUND Prolonged storage of red blood cells 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 biorheological parameters. METHODS Red blood cells stored hypothermically for 6–7 weeks were washed in a 1% human serum albumin solution. Red cell deformability was measured with osmotic gradient ektacytometry. The viscosity of red cell suspensions were measured with a Couette-type viscometer. The flow behaviour of red cells suspended at 40% hematocrit was tested with an artificial microvascular network. RESULTS Washing in 1% albumin reduced higher degrees of echinocytes and increased the frequency of discocytes, thereby shifting the morphological index towards discocytosis. Washing also reduced red cell swelling. This shape recovery was not seen after washing in saline, buffer or plasma. Red cell shape normalisation 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 artificial microvascular network. CONCLUSIONS Washing of stored red blood cells in a 1% human serum albumin 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 red cells, and thus potentially reducing the likelihood of adverse clinical outcomes associated with transfusion of blood stored for longer periods of time.

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.

BACKGROUND The shape of human red blood cells (RBCs) deteriorates progressively throughout hypothermic storage, with echinocytosis being the most prevalent pathway of this morphological 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 leukocyte-reduced, re-suspended 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, sphero-echinocyte 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 statistically 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 artificial microvascular network. Echinocytosis induced by hypothermic storage could therefore be responsible for a similarly substantial impairment of deformability previously observed for stored RBCs.

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. MCV (mean corpuscular volume) and MCHC (mean corpuscular hemoglobin concentration), were determined using centrifuged microhematocrit. RBC suspensions at constant cell numbers were used to measure viscosity at shear rates ranging from 0.11 to 69.5 s−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 towards 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.

BackgroundThe 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 FindingsIn 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.ConclusionsOur 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 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, 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/hour; accuracy: 91.9% low-resolution, 75.3% high-resolution). Diameter mean and standard deviation by morphology class: discocyte 7.80±0.49μm, echinocyte 1 7.61±0.63μm, echinocyte 2 7.02±0.61μm, echinocyte 3 6.47±0.42μm, sphero-echinocyte 6.01±0.26μm, spherocyte 6.02±0.27μm, stomatocyte 1 6.95±0.61μm, stomatocyte 2 7.32 ± 0.47μm. Conclusion 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.

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 (R2 = 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 = 4). The paper-based assay can be completed in less than 35 minutes 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.