Imaging Flow Cytometry Documents Incomplete Resistance Of F-Cells To Hypoxia-Induced Sickling In Blood Samples From Patients With Sickle Cell Anemia

• Published in: Blood 2013-11, Vol.122 (21), p.183-183
• Main Authors: Fertrin, Kleber Yotsumoto, van Beers, Eduard J., Samsel, Leigh, Mendelsohn, Laurel G., Saiyed, Rehan, Nichols, James S., Hepp, David A., Brantner, Christine A., Daniels, Mathew P., McCoy, J. Philip, Kato, Gregory J.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V122.21.183.183

Fetal hemoglobin (HbF) production induced by hydroxyurea is the mainstay of treatment for sickle cell anemia (SCA). Increased HbF production correlates with a higher number of HbF-containing red blood cells (RBCs) called F-cells. Successful treatment with hydroxyurea is associated with an increased number of F-cells, less hemolysis, improvement of anemia, and decreased frequency of vaso-occlusive crises in SCA patients. Comparison of in vitro sickling among blood specimens from sickle mouse models and from patients with different HbF levels has provided compelling evidence that increasing the percentage of circulating F-cells is associated with improvement of hemolytic biomarkers. While it has been demonstrated that higher HbF content prolongs sickle RBC survival, there is only indirect evidence of the response to hypoxia of F-cells compared to non-F-cells. We investigated the influence of HbF content on sickling through our recently developed Sickle Imaging Flow Cytometry Assay (SIFCA). SIFCA allows simultaneous analysis of both expression of intracellular proteins and morphological features of each cell in a robust, reproducible, operator-independent sickling assay. Peripheral venous blood samples were collected upon written consent from adult SCA patients with a wide range of HbF percentages (HbF range 2.0-26.9%) (n=15, nine on hydroxyurea treatment). RBC pellets were used to prepare 1% suspensions that were subjected to deoxygenation for 2 hours at 2% oxygen. RBCs were then labeled for HbF using a standard protocol for F-cell quantitation and a minimum of 20,000 cells were analyzed by imaging flow cytometry (ImageStreamX Mk II, Amnis Corporation), allowing the correlation between shape change and intensity of HbF expression for each RBC. We confirmed previous observations using conventional flow cytometry that F-cell count percentages significantly correlate with mean HbF determined by HPLC (r2P=0.9700, 95% CI 0.9098-0.9902, P