Sickle cell disease is a destructive genetic disorder characterized by the formation of fibrils of deoxygenated hemoglobin, leading to the red blood cell (RBC) morphology changes that underlie the clinical manifestations of this disease. Using cryogenic soft X-ray tomography (SXT), we characterized the morphology of sickled RBCs in terms of volume and the number of protrusions per cell. We were able to identify statistically a relationship between the number of protrusions and the volume of the cell, which is known to correlate to the severity of sickling. This structural polymorphism allows for the classification of the stages of the sickling process. Recent studies have shown that elevated sphingosine kinase 1 (Sphk1)-mediated sphingosine 1-phosphate production contributes to sickling. Here, we further demonstrate that compound 5C, an inhibitor of Sphk1, has anti-sickling properties. Additionally, the variation in cellular morphology upon treatment suggests that this drug acts to delay the sickling process. SXT is an effective tool that can be used to identify the morphology of the sickling process and assess the effectiveness of potential therapeutics.
The authors declare no competing or financial interests.
M.C.D., Y.X., C.A.L. and W.C. designed the study. Y.Z. prepared the samples and performed the light microscopy and survival experiments. B.P.C., E.A.S., R.B. and M.C.D. froze and imaged the samples using SXT. M.C.D. processed the SXT image data, staged the cells, performed the volumetric analysis and wrote the manuscript. M.C.D. and M.F.S. analyzed the data. R.H.R. and M.C.D. performed statistical tests. All authors reviewed the manuscript before submission.
This research has been supported, in whole or in part, by National Institutes of Health [grant number PN2EY016525 to W.C., P41GM103832 to W.C. and P41GM103445 to C.A.L.]; and by the U.S. Department of Energy, Office of Biological and Environmental Research [grant number DE-AC02-05CH11231 to C.A.L.]; and through a National Institute of Biomedical Imaging and Bioengineering training grant [grant number T32EB009270 to M.C.D.] awarded to the Keck Center of the Gulf Coast Consortia. Deposited in PMC for release after 12 months.
Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.189225.supplemental
- Received March 11, 2016.
- Accepted August 1, 2016.
- © 2016. Published by The Company of Biologists Ltd