Stored red blood cells (RBCs) are needed for life-saving blood transfusions, but they undergo continuous degradation. RBC storage lesions are often assessed by microscopic examination or biochemical and biophysical assays, which are complex, time-consuming, and destructive to fragile cells. Here we demonstrate the use of label-free imaging flow cytometry and deep learning to characterize RBC lesions. Using brightfield images, a trained neural network achieved 76.7% agreement with experts in classifying seven clinically relevant RBC morphologies associated with storage lesions, comparable to 82.5% agreement between different experts. Given that human observation and classification may not optimally discern RBC quality, we went further and eliminated subjective human annotation in the training step by training a weakly supervised neural network using only storage duration times. The feature space extracted by this network revealed a chronological progression of morphological changes that better predicted blood quality, as measured by physiological hemolytic assay readouts, than the conventional expert-assessed morphology classification system. With further training and clinical testing across multiple sites, protocols, and instruments, deep learning and label-free imaging flow cytometry might be used to routinely and objectively assess RBC storage lesions. This would automate a complex protocol, minimize laboratory sample handling and preparation, and reduce the impact of procedural errors and discrepancies between facilities and blood donors. The chronology-based machine-learning approach may also improve upon humans’ assessment of morphological changes in other biomedically important progressions, such as differentiation and metastasis.

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

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...

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