22 February 2017 Application of image flow cytometry for the characterization of red blood cell morphology
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Red blood cells (RBCs) stored in hypothermic environments for the purpose of transfusion have been documented to undergo structural and functional changes over time. One sign of the so-called RBC storage lesion is irreversible damage to the cell membrane. Consequently, RBCs undergo a morphological transformation from regular, deformable biconcave discocytes to rigid spheroechinocytes. The spherically shaped RBCs lack the deformability to efficiently enter microvasculature, thereby reducing the capacity of RBCs to oxygenate tissue. Blood banks currently rely on microscope techniques that include fixing, staining and cell counting in order to morphologically characterize RBC samples; these methods are labor intensive and highly subjective. This study presents a novel, high-throughput RBC morphology characterization technique using image flow cytometry (IFC). An image segmentation template was developed to process 100,000 images acquired from the IFC system and output the relative spheroechinocyte percentage. The technique was applied on samples extracted from two blood bags to monitor the morphological changes of the RBCs during in vitro hypothermic storage. The study found that, for a given sample of RBCs, the IFC method was twice as fast in data acquisition, and analyzed 250-350 times more RBCs than the conventional method. Over the lifespan of the blood bags, the mean spheroechinocyte population increased by 37%. Future work will focus on expanding the template to segregate RBC images into more subpopulations for the validation of the IFC method against conventional techniques; the expanded template will aid in establishing quantitative links between spheroechinocyte increase and other RBC storage lesion characteristics.
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Ruben N. Pinto, Ruben N. Pinto, Joseph A. Sebastian, Joseph A. Sebastian, Michael Parsons, Michael Parsons, Tim C. Chang, Tim C. Chang, Jason P. Acker, Jason P. Acker, Michael C. Kolios, Michael C. Kolios, } "Application of image flow cytometry for the characterization of red blood cell morphology", Proc. SPIE 10076, High-Speed Biomedical Imaging and Spectroscopy: Toward Big Data Instrumentation and Management II, 100761F (22 February 2017); doi: 10.1117/12.2253583; https://doi.org/10.1117/12.2253583

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