Oxygen gradient ektacytometry-derived biomarkers are associated with acute complications in sickle cell disease.

ABSTRACT: We investigated the potential of the point of sickling (PoS; the pO2 tension at which red cells start to sickle), determined by oxygen gradient ektacytometry to serve as a biomarker associated with the incidence of acute sickle cell disease-related complications in 177 children and 50 adults. In the pediatric cohort, for every 10 mmHg increase in PoS reflecting a greater likelihood of sickling, the likelihood of an individual experiencing >1 type of acute complication increased; the adjusted odds ratio (aOR) was 1.65. For every 0.1 increase in minimum elongation index (EImin; reflecting improved red blood cell deformability at hypoxia), the aOR was 0.50. In the adult cohort, for every 10 mmHg increase in PoS, we found an aOR of 3.00, although this was not significant after correcting for multiple testing. There was a trend for an association between higher PoS and greater likelihood of vaso-occlusive episodes (VOEs; children aOR, 1.35; adults aOR, 2.22). In children, only EImin was associated with VOEs (aOR, 0.68). When data of both cohorts were pooled, significant associations with PoS and/or EImin were found for all acute complications, independently and when >1 type of acute complication was assessed. These findings indicate that oxygen gradient ektacytometry generates novel biomarkers and provides a rationale for further development of these biomarkers in the assessment of clinical severity, evaluation of novel therapies, and as surrogate clinical trial end points. These biomarkers may be useful in assessing efficacy of novel therapies like pyruvate kinase activators, voxelotor, and L-glutamine.

Vascular pathophysiology of sickle cell disease.

Sickle cell disease (SCD) is an hereditary disorder characterized by the production of an abnormal hemoglobin called hemoglobin S (HbS). HbS may polymerize in deoxygenated conditions, which leads to red blood cell (RBC) sickling. Sickled RBCs are more rigid and fragile, and prone to lysis. SCD patients exhibit various acute and/or chronic complications, which may affect several organs. The clinical presentation of SCD is highly variable from one patient to another and cannot be only explained by RBC sickling. Increased blood viscosity, caused by the presence of RBCs with abnormal deformability and aggregation, may increase vascular resistance and increase the risk of acute and chronic vascular complications. Chronic hemolysis results in decreased nitric oxide (NO) bioavailability which may compromise vasodilation and participate to the development of chronic vasculopathy. Furthermore, chronic hemolysis is responsible for increased inflammation and oxidative stress, which affect the vascular system and may promote the adhesion of circulating cells to endothelial cells. Extracellular vesicles and especially RBC microparticles (massively released in the context of SCD) are also at the origin of vascular damages and increased white blood cells adhesion to the endothelium, which may trigger vaso-occlusive crisis and other vascular-related complications. This review highlights the fact that SCD should not only be considered as a hematological disorder but also as a vascular disease.

A preliminary study of phosphodiesterases and adenylyl cyclase signaling pathway on red blood cell deformability of sickle cell patients.

Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by chronic anemia, intravascular hemolysis, and the occurrence of vaso-occlusive crises due to the mechanical obstruction of the microcirculation by poorly deformable red blood cells (RBCs). RBC deformability is a key factor in the pathogenesis of SCD, and is affected by various factors. In this study, we investigated the effects of adenylyl cyclase (AC) signaling pathway modulation and different phosphodiesterase (PDE) modulatory molecules on the deformability and mechanical stress responses of RBC from SCD patients (HbSS genotype) by applying 5 Pa shear stress with an ektacytometer (LORRCA). We evaluated RBC deformability before and after the application of shear stress. AC stimulation with Forskolin had distinct effects on RBC deformability depending on the application of 5 Pa shear stress. RBC deformability was increased by Forskolin before shear stress application but decreased after 5 Pa shear stress. AC inhibition with SQ22536 and protein kinase A (PKA) inhibition with H89 increased RBC deformability before and after the shear stress application. Non-selective PDE inhibition with Pentoxifylline increased RBC deformability. However, modulation of the different PDE types had distinct effects on RBC deformability, with PDE1 inhibition by Vinpocetine increasing deformability while PDE4 inhibition by Rolipram decreased RBC deformability after the shear stress application. The effects of the drugs varied greatly between patients suggesting some could benefit from one drug while others not. Developing drugs targeting the AC signaling pathway could have clinical applications for SCD, but more researches with larger patient cohorts are needed to identify the differences in the responses of sickle RBCs.

Increased retention of functional mitochondria in mature sickle red blood cells is associated with increased sickling tendency, hemolysis and oxidative stress.

Abnormal retention of mitochondria in mature red blood cells (RBC) has been recently reported in sickle cell anemia (SCA) but their functionality and their role in the pathophysiology of SCA remain unknown. The presence of mitochondria within RBC was determined by flow cytometry in 61 SCA patients and ten healthy donors. Patients were classified according to the percentage of mature RBC with mitochondria contained in the whole RBC population: low (0-4%), moderate (>4% and <8%), or high level (>8%). RBC rheological, hematological, senescence and oxidative stress markers were compared between the three groups. RBC senescence and oxidative stress markers were also compared between mature RBC containing mitochondria and those without. The functionality of residual mitochondria in sickle RBC was measured by high-resolution respirometry assay and showed detectable mitochondrial oxygen consumption in sickle mature RBC but not in healthy RBC. Increased levels of mitochondrial reactive oxygen species were observed in mature sickle RBC when incubated with Antimycin A versus without. In addition, mature RBC retaining mitochondria exhibited greater levels of reactive oxygen species compared to RBC without mitochondria, as well as greater Ca2+, lower CD47 and greater phosphatidylserine exposure. Hematocrit and RBC deformability were lower, and the propensity of RBC to sickle under deoxygenation was higher, in the SCA group with a high percentage of mitochondria retention in mature RBC. This study showed the presence of functional mitochondria in mature sickle RBC, which could favor RBC sickling and accelerate RBC senescence, leading to increased cellular fragility and hemolysis.

Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner.

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca2+ ) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1-10 µM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca2+ influx. Yoda1 induced Ca2+ -dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo-/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease.

Effects of a Maximal Exercise Followed by a Submaximal Exercise Performed in Normobaric Hypoxia (2500 m), on Blood Rheology, Red Blood Cell Senescence, and Coagulation in Well-Trained Cyclists.

Acute normoxic exercise impacts the rheological properties of red blood cells (RBC) and their senescence state; however, there is a lack of data on the effects of exercise performed in hypoxia on RBC properties. This crossover study compared the effects of acute hypoxia vs. normoxia on blood rheology, RBC senescence, and coagulation during exercise. Nine trained male cyclists completed both a session in normoxia (FiO2 = 21%) and hypoxia (FiO2 = 15.3% ≈ 2500 m). The two sessions were randomly performed, separated by one week, and consisted of an incremental and maximal exercise followed by a 20 min exercise at the first ventilatory threshold (VT1) on a home-trainer. Blood samples were taken before and after exercise to analyze hematological parameters, blood rheology (hematocrit, blood viscosity, RBC deformability and aggregation), RBC senescence markers (phosphatidylserine (PS) and CD47 exposure, intraerythrocyte reactive oxygen species (ROS), and calcium content), and blood clot viscoelastic properties. Hemoglobin oxygen saturation (SpO2) and blood lactate were also measured. In both conditions, exercise induced an increase in blood viscosity, hematocrit, intraerythrocyte calcium and ROS content, and blood lactate concentration. We also observed an increase in blood clot amplitude, and a significant drop in SpO2 during exercise in the two conditions. RBC aggregation and CD47 exposure were not modified. Exercise in hypoxia induced a slight decrease in RBC deformability which could be related to the slight increase in mean corpuscular hemoglobin concentration (MCHC). However, the values of RBC deformability and MCHC after the exercise performed in hypoxia remained in the normal range of values. In conclusion, acute hypoxia does not amplify the RBC and coagulation changes induced by an exercise bout.

Sublingual Microcirculation Specificity of Sickle Cell Patients: Morphology of the Microvascular Bed, Blood Rheology, and Local Hemodynamics.

Patients with sickle cell disease (SCD) have poorly deformable red blood cells (RBC) that may impede blood flow into microcirculation. Very few studies have been able to directly visualize microcirculation in humans with SCD. Sublingual video microscopy was performed in eight healthy (HbAA genotype) and four sickle cell individuals (HbSS genotype). Their hematocrit, blood viscosity, red blood cell deformability, and aggregation were individually determined through blood sample collections. Their microcirculation morphology (vessel density and diameter) and microcirculation hemodynamics (local velocity, local viscosity, and local red blood cell deformability) were investigated. The De Backer score was higher (15.9 mm-1) in HbSS individuals compared to HbAA individuals (11.1 mm-1). RBC deformability, derived from their local hemodynamic condition, was lower in HbSS individuals compared to HbAA individuals for vessels < 20 µm. Despite the presence of more rigid RBCs in HbSS individuals, their lower hematocrit caused their viscosity to be lower in microcirculation compared to that of HbAA individuals. The shear stress for all the vessel diameters was not different between HbSS and HbAA individuals. The local velocity and shear rates tended to be higher in HbSS individuals than in HbAA individuals, notably so in the smallest vessels, which could limit RBC entrapment into microcirculation. Our study offered a novel approach to studying the pathophysiological mechanisms of SCD with new biological/physiological markers that could be useful for characterizing the disease activity.

Influence of storage and buffer composition on the mechanical behavior of flowing red blood cells.

On-chip study of blood flow has emerged as a powerful tool to assess the contribution of each component of blood to its overall function. Blood has indeed many functions, from gas and nutrient transport to immune response and thermal regulation. Red blood cells play a central role therein, in particular through their specific mechanical properties, which directly influence pressure regulation, oxygen perfusion, or platelet and white cell segregation toward endothelial walls. As the bloom of in-vitro studies has led to the apparition of various storage and sample preparation protocols, we address the question of the robustness of the results involving cell mechanical behavior against this diversity. The effects of three conservation media (EDTA, citrate, and glucose-albumin-sodium-phosphate) and storage time on the red blood cell mechanical behavior are assessed under different flow conditions: cell deformability by ektacytometry, shape recovery of cells flowing out of a microfluidic constriction, and cell-flipping dynamics under shear flow. The impact of buffer solutions (phosphate-buffered saline and density-matched suspension using iodixanol/Optiprep) are also studied by investigating individual cell-flipping dynamics, relative viscosity of cell suspensions, and cell structuration under Poiseuille flow. Our results reveal that storing blood samples up to 7 days after withdrawal and suspending them in adequate density-matched buffer solutions has, in most experiments, a moderate effect on the overall mechanical response, with a possible rapid evolution in the first 3 days after sample collection.

Shear-Stress-Gradient and Oxygen-Gradient Ektacytometry in Sickle Cell Patients at Steady State and during Vaso-Occlusive Crises.

Oxygen gradient ektacytometry (oxygenscan) measures the changes in red blood cell (RBC) deformability in normoxia and during deoxygenation. We investigated the changes in RBC deformability, measured by both oxygenscan and classical shear-stress-gradient ektacytometry, in 10 patients with sickle cell disease (SCD) during vaso-occlusive crisis (VOC) versus steady state. Oxygenscan and shear-stress-gradient ektacytometry parameters were also measured in 38 SCD patients at steady state on two different occasions. Shear-stress-gradient ektacytometry parameters, maximal RBC deformability at normoxia and the minimum RBC deformability during deoxygenation were lower during VOC compared to steady state. The oxygen partial pressure at which RBCs started to sickle (PoS) was not significantly affected by VOC, but the results were very heterogeneous: the PoS increased in 5 in 10 patients and decreased in 4 in 10 patients. Both oxygenscan and shear-stress-gradient ektacytometry parameters remained unchanged in patients at steady state between two sets of measurements, performed at 17 ± 8 months intervals. In conclusion, the present study showed that both oxygen gradient ektacytometry and shear-stress-gradient ektacytometry are sensitive to disease activity in SCD, and that both techniques give comparable results; however, the oxygen-dependent propensity of RBCs to sickle was highly variable during VOC.

Increased blood viscosity and red blood cell aggregation in patients with COVID-19.

The aim of this study was to (1) analyze blood viscosity, red blood cell (RBC) deformability, and aggregation in hospitalized patients with Coronavirus disease 19 (COVID-19); (2) test the associations between impaired blood rheology and blood coagulation; and (3) test the associations between impaired blood rheology and several indicators of clinical severity. A total of 172 patients with COVID-19, hospitalized in COVID-unit of the Internal Medicine Department (Lyon, France) participated in this study between January and May 2021. Clinical parameters were collected for each patient. Routine hematological/biochemical parameters, blood viscosity, RBC deformability and aggregation, and RBC senescence markers were measured on the first day of hospitalization. A control group of 38 healthy individuals was constituted to compare the blood rheological and RBC profile. Rotational thromboelastography was performed in 76 patients to study clot formation dynamics. Our study demonstrated that patients with COVID-19 had increased blood viscosity despite lower hematocrit than healthy individuals, as well as increased RBC aggregation. In-vitro experiments demonstrated a strong contribution of plasma fibrinogen in this RBC hyper-aggregation. RBC aggregation correlated positively with clot firmness, negatively with clot formation time, and positively with the length of hospitalization. Patients with oxygen supplementation had higher RBC aggregation and blood viscosity than those without, and patients with pulmonary lesions had higher RBC aggregation and enhanced coagulation than those without. This study is the first to demonstrate blood hyper-viscosity and RBC hyper-aggregation in a large cohort of patients with COVID-19 and describe associations with enhanced coagulation and clinical outcomes.

Nocturnal Hypoxemia Rather Than Obstructive Sleep Apnea Is Associated With Decreased Red Blood Cell Deformability and Enhanced Hemolysis in Patients With Sickle Cell Disease.

Background: Although obstructive sleep apnea (OSA) could act as a modulator of clinical severity in sickle cell disease (SCD), few studies focused on the associations between the two diseases. Research Question: The aims of this study were: (1) to explore the associations between OSA, nocturnal oxyhemoglobin saturation (SpO2) and the history of several acute/chronic complications, (2) to investigate the impact of OSA and nocturnal SpO2 on several biomarkers (hematological, blood rheological, and coagulation) in patients with SCD. Study Design and Methods: Forty-three homozygous SCD patients underwent a complete polysomnography recording followed by blood sampling. Results: The proportion of patients suffering from nocturnal hypoxemia did not differ between those with and those without OSA. No association between OSA and clinical severity was found. Nocturnal hypoxemia was associated with a higher proportion of patients with hemolytic complications (glomerulopathy, leg ulcer, priapism, or pulmonary hypertension). In addition, nocturnal hypoxemia was accompanied by a decrease in RBC deformability, enhanced hemolysis and more severe anemia. Interpretation: Nocturnal hypoxemia in SCD patients could be responsible for changes in RBC deformability resulting in enhanced hemolysis leading to the development of complications such as leg ulcers, priapism, pulmonary hypertension or glomerulopathy. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT03753854.

Comparisons of oxygen gradient ektacytometry parameters between sickle cell patients with or without α-thalassaemia.

The present study tested the impact of α-thalassaemia on oxygen gradient ektacytometry in sickle cell anaemia (SCA). Three SCA groups were compared: (i) no α-thalassaemia (four α-genes, n = 62), (ii) silent α-thalassaemia (three α-genes, n = 35) and (iii) homozygous α-thalassaemia (two α-genes, n = 12). Red blood cell (RBC) deformability measured in normoxia was not different between the three groups. The lowest RBC deformability reached at low oxygen partial pressure (pO2 ) was greater and the pO2 at which RBC started to sickle was lower in the two α-genes group compared to the other groups. Our present study showed an effect of α-thalassaemia on oxygen gradient ektacytometry in SCA.

Is Skeletal Muscle Dysfunction a Limiting Factor of Exercise Functional Capacity in Patients with Sickle Cell Disease?

Patients with sickle cell disease (SCD) have reduced functional capacity due to anemia and cardio-respiratory abnormalities. Recent studies also suggest the presence of muscle dysfunction. However, the interaction between exercise capacity and muscle function is currently unknown in SCD. The aim of this study was to explore how muscle dysfunction may explain the reduced functional capacity. Nineteen African healthy subjects (AA), and 24 sickle cell anemia (SS) and 18 sickle cell hemoglobin C (SC) patients were recruited. Maximal isometric torque (Tmax) was measured before and after a self-paced 6-min walk test (6-MWT). Electromyographic activity of the Vastus Lateralis was recorded. The 6-MWT distance was reduced in SS (p < 0.05) and SC (p < 0.01) patients compared to AA subjects. However, Tmax and root mean square value were not modified by the 6-MWT, showing no skeletal muscle fatigue in all groups. In a multiple linear regression model, genotype, step frequency and hematocrit were independent predictors of the 6-MWT distance in SCD patients. Our results suggest that the 6-MWT performance might be primarily explained by anemia and the self-paced step frequency in SCD patients attempting to limit metabolic cost and fatigue, which could explain the absence of muscle fatigue.