Spherocytic shift of red blood cells during storage provides a quantitative whole cell–based marker of the storage lesion

Roussel, Camille; Dussiot, Michaël; Marin, Mickaël; Morel, Alexandre; Ndour, Papa Alioune; Duez, Julien; Kim, Caroline Le Van; Hermine, Olivier; Colin, Yves; Buffet, Pierre; Amireault, Pascal

doi:10.1111/trf.14015

Cited by 61 publications

(98 citation statements)

References 47 publications

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“…2) were constructed with twodimensional thresholds (i.e., ROI-based gating), which helped us achieve more accurate delineations of less discernable subpopulations. RBC morphology characterization with the Imag-eStreamX ® /IDEAS ® system has been previously investigated by Roussel et al; they observed a strong correlation between the increase in small-diameter RBCs (inferred as SECs) captured on the IFC platform and RBC storage lesion populations observed through LM (42). In comparison, the novelty in our IFC template lies in the ability to distinguish, directly, RBC subclasses based on the standard LM morphology classification guidelines (19,20)).…”

Section: Comparative Analysismentioning

confidence: 76%

“…Flow morphometry techniques, such as the fluidic system by Sierra et al, acquire high-throughput LM images of RBCs in flow suspension (41). RBC morphology characterization with the Imag-eStreamX ® /IDEAS ® system has been previously investigated by Roussel et al; they observed a strong correlation between the increase in small-diameter RBCs (inferred as SECs) captured on the IFC platform and RBC storage lesion populations observed through LM (42). In comparison, our binary trees (Fig.…”

Section: Comparative Analysismentioning

confidence: 99%

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

et al. 2019

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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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Section: Comparative Analysismentioning

confidence: 76%

Section: Comparative Analysismentioning

confidence: 99%

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

et al. 2019

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“…Measurements reported elsewhere on the extent of the DEHP interaction with the RBC membrane in transfusion bags [3] suggest that the DEHP-to-lipid ratio is sufficiently high to affect the lipid-based membrane physical properties [39]; the ratio in the stomatocytes seen here may be in a similar range, as suggested by the calculation of stomatocyte counts formed after exposure to stirred DEHP. The RBC suspensions in transfusion bags have been shown to have a relatively high proportion of stomatocytes over the majority of the storage period [45]. This may be due to a combination of factors including the pH of the storage medium.…”

Section: Discussionmentioning

confidence: 99%

The Blood Bag Plasticizer Di-2-Ethylhexylphthalate Causes Red Blood Cells to Form Stomatocytes, Possibly by Inducing Lipid Flip-Flop

Melzak

Uhlig

Kirschhöfer

et al. 2018

Transfus Med Hemother

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Background: During storage of red blood cell (RBC) concentrates, the plasticizer di-2-ethylhexylphthalate (DEHP) that keeps the blood bags soft leaches out and can be taken up by the RBCs. DEHP is known to be beneficial for the RBC storage quality, but the molecular mechanisms of the action are unknown. Methods: Aqueous suspensions of DEHP were added to RBCs in buffer. The morphological effects were observed on RBCs from 5 donors. Flow cytometry with annexin A5 binding was used to measure the exposed phosphatidylserine. Results: DEHP induced the formation of stomatocytes at concentrations as low as ng/ml, provided that the cell suspension was also sufficiently dilute. Some spherocytes, which were susceptible to lysis, were also formed; after lysis, RBC ghosts were seen to continue the transition to the cup-shaped stomatocyte form. Incubation with DEHP increased the exposed phosphatidylserine, an effect that was also observed in the presence of vanadate, which inhibits the ATP-dependent translocases that maintain the membrane's lipid asymmetry. Conclusions: DEHP can have an active effect on RBC shape, instead of just preventing the storage-related shape changes. The effect appears to be mediated by increased flip-flop of lipids between the leaflets of the RBC membrane.

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“…The morphology and size (i.e., projected surface area) of RBC are related to their ability to persist in circulation through their impact on the surface-to-volume ratio (Mohandas et al, 1980;Jaureguiberry et al, 2014;Pivkin et al, 2016). As an illustration, IFM has recently revealed that storage of RBC in blood bank conditions induces a spherocytic shift of some RBC due to a surface area loss (Roussel et al, 2017(Roussel et al, , 2018. These "storage-induced micro-spherocytes" are removed from the circulation (Roussel, 2019).…”

Section: Imaging Flow Cytometrymentioning

confidence: 99%

Methods to Investigate the Deformability of RBC During Malaria

et al. 2020

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Despite a 30% decline in mortality since 2000, malaria still affected 219 million subjects and caused 435,000 deaths in 2017. Red blood cells (RBC) host Plasmodium parasites that cause malaria, of which Plasmodium falciparum is the most pathogenic. The deformability of RBC is markedly modified by invasion and development of P. falciparum. Surface membrane area is potentially impacted by parasite entry and development, the cytoskeleton is modified by parasite proteins and cytosol viscosity is altered by parasite metabolism. RBC hosting mature parasites (second half of the asexual erythrocytic cycle) are abnormally stiff but the main reason for their absence from the circulation is their adherence to endothelial cells, mediated by parasite proteins exposed at the infected-RBC surface. By contrast, the circulation of non-adherent rings and gametocytes, depends predominantly on deformability. Altered deformability of rings and of uninfected-RBC altered by malaria infection is an important determinant of malaria pathogenesis. It also impacts the response to antimalarial therapy. Unlike conventional antimalarials that target mature stages, currently recommended firstline artemisinin derivatives and the emerging spiroindolones act on circulating rings. Methods to investigate the deformability of RBC are therefore critical to understand the clearance of infected-and uninfected-RBC in malaria. Herein, we review the main methods to assess the deformability of P. falciparum infected-RBC, and their contribution to the understanding of how P. falciparum infection causes disease, how the parasite is transmitted and how antimalarial drugs induce parasite clearance.

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Spherocytic shift of red blood cells during storage provides a quantitative whole cell–based marker of the storage lesion

Cited by 61 publications

References 47 publications

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

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

The Blood Bag Plasticizer Di-2-Ethylhexylphthalate Causes Red Blood Cells to Form Stomatocytes, Possibly by Inducing Lipid Flip-Flop

Methods to Investigate the Deformability of RBC During Malaria

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