BackgroundBone changes are common in sickle cell disease, but the pathogenesis is not fully understood. Tartrate-resistant acid phosphatase (TRACP) type 5b is produced by bone-resorbing osteoclasts. In other forms of hemolytic anemia, increased iron stores are associated with osteoporosis. We hypothesized that transfusional iron overload would be associated with increased osteoclast activity in patients with sickle cell disease. Design and MethodsWe examined tartrate-resistant acid phosphatase 5b concentrations in patients with sickle cell disease and normal controls of similar age and sex distribution at steady state. Serum tartrateresistant acid phosphatase 5b concentration was measured using an immunocapture enzyme assay and plasma concentrations of other cytokines were assayed using the Bio-Plex suspension array system. Tricuspid regurgitation velocity, an indirect measure of systolic pulmonary artery pressure, was determined by echocardiography. ResultsTartrate-resistant acid phosphatase 5b concentrations were higher in 58 adults with sickle cell disease than in 22 controls (medians of 4.4 versus 2.4 U/L, respectively; P=0.0001). Among the patients with sickle cell disease, tartrate-resistant acid phosphatase 5b independently correlated with blood urea nitrogen (standardized beta=0.40, P=0.003), interleukin-8 (standardized beta=0.30, P=0.020), and chemokine C-C motif ligand 5 (standardized beta=-0.28, P=0.031) concentrations, but not with serum ferritin concentration. Frequent blood transfusions (>10 units in life time) were not associated with higher tartrate-resistant acid phosphatase 5b levels in multivariate analysis. There were strong correlations among tartrate-resistant acid phosphatase 5b, alkaline phosphatase and tricuspid regurgitation velocity (r>0.35, P<0.001). ConclusionsPatients with sickle cell disease have increased osteoclast activity as reflected by serum tartrateresistant acid phosphatase 5b concentrations. Our results may support a potential role of inflammation rather than increased iron stores in stimulating osteoclast activity in sickle cell disease. The positive relationships among tartrate-resistant acid phosphatase 5b, alkaline phosphatase and tricuspid regurgitation velocity raise the possibility of a common pathway in the pulmonary and bone complications of sickle cell disease.Key words: sickle cell disease, osteoclast activity, bone turnover, inflammation, TRACP 5b, pulmonary complications.Citation: Nouraie M, Cheng K, Niu X, Moore-King E, Fadojutimi-Akinsi MF, Minniti CP, Sable C, Rana S, Dham N, Campbell A, Ensing G, Kato GJ, Gladwin MT, Castro OL, and Gordeuk VR. Predictors of osteoclast activity in sickle cell disease patients. Haematologica 2011;96(8):1092-1098. doi:10.3324/haematol.2011 This is an open-access paper. Predictors of osteoclast activity in patients with sickle cell disease

Endothelial activation and sickle red blood cell (RBC) adhesion are central to the pathogenesis of sickle cell disease (SCD). Quantitatively, RBC-derived extracellular vesicles (REVs) are more abundant from SS RBCs compared with healthy RBCs (AA RBCs). Sickle RBC-derived REVs (SS REVs) are known to promote endothelial cell (EC) activation through cell signalling and transcriptional regulation at longer terms. However, the SS REV-mediated short-term non-transcriptional response of EC is unclear. Here, we examined the impact of SS REVs on acute microvascular EC activation and RBC adhesion at 2 h. Compared with AA REVs, SS REVs promoted human pulmonary microvascular ECs (HPMEC) activation indicated by increased von Willebrand factor (VWF) expression. Under microfluidic conditions, we found abnormal SS RBC adhesion to HPMECs exposed to SS REVs. This enhanced SS RBC adhesion was reduced by haeme binding protein haemopexin or VWF cleaving protease ADAMTS13 to a level similar to HPMECs treated with AA REVs. Consistent with these observations, haemin-or SS REV-induced microvascular stasis in SS mice with implanted dorsal skin-fold chambers that was inhibited by ADAMTS13. The adhesion induced by SS REVs was variable and was higher with SS RBCs from patients with increased markers of haemolysis (lactate dehydrogenase and reticulocyte count) or a concomitant clinical diagnosis of deep vein thrombosis. Our results emphasise the critical contribution made by REVs to the pathophysiology of SCD by triggering acute microvascular EC activation and abnormal RBC adhesion. These findings may

A point-of-care diagnostic technology and approach is presented to perform both anemia detection and hemoglobin variant identification in a single test using paper-based microchip electrophoresis.

Endothelial activation and sickle red blood cell (RBC) adhesion are central to the pathogenesis of sickle cell disease (SCD). Quantitatively, RBC-derived extracellular vesicles, REVs, are more abundant from SS RBCs compared with healthy RBCs (AA RBCs). Sickle RBC-derived REVs (SS REVs) are known to promote endothelial cell (EC) activation through cell signaling and transcriptional regulation at longer terms. However, the SS REV-mediated short term non transcriptional response of EC is unclear. Here, we examined the impact of SS REVs on acute microvascular EC activation and RBC adhesion at 2 hours. Compared with AA REVs, SS REVs promoted human pulmonary microvascular endothelial cells (HPMEC) activation indicated by increased von Willebrand Factor (vWF) expression. Under microfluidic conditions, we found abnormal SS RBC adhesion to HPMECs exposed to SS REVs. This enhanced SS RBC adhesion was reduced by vWF cleaving protease ADAMTS13 to a level similar to HPMECs treated with AA REVs. Consistent with these observations, studies in SS mice with implanted dorsal skin-fold chambers found hemin-induced stasis was inhibited by ADAMTS13. The adhesion induced by SS REVs was variable, and was higher with SS RBCs from patients with increased markers of hemolysis (LDH and reticulocyte count) or a concomitant clinical diagnosis of deep vein thrombosis. Our results emphasize the critical contribution made by REVs to the pathophysiology of SCD by triggering acute microvascular EC activation and abnormal RBC adhesion. These findings may help to better understand acute pathophysiological mechanism of SCD and thereby the development of new treatment strategies using vWF as a potential target.

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Introduction: Anemia affects a third of the world's population with the heaviest burden borne by women and children. Anemia leads to preventable impaired development in children, as well as high morbidity and early mortality among sufferers. Inherited hemoglobin (Hb) disorders, such as sickle cell disease (SCD), are associated with chronic hemolytic anemia causing high morbidity and mortality. Anemia and SCD are inherently associated and are both prevalent in the same regions of the world including sub-Saharan Africa, India, and south-east Asia. Anemia and SCD-related complications can be mitigated by screening, early diagnosis followed by timely intervention. Anemia treatment depends on the accurate characterization of the cause, such as inherited Hb disorders. Meanwhile, Hb disorders or SCD treatments, such as hydroxyurea therapy, requires close monitoring of blood Hb level and the patient's anemia status over time. As a result, it is crucially important to perform integrated detection and monitoring of blood Hb level, anemia status, and Hb variants, especially in areas where anemia and inherited Hb disorders are the most prevalent. Blood Hb level (in g/dL) is used as the main indicator of anemia, while the presence of Hb variants (e.g., sickle Hb or HbS) in blood is the primary indicator of an inherited disorder. The current clinical standards for anemia testing and Hb variant identification are complete blood count (CBC) and High-Performance Liquid Chromatography (HPLC), respectively. State-of-the-art laboratory infrastructure and trained personnel are required for these laboratory tests. However, these resources are typically scarce in low- and middle-income countries, where anemia and Hb disorders are the most prevalent. As a result, there is a dire need for high accuracy portable point-of-care (POC) devices to perform integrated anemia and Hb variant tests with affordable cost and high throughput. Methods: In 2019, the World Health Organization (WHO) listed Hb electrophoresis as an essential in vitro diagnostic (IVD) technology for diagnosing SCD and sickle cell trait. We have leveraged the common Hb electrophoresis method and developed a POC microchip electrophoresis test, Hemoglobin Variant/Anemia (HbVA). This technology is being commercialized under the product name "Gazelle" by Hemex Health Inc. for Hb variant identification with integrated anemia detection (Fig. 1A&B). We hypothesized that computer vision and deep learning will enhance the accuracy and reproducibility of blood Hb level prediction and anemia detection in cellulose acetate based Hb electrophoresis, which is a clinical standard test for Hb variant screening and diagnosis worldwide (Fig. 1C). To test this hypothesis, we integrated, for the first time, a new, computer vision and artificial neural network (ANN) based deep learning imaging and data analysis algorithm, to Hb electrophoresis. Here, we show the feasibility of this new, computer vision and deep learning enabled diagnostic approach via testing of 46 subjects, including individuals with anemia and homozygous (HbSS) or heterozygous (HbSC or Sβ-thalassemia) SCD. Results and Discussion: HbVA computer vision tracked the electrophoresis process real-time and the deep learning neural network algorithm determined Hb levels which demonstrated significant correlation with a Pearson Correlation Coefficient of 0.95 compared to the results of reference standard CBC (Fig.1D). Furthermore, HbVA demonstrated high reproducibly with a mean absolute error of 0.55 g/dL and a bias of -0.10 g/dL (95% limits of agreement: 1.5 g/dL) according to Bland-Altman analysis (Fig. 1E). Anemia determination was achieved with 100% sensitivity and 92.3% specificity with a receiver operating characteristic area under the curve (AUC) of 0.99 (Fig. 1F). Within the same test, subjects with SCD were identified with 100% sensitivity and specificity (Fig. 1G). Overall, the results suggested that computer vision and deep learning methods can be used to extract new information from Hb electrophoresis, enabling, for the first time, reproducible, accurate, and integrated blood Hb level prediction, anemia detection, and Hb variant identification in a single affordable test at the POC. Disclosures An: Hemex Health, Inc.: Patents & Royalties. Hasan:Hemex Health, Inc.: Patents & Royalties. Ahuja:Genentech: Consultancy; Sanofi-Genzyme: Consultancy; XaTec Inc.: Consultancy; XaTec Inc.: Research Funding; XaTec Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Genentech: Honoraria; Sanofi-Genzyme: Honoraria. Little:GBT: Research Funding; Bluebird Bio: Research Funding; BioChip Labs: Patents & Royalties: SCD Biochip (patent, no royalties); Hemex Health, Inc.: Patents & Royalties: Microfluidic electropheresis (patent, no royalties); NHLBI: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees. Gurkan:Hemex Health, Inc.: Consultancy, Current Employment, Patents & Royalties, Research Funding; BioChip Labs: Patents & Royalties; Xatek Inc.: Patents & Royalties; Dx Now Inc.: Patents & Royalties.

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1534 Poster Board I-557 Background Low bone mass density affects more than 65% of adult sickle cell disease patients and correlates with lower hemoglobin and higher ferritin concentrations (1). Increased iron supply promotes osteoclast differentiation and bone resorption (2). Proinflammatory cytokines also promote bone resorption (3). Tartrate resistant acid phosphatase isoform 5b (TRACP-5b) is produced only by activated osteoclasts and therefore serves as a marker of bone resorption (4). Sickle cell disease is a condition of chronic inflammation and patients often suffer from transfusional iron overload as well. In this study we aimed to determine the predictors of bone resorption in patients with sickle cell disease by measuring circulating levels of TRACP-5b. Methods Fifty-nine adult sickle cell disease patients and 22 apparently healthy controls were recruited at Howard University Hospital. Patients were at steady state with no crisis, hospitalization or blood transfusion in the last 3 weeks. Clinical and laboratory information was collected at the time of recruitment and TRACP-5b was measured in non-fasting serum samples using an enzyme immuno assay kit (Quidel, San Diego, CA). Serum concentrations of inflammatory cytokines and growth factors were measured by Multiplex assay (Bio-Rad, Hercules, CA).. Results Sickle cell disease patients had elevated concentrations of TRACP-5b compared to controls (median values of 4.4 vs. 2.4 U/l, P < 0.0001). Among the patients, TRACP-5b concentrations correlated positively with number of blood transfusions (r = 0.19) and serum concentrations of alkaline phosphatase (r=0.46), endothelin-1 (r=0.39), interleukin-8 (r= 0.38), and interleukin-6 (r=0.25). TRACP-5b correlated negatively with RANTES (r = -0.42) and PDGF (r = -0.31). It did not correlate significantly with serum ferritin (r = -0.03), LDH (r = 0.13) or hemoglobin concentration (r = 0.11). Interestingly, TRACP-5b correlated positively with tricuspid regurgitation velocity, which reflects systolic pulmonary artery pressure (r = 0.30). Conclusion Sickle cell patients have elevated steady-state osteoclast activity as reflected in serum TRACP-5b concentrations. Multiple blood transfusions and inflammation are associated findings. Among patients, higher TRACP-5b concentrations are associated with lower concentrations of RANTES and PDGF-BB, factors that influence function of osteoblasts. Further studies are needed to investigate whether common pathways may be involved in osteoclast activation and pulmonary changes in sickle cell disease. Supported by grants number 2 R25 HL003679-08 and 1 R01 HL079912-02 and 1U54HL090508-01 from NHLBI, by Howard University GCRC grant no 2MOI RR10284-10 from NCRR, NIH, Bethesda, MD, and by the intramural research program of the National Institutes of Health. Disclosures Gordeuk: Biomarin: Research Funding; TRF Pharma: Research Funding; Merck: Research Funding; Novartis: Speakers Bureau.