Label‐Free Analysis of Red Blood Cell Storage Lesions Using Imaging Flow Cytometry

Abstract

Deleterious changes, collectively termed as storage lesions, alter the characteristics of red blood cell (RBC) morphology during in vitro storage. Due to gradual loss of cellular membrane, RBCs lose their original biconcave disk shape and begin a process of spherical deformation that is characterized by well-defined morphological criteria. At the spheroechinocyte stage, the structure of RBC is irreversibly damaged and lacks the elasticity necessary to efficiently deliver oxygen. Quantifying the prevalence of spheroechinocytes could provide an important morphological measure of the quality of stored blood products. Unlike the conventional RBC morphology characterization assay involving light microscopy, we introduce a label-free assay using imaging flow cytometry (IFC). The technique captures 100,000 images of a sample and calculates a relative measure of spheroechinocyte population in a fraction of the time required by the conventional method. A comparative method study, measuring a morphological index for 11 RCC units through storage, found that the two techniques measured similar trends with IFC reporting the metric at an average of 3.9% higher. We monitored 18 RCC units between Weeks 1 and 6 of storage and found that the spheroechinocyte population increased by an average of 26.2%. The large (3.5-64.1%) variation between the units' spheroechinocyte population percentage at Week 1 suggests a possible dependence of blood product quality on donor characteristics. Given our method's ability to rapidly monitor large samples and refine morphological characterization beyond conventional methods, we believe our technique offers good potential for studying the underlying relationships between RBC morphology and blood storage lesions.Key terms deformability; erythrocyte; imaging flow cytometry; red blood cell morphology; storage lesion CURRENTLY, blood banks and hospital laboratories are devoid of convenient, label-free techniques to measure the morphological distribution of red blood cells (RBCs) stored in vitro. Parameters such as membrane shape and deformability are critical for understanding an RBC's functional efficacy as an oxygen transporter (1). Microscopy-related assays, considered the gold standard for RBC morphology analysis, stain and smear samples prior to manual characterization. High-throughput assays that can measure RBCs within environments closely resembling in vivo conditions could obtain a more insightful assessment of the morphology distribution.RBCs are stored in vitro primarily to enhance the oxygen carrying capacity of vulnerable patients via transfusions (2). Blood banks extract RBCs from donated blood using customized separation techniques and suspend the cells in a preservative fluid to increase its shelf-life (3). During storage of this red cell concentrate (RCC), typically set at a maximum of 6 weeks, RBCs undergo a sequence of significant biochemical and biomechanical changes, which have been collectively termed hypothermic storage lesions (4,5). RBC storage lesions cause the formation of m...

Objective assessment of stored blood quality by deep learning

“…We collected blood samples from healthy volunteers at two sites on different continents (Canadian Blood Services, hereafter “Canadian,” and the University Hospital of Geneva, hereafter “Swiss”) and processed red cell units using standard blood bank protocols ( 25 ), followed by IFC analysis every 3 to 7 d until expiration at 6 wk ( SI Appendix , Figs. S1 A and S2 ) ( 25 – 27 ). Five researchers annotated ∼52,700 RBCs spanning across the blood units ( SI Appendix , Fig.…”

“…The instrument-associated analysis software IDEAS v6.2 was used to preliminarily process the acquired IFC data to remove out-of-focus cells, artifacts, debris, and clumped objects, as previously described ( 25 – 27 ). Images of in-focus single cells were then used for manual annotation and downstream deep-learning analysis.…”

Donor‐dependent aging of young and old red blood cell subpopulations: Metabolic and functional heterogeneity

“…For RBC morphology index (MI) assessment, 5 μL of each RBC sample was suspended in 200 μL of 1× Dulbeccoʼs PBS (DPBS; Sigma‐Aldrich). A sequentially numbered set of individually captured RBC bright‐field images (195 ± 34 images per sample) was manually assigned by a human operator to six morphology subclasses: smooth discs (SDCs), crenated discs (CDCs), crenated discoids (CDDs), crenated spheroids (CSDs), crenated spheres (CSEs), smooth spheres (SSEs), and multiplied by fractional weights 43,44 : …”

Determination of cell nucleus-to-cytoplasmic ratio using imaging flow cytometry and a combined ultrasound and photoacoustic technique: a comparison study

“…[11][12][13] Imaging of single cells in suspension is advantageous as it mimics biological cells in flow, such as those that would be found in the blood. 14,15 IFC analysis software provides analytic tools that utilize feature-based image-gating strategies to characterize cell size, shape, and intensity patterns. 16 IFC incorporates the highquality image resolution and morphological content of optical microscopy while maintaining the high-throughput, automation, and population statistics of conventional FC.…”