Imaging flow cytometry (IFC) might be a time-effective alternative approach to identify and analyze red blood cells (RBCs) in sickle cell disease (SCD), according to results of a new study published in Cytometry Part A.

“This technique might be useful for evaluating patients with different clinical presentations, such as acute crises, and to assess effectiveness of therapy,” the authors said. Using IFC, they were able to automate the quantification of cells from SCD blood.

Patients with SCD had a higher percentage of poikilocytes (0.984% vs 0.04%) and CD71-positive RBCs (2.02% vs 0.09%) than healthy donors. The poikilocytes identified in healthy donors were probably an artifact of sample preparation, the authors said.

Moreover, they identified 2 subpopulations of CD71-positive RBCs from patients with SCD: 1 with high CD71 expression and a puckered morphology and another with lower CD71 expression and a biconcave morphology. They hypothesized that the latter represented a later stage of differentiation.

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Also, some RBCs with puckered morphologies were actually identified as nucleated RBCs, due to the strong positive staining for 4′-6 diamidino-2-phenylindole and α4 integrin.

The authors examined the morphologies of dense cells, an important subpopulation of poikilocytes represented dense cells, isolated through Percoll density gradients. The morphologies of these cells were similar to those of the poikilocytes. However, they appeared more contracted and flattened.

When compared to conventional flow cytometry, IFC performed better in identifying phosphatidylserine (PS)-expressing RBCs. These cells were subdivided into 2 subpopulations: 1 with diffuse PS expression, primarily composed of RBC ghosts, and another with lower overall PS expression, which localized in intense dots overlying Howell-Jolly bodies.

IFC combines flow cytometry and microscopy, thereby allowing for a rapid-throughput analysis of cellular morphology while examining cell-surface markers simultaneously.


Özpolat T, Chang TC, Wu X, et al. Phenotypic analysis of erythrocytes in sickle cell disease using imaging flow cytometry. Cytom Part A. Published online January 31, 2022. doi:10.1002/cyto.a.24536