Abstract
Red blood cells (RBC) ability to circulate is closely related to their surface area-to-volume ratio. A decrease in this ratio induces a decrease in RBC deformability that can lead to their retention and elimination in the spleen. We recently showed that a subpopulation of “small RBC” with reduced projected surface area accumulated upon storage in blood bank concentrates, but data on the volume of these altered RBC are lacking. So far, single cell measurement of RBC volume has remained a challenging task achieved by a few sophisticated methods some being subject to potential artifacts. We aimed to develop a reproducible and ergonomic method to assess simultaneously RBC volume and morphology at the single cell level. We adapted the fluorescence exclusion measurement of volume in nucleated cells to the measurement of RBC volume. This method requires no pre-treatment of the cell and can be performed in physiological or experimental buffer. In addition to RBC volume assessment, brightfield images enabling a precise definition of the morphology and the measurement of projected surface area can be generated simultaneously. We first verified that fluorescence exclusion is precise, reproducible and can quantify volume modifications following morphological changes induced by heating or incubation in non-physiological medium. We then used the method to characterize RBC stored for 42 days in SAG-M in blood bank conditions. Simultaneous determination of the volume, projected surface area and morphology allowed to evaluate the surface area-to-volume ratio of individual RBC upon storage. We observed a similar surface area-to-volume ratio in discocytes (D) and echinocytes I (EI), which decreased in EII (7%) and EIII (24%), sphero-echinocytes (SE; 41%) and spherocytes (S; 47%). If RBC dimensions determine indeed the ability of RBC to cross the spleen, these modifications are expected to induce the rapid splenic entrapment of the most morphologically altered RBC (EIII, SE, and S) and further support the hypothesis of a rapid clearance of the “small RBC” subpopulation by the spleen following transfusion.
Highlights
Surface area-to-volume ratio is a major determinant of red blood cell (RBC) deformability and ability to circulate [1]
This method is reproducible and sensitive and allows the simultaneous measurement of volume and projected surface area together with a detailed determination of RBC morphology at a single cell level. Several methods such as automated complete blood count and impedance-based Coulter-Counter have been described to measure cell volume, but while of major clinical impact, they have disadvantages regarding mechanistic exploration of RBC. These high-throughput methods provide reproducible data but volume can only be measured on global populations of RBC and not at a single cell level [32]
Light scattering in flow cytometry is a high-throughput method which is frequently used because of the wide availability of flow cytometers
Summary
Surface area-to-volume ratio is a major determinant of red blood cell (RBC) deformability and ability to circulate [1]. We recently showed that alterations in the morphology of stored RBC was accompanied by a decrease in the projected surface area that only impacts a subpopulation of RBC [10]. The proportion of this subpopulation increases upon storage but is highly variable between donors. Determination of the projected surface area of these stored RBC was conducted using imaging flow cytometry, a high throughput tool allowing a detailed and objective quantification of cell morphology and dimensions [15]. Imaging flow cytometry cannot determine cell volume misses the key pathophysiological feature of RBC: surface area-to-volume ratio
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