Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

Abstract

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO 2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO 2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO 2 at which maximum EI (EI max) shows a 5% decrease, and minimum EI during deoxygenation (EI min) are the most informative, reflecting an individual patient's pO 2 at which sickling starts and the minimal deformability of a patient's red blood cells, respectively. PoS is associated with an individual patient's hemoglobin affinity for oxygen, whereas EI min shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Automated Oxygen Gradient Ektacytometry: A Novel Biomarker in Sickle Cell Anemia

“…The Point of Sickling (PoS) is the pO 2 at which the EI max decreases by 5% as deoxygenation proceeds. The PoS represents the patient-specific pO 2 , where HbS polymerization is accelerated and drives the sickling of RBCs that are deformable at normoxia (Rab et al, 2019a). Recovery is calculated as the percentage of EI max reached during reoxygenation and represents the capacity to reverse sickling with reoxygenation.…”

“…As sickling is caused by deoxygenation, the study of RBC deformability under varying oxygen saturation is promising to be of physiologic relevance. Oxygen gradient ektacytometry, referred to as the oxygenscan, is a next-generation functional assay that measures RBC deformability through an automated cycle of deoxygenation and reoxygenation, demonstrating characteristic features of sickle RBCs (Rab et al, 2019a). As use of the oxygenscan is increasing in sickle cell research, there is a need for standardization of methodology, clinical validation, and correlation with known SCA biomarkers such as fetal hemoglobin (HbF) and dense red blood cells (DRBCs; Rab et al, 2019bRab et al, , 2020a.…”

In Vitro Anti Sickling Activity of HyphaeneThebaica Fruits Extract on Sickle Cell

“…[4] phytochemical components show medicinal values of these plants, which produce de nite and various physiological action on human body. Some of the most important of these components are avonoids, alkaloids and phenolic compounds [5]. Preparation of the aqueous extract of doum fruits 40 g of the doum fruits was extracted by soaking in 200 ml hot distilled water for about four hours with continuous steering.…”

“…Then after cooled, extract was ltered using lter paper and stored till used. Concentration was calculated by dried 2 ml of the extract in a Petri dish using water [5].as followed:…”

“…Extraction carried out for three days with daily ltration and evaporation the solvent under reduced pressure using rotary evaporator apparatus. Sample extract was allowed to air in evaporating dish till complete dryness and the yield 9.92 % were calculated [5] .as follows: Weight of extract obtained / weight of plant sample X 100.…”