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Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry.
J Vis Exp. 2019 11 05JV

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 pO2 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 pO2. 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 pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient's pO2 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 EImin 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.

Authors+Show Affiliations

Laboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht University; Van Creveldkliniek, University Medical Center Utrecht, Utrecht University; M.A.E.Rab@umcutrecht.nl.Laboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht University.Laboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht University.Department of Pediatrics, Division of Hematology/Oncology, Baylor College of Medicine.Department of Pediatrics, Division of Hematology/Oncology, Baylor College of Medicine.Van Creveldkliniek, University Medical Center Utrecht, Utrecht University.Laboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht University.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Video-Audio Media

Language

eng

PubMed ID

31762454

Citation

Rab, Minke A E., et al. "Characterization of Sickling During Controlled Automated Deoxygenation With Oxygen Gradient Ektacytometry." Journal of Visualized Experiments : JoVE, 2019.
Rab MAE, van Oirschot BA, Bos J, et al. Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry. J Vis Exp. 2019.
Rab, M. A. E., van Oirschot, B. A., Bos, J., Kanne, C. K., Sheehan, V. A., van Beers, E. J., & van Wijk, R. (2019). Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry. Journal of Visualized Experiments : JoVE, (153). https://doi.org/10.3791/60213
Rab MAE, et al. Characterization of Sickling During Controlled Automated Deoxygenation With Oxygen Gradient Ektacytometry. J Vis Exp. 2019 11 5;(153) PubMed PMID: 31762454.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry. AU - Rab,Minke A E, AU - van Oirschot,Brigitte A, AU - Bos,Jennifer, AU - Kanne,Celeste K, AU - Sheehan,Vivien A, AU - van Beers,Eduard J, AU - van Wijk,Richard, Y1 - 2019/11/05/ PY - 2019/11/26/entrez PY - 2019/11/26/pubmed PY - 2020/7/7/medline JF - Journal of visualized experiments : JoVE JO - J Vis Exp IS - 153 N2 - 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 pO2 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 pO2. 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 pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient's pO2 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 EImin 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. SN - 1940-087X UR - https://www.unboundmedicine.com/medline/citation/31762454/Characterization_of_Sickling_During_Controlled_Automated_Deoxygenation_with_Oxygen_Gradient_Ektacytometry_ L2 - https://doi.org/10.3791/60213 DB - PRIME DP - Unbound Medicine ER -