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.

Dimethyl fumarate increases fetal hemoglobin, provides heme detoxification, and corrects anemia in sickle cell disease

“…Postfixation cells were washed and analyzed for shape change using the Amnis ImageStream X Mark II Imaging Flow Cytometer (MilliporeSigma). Shape change was quantitated using IDEAS application software (MilliporeSigma) using a modified protocol (SIFCA) provided by Gregory J. Kato (Vascular Medical Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA) (49,50).…”

Treating sickle cell disease by targeting HbS polymerization

“…This agent is an aldehyde that forms a reversible Schiff base linkage primarily with the N-terminal amino group of a-globin resulting in a dose-dependent increase in oxygen affinity. [102][103][104] A significant concern with aldehyde drugs is their potential to covalently modify other cellular and plasma proteins. More recently, a polyaromatic aldehyde, GBT440, has been developed that also binds via a Schiff base to the a-globin N terminus with enhancement of oxygen affinity similar to Aes-103, but with higher specificity and at much lower concentrations.…”

Intravascular hemolysis and the pathophysiology of sickle cell disease

“…Erythrocyte injury leads to extra-and intravascular hemolysis, endothelial dysfunction and vasculopathy, and occlusion of small and large blood vessels, producing tissue ischemia/reperfusion injury and inflammation. Damage to circulating erythrocytes occurs with wide diversity amongst individuals (1). This heterogeneity arises from differences in intrinsic characteristics of sickle erythrocytes, like heterocellular fetal hemoglobin (HbF) distribution, HbS concentration (2), hydration, and density (3,4), and the cell's environmental transitions from macro-to microcirculation, laminar to turbulent flow, normoxia to hypoxia, isotonic to hypertonic environment, and acidotic to alkalotic milieu.…”