title: Imaging flow cytometry for automated detection of hypoxia-induced erythrocyte shape change in sickle cell disease.
abstract: In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient's disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged "sickled" and "normal" erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R = 0.98, P ≤ 0.001), negatively with fetal hemoglobin (HbF) levels (R = -0.558, P = 0.027), negatively with pH (R = -0.688, P = 0.026), negatively with pretreatment with the antisickling agent, Aes-103 (5-hydroxymethyl-2-furfural) (R = -0.766, P = 0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R = 0.799, P = 0.002). This study shows proof of principle that the automated, operator-independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes-103. SIFCA is a new method that may be useful in sickle cell drug development.
authors.affiliation: Hematology Branch and the Electron Microscopy Core Facility and Flow Cytometry Core Facility, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland.
authors.forename: Eduard J
authors.initials: EJ
authors.lastname: van Beers
authors.forename: Leigh
authors.initials: L
authors.lastname: Samsel
authors.forename: Laurel
authors.initials: L
authors.lastname: Mendelsohn
authors.forename: Rehan
authors.initials: R
authors.lastname: Saiyed
authors.forename: Kleber Y
authors.initials: KY
authors.lastname: Fertrin
authors.forename: Christine A
authors.initials: CA
authors.lastname: Brantner
authors.forename: Mathew P
authors.initials: MP
authors.lastname: Daniels
authors.forename: James
authors.initials: J
authors.lastname: Nichols
authors.forename: J Philip
authors.initials: JP
authors.lastname: McCoy
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kato
mesh_terms: D000755:Anemia, Sickle Cell / Q000097:blood* / Q000473:pathology; D001331:Automation / Q000379:methods; D015687:Cell Hypoxia / Q000502:physiology*; D004912:Erythrocytes / Q000473:pathology*; D004913:Erythrocytes, Abnormal / Q000473:pathology*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D010100:Oxygen / Q000097:blood
chemical_list: D010100:Oxygen