Quantifying morphological heterogeneity: a study of more than 1 000 000 individual stored red blood cells

Abstract

Background and Objectives The morphology of red blood cells (RBCs) deteriorates progressively during hypothermic storage. The degree of deterioration varies between individual cells, resulting in a highly heterogeneous population of cells contained within each RBC unit. Current techniques capable of categorizing the morphology of individual stored RBCs are manual, laborious, error-prone procedures that limit the number of cells that can be studied. Our objective was to create a simple, automated system for high-throughput RBC morphology classification. Materials and Methods A simple microfluidic device, designed to enable rapid, consistent acquisition of images of optimally oriented RBCs, was fabricated using soft lithography. A custom image analysis algorithm was developed to categorize the morphology of each individual RBC in the acquired images. The system was used to determine morphology of individual RBCs in several RBC units stored hypothermically for 6–8 weeks. Results The system was used to automatically determine the distribution of cell diameter within each morphological class for >1,000,000 individual stored RBCs (speed: >10,000 cells/hour; accuracy: 91.9% low-resolution, 75.3% high-resolution). Diameter mean and standard deviation by morphology class: discocyte 7.80±0.49μm, echinocyte 1 7.61±0.63μm, echinocyte 2 7.02±0.61μm, echinocyte 3 6.47±0.42μm, sphero-echinocyte 6.01±0.26μm, spherocyte 6.02±0.27μm, stomatocyte 1 6.95±0.61μm, stomatocyte 2 7.32 ± 0.47μm. Conclusion The automated morphology classification procedure described in this study is significantly simpler, faster and less subjective than conventional manual procedures. The ability to evaluate the morphology of individual RBCs automatically, rapidly and in statistically significant numbers enabled us to perform the most extensive study of stored RBC morphology to date.

Shape matters: the effect of red blood cell shape on perfusion of an artificial microvascular network

“…The maximum mean decrease in the AMVN perfusion rate we measured in this study was approximately 30% for the sample that mostly consisted of spherocytes (the endpoint of the echinocytic transformation). Echinocytosis is by far the most common type of RBC shape deterioration occurring during hypothermic storage and therefore our results could have particularly important implications in the context of blood storage and transfusion. Interestingly, in our previous studies we measured a 20% to 25% difference in the AMVN perfusion rate between fresh RBCs (mostly discocytes) and RBCs stored hypothermically for 6 weeks .…”

“…Interestingly, in our previous studies we measured a 20% to 25% difference in the AMVN perfusion rate between fresh RBCs (mostly discocytes) and RBCs stored hypothermically for 6 weeks. 20,27 Because 6-week-old blood is a heterogeneous mixture composed predominantly of cells in early stages of echinocytosis (which may recover their shape in vivo, after transfusion, 36 or via washing 20 ), some discocytes, and a sizable fraction of irreparably damaged late-stage echinocytes and spherocytes, 26 the shape change could explain a large fraction of the decline in the AMVN perfusion rate we have previously measured for hypothermically stored blood.…”

“…Figure 2 illustrates the changes in RBC morphology induced by exposure to different SA concentrations. The specific concentrations of SA were chosen so that the resulting morphologies approximated different degrees of echinocytosis, 26,31 namely, echinocyte I (an irregularly contoured discocyte with up to five protrusions) for 9 mmol/L SA, echinocyte II (a flat RBC with a few spicules) for 18 mmol/L SA, echinocyte III (an ovaloid or spherical RBC with many spicules) for 27 mmol/L SA, spheroechinocyte (a spherical RBC with multiple short and thin spicules) for 36 mmol/L SA, and finally spherocyte (a completely spherical RBC) for 54 mmol/L SA. Figure 3 shows the characteristic images of RBCs with each of the induced morphologies as they perfused the arteriole and capillary portions of the AMVN (Fig.…”

“…Microvesiculation results in a disproportionately larger loss of cell membrane than cell volume, increasing sphericity of the cell. Stored RBCs gradually transition from normal, biconcave discocytes, through distinct intermediate echinocytic stages (echinocyte I, echinocyte II, echinocyte III, spheroechinocyte) to spherocytes and ultimately undergo lysis . The rate and degree of this morphologic deterioration varies greatly between individual RBCs, resulting in a highly heterogeneous population of cells with different morphologies within each stored RBC unit.…”

Flow morphometry to assess the red blood cell storage lesion

“…RBCs in the first stage are called discocytes (D) since they can be described as smooth biconcave discs. This scheme with three morphological classes constitutes a lower classification resolution as compared to previous work (8,10,16). This stage can be characterized by the appearance of increasing numbers of bumps, which gradually become more numerous and finer, while the cells become more spherical.…”