Imaging flow cytometry for detection of red blood cell inclusions: A quantitative approach to assess splenic filtration function in sickle cell disease

Abstract

Abstract Introduction. The spleen plays a critical role in maintaining red blood cell (RBC) homeostasis by removing intracellular inclusions such as micronuclei (Howell–Jolly bodies, HJB) and vacuoles. HJB are nuclear remnants detectable by flow cytometry or blood smear, while vacuoles—quantified as “red cell pits”—are cytoplasmic spaces identified through phase-contrast microscopy (PCM). In individuals with impaired splenic function, such as those with sickle cell disease (SCD), these inclusions persist in circulation and serve as qualitative or semi-quantitative markers of splenic dysfunction. This study explores imaging flow cytometry (IFC) technology as a novel, high-throughput, and non-invasive method for assessing splenic function by detecting RBC inclusions. Methods. To streamline the workflow and minimize sample requirements, staining protocols were optimized to use only 2 µL of whole blood, eliminating the need for preparatory steps such as RNA digestion and buffy coat removal. White blood cells (WBCs) were excluded during the analysis of the IFC data using CD45 staining. Nucleic acid-containing inclusions were identified using the cell-permeant SYTO™ Deep Red Nucleic Acid Stain (SYTO-DR) which has preferential affinity for dsDNA versus RNA. Vacuoles were stained with the amine-reactive dye Cell Trace™ Yellow (CTY), as previously described (Sissoko et al, AJH 2022). Inclusions were identified from 50,000 events collected using the ImageStream®X system and analyzed with IDEAS® software. HJB were detected using a mask based on Modulation and H-Entropy features of the SYTO-DR signal, while vacuoles were identified using a spot count mask in CTY-positive cells. Inclusion quantification was compared with manual pitted RBC counts on 500 RBC visualized by PCM. Inclusions were assessed in samples from healthy controls and SCD patients (SS genotype) receiving hydroxyurea therapy, stratified by fetal hemoglobin (HbF) levels—high (≥25%) and low/medium (<25%)—as determined by Hb electrophoresis using the Sebia Capillarys™ system. Additional samples were obtained from individuals with SCD who had undergone splenectomy. Results. Both types of inclusions were verified through visual inspection of single-cell images during IFC data analysis. HJB appeared as distinct, solitary DNA inclusions in cells with low RNA and CD71 signal intensities, allowing clear differentiation between mature RBCs and reticulocytes. In healthy controls (HCs), inclusions were rare (median HJB = 0%; vacuoles = 1.6%). In contrast, significantly higher inclusion frequencies were observed in patients with low/medium HbF levels (HJB = 0.19%; vacuoles = 24%) and in individuals with splenectomy (HJB = 0.22%; vacuoles = 34%), with no statistically significant difference. Most individuals with ≥25% HbF exhibited normal or reduced inclusion levels (HJB = 0%; vacuoles = 11%), although a small subset of individuals (~18%) showed persistently elevated counts. Overall, HbF was negatively correlated with inclusions (HJB: r = –0.35; vacuoles: r = –0.56), indicating a protective effect of HbF on splenic function. Age was positively and moderately correlated with both HJB (r = 0.40) and vacuoles (r = 0.61). Strong correlations were observed between HJB (r = 0.7) and vacuoles (r = 0.9) with pitted RBC counts. HJB were consistently detectable when pitted RBC counts approached ~10% (Slope= 0), while vacuoles (Slope=0.9) were detectable at even lower pit counts, suggesting greater sensitivity for early splenic dysfunction. Conclusion. We present an innovative alternative method for quantifying RBC inclusions using IFC and streamlined, objective data analysis, improving on the subjective nature of manual microscopic examination. Both HJB and vacuoles serve as reliable surrogate markers for pitted RBC counts, despite being detected by markedly different methods. Upon clinical validation, this assay could become a valuable diagnostic tool for assessing the degree of splenic dysfunction. It offers a promising approach for individualized patient monitoring and for evaluating the efficacy and safety of therapies aimed at preserving or restoring splenic filtration function in SCD.