Sickle cell disease (SCD) is a hematological disorder leading to blood vessel occlusion accompanied by painful episodes and even death. Red blood cells (RBCs) of SCD patients have diverse shapes that reveal important biomechanical and bio-rheological characteristics, e.g. their density, fragility, adhesive properties, etc. Hence, having an objective and effective way of RBC shape quantification and classification will lead to better insights and eventual better prognosis of the disease. To this end, we have developed an automated, high-throughput, ex-vivo RBC shape classification framework that consists of three stages. First, we present an automatic hierarchical RBC extraction method to detect the RBC region (ROI) from the background, and then separate touching RBCs in the ROI images by applying an improved random walk method based on automatic seed generation. Second, we apply a mask-based RBC patch-size normalization method to normalize the variant size of segmented single RBC patches into uniform size. Third, we employ deep convolutional neural networks (CNNs) to realize RBC classification; the alternating convolution and pooling operations can deal with non-linear and complex patterns. Furthermore, we investigate the specific shape factor quantification for the classified RBC image data in order to develop a general multiscale shape analysis. We perform several experiments on raw microscopy image datasets from 8 SCD patients (over 7,000 single RBC images) through a 5-fold cross validation method both for oxygenated and deoxygenated RBCs. We demonstrate that the proposed framework can successfully classify sickle shape RBCs in an automated manner with high accuracy, and we also provide the corresponding shape factor analysis, which can be used synergistically with the CNN analysis for more robust predictions. Moreover, the trained deep CNN exhibits good performance even for a deoxygenated dataset and distinguishes the subtle differences in texture alteration inside the oxygenated and deoxygenated RBCs.

SignificancePatients with sickle cell disease (SCD) suffer from painful vasoocclusive crises. Polymerization of sickle hemoglobin (HbS) in RBCs is generally considered a major contributor to such crisis events. Here, we present the simultaneous and synergistic coupling of adhesion and HbS polymerization. We show that the age of RBCs in circulation plays an important role in mediating this synergistic effect on blood rheology and clinical symptoms. In particular, the youngest RBCs exhibit unique adhesion dynamics, whereby polymerized HbS fiber bundles grow from cell surfaces to serve as sites of cytoadherence. Our molecular-level simulations show how the attachment and dissociation of molecular bonds influence adhesion dynamics. These results provide a framework that could elucidate the mechanistic basis of SCD vasoocclusive pain crises.

Hydroxyurea (HU) has been used clinically to reduce the frequency of painful crisis and the need for blood transfusion in sickle cell disease (SCD) patients. However, the mechanisms underlying such beneficial effects of HU treatment are still not fully understood. Studies have indicated a weak correlation between clinical outcome and molecular markers, and the scientific quest to develop companion biophysical markers have mostly targeted studies of blood properties under hypoxia. Using a common-path interferometric technique, we measure biomechanical and morphological properties of individual red blood cells in SCD patients as a function of cell density, and investigate the correlation of these biophysical properties with drug intake as well as other clinically measured parameters. Our results show that patient-specific HU effects on the cellular biophysical properties are detectable at normoxia, and that these properties are strongly correlated with the clinically measured mean cellular volume rather than fetal hemoglobin level.

Background Transfusion of packed erythrocytes stored for a long duration is associated with increased pulmonary arterial pressure and vascular resistance. Prolonged storage decreases erythrocyte deformability, and older erythrocytes are rapidly removed from the circulation after transfusion. The authors studied whether treating stored packed ovine erythrocytes with NO before transfusion could prevent pulmonary vasoconstriction, enhance erythrocyte deformability, and prolong erythrocyte survival after transfusion. Methods Ovine leukoreduced packed erythrocytes were treated before transfusion with either NO gas or a short-lived NO donor. Sheep were transfused with autologous packed erythrocytes, which were stored at 4°C for either 2 (“fresh blood”) or 40 days (“stored blood”). Pulmonary and systemic hemodynamic parameters were monitored before, during, and after transfusion. Transfused erythrocytes were labeled with biotin to measure their circulating lifespan. Erythrocyte deformability was assessed before and after NO treatment using a microfluidic device. Results NO treatment improved the deformability of stored erythrocytes and increased the number of stored erythrocytes circulating at 1 and 24 h after transfusion. NO treatment prevented transfusion-associated pulmonary hypertension (mean pulmonary arterial pressure at 30 min of 21 ± 1 vs. 15 ± 1 mmHg in control and NO–treated packed erythrocytes, P < 0.0001). Washing stored packed erythrocytes before transfusion did not prevent pulmonary hypertension. Conclusions NO treatment of stored packed erythrocytes before transfusion oxidizes cell-free oxyhemoglobin to methemoglobin, prevents subsequent NO scavenging in the pulmonary vasculature, and limits pulmonary hypertension. NO treatment increases erythrocyte deformability and erythrocyte survival after transfusion. NO treatment might provide a promising therapeutic approach to prevent pulmonary hypertension and extend erythrocyte survival.

Transient regulation of Plasmodium numbers below the density that induces fever has been observed in chronic malaria infections in humans. This species transcending control cannot be explained by immunity alone. Using an in vitro system we have observed density dependent regulation of malaria population size as a mechanism to possibly explain these in vivo observations. Specifically, Plasmodium falciparum blood stages from a high but not low‐density environment exhibited unique phenotypic changes during the late trophozoite (LT) and schizont stages of the intraerythrocytic cycle. These included in order of appearance: failure of schizonts to mature and merozoites to replicate, apoptotic‐like morphological changes including shrinking, loss of mitochondrial membrane potential, and blebbing with eventual release of aberrant parasites from infected erythrocytes. This unique death phenotype was triggered in a stage‐specific manner by sensing of a high‐density culture environment. Conditions of glucose starvation, nutrient depletion, and high lactate could not induce the phenotype. A high‐density culture environment induced rapid global changes in the parasite transcriptome including differential expression of genes involved in cell remodeling, clonal antigenic variation, metabolism, and cell death pathways including an apoptosis‐associated metacaspase gene. This transcriptional profile was also characterized by concomitant expression of asexual and sexual stage‐specific genes. The data show strong evidence to support our hypothesis that density sensing exists in P. falciparum. They indicate that an apoptotic‐like mechanism may play a role in P. falciparum density regulation, which, as in yeast, has features quite distinguishable from mammalian apoptosis. Database Gene expression data are available in the GEO databases under the accession number http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE91188.