Assessment of erythrocyte shape by flow cytometry techniques

Piagnerelli, Michaël; Boudjeltia, Karim Zouaoui; Brohee, D.; Vereerstraeten, A; Piro, P.; Vincent, Jean‐Louis; Vanhaeverbeek, Michel

doi:10.1136/jcp.2006.037523

Cited by 68 publications

(76 citation statements)

References 34 publications

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“…The samples described in Table 1 were also examined by FACS, and representative histograms are shown in Figure 5. Control RBC (no enzyme and no cold shock) (Figure 5A) gave rise to a characteristic asymmetric histogram, with a shoulder at the high-scattering side, that is attributed to the discoidal shape of these cells (19). A smaller peak, at lower scattering intensity, is probably due to erythrocyte “ghosts”; see below.…”

Section: Resultsmentioning

confidence: 99%

Ceramide-Enriched Membrane Domains in Red Blood Cells and the Mechanism of Sphingomyelinase-Induced Hot−Cold Hemolysis

et al. 2008

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Hot-cold hemolysis is the phenomenon whereby red blood cells, preincubated at 37 °C in the presence of certain agents, undergo rapid hemolysis when transferred to 4 °C. The mechanism of this phenomenon is not understood. PlcHR 2 , a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, that is the prototype of a new phosphatase superfamily, induces hot-cold hemolysis. We found that the sphingomyelinase, but not the phospholipase C activity, is essential for hot-cold hemolysis because the phenomenon occurs not only in human erythrocytes that contain both phosphatidylcholine (PC) and sphingomyelin (SM) but also in goat erythrocytes, which lack PC. However, in horse erythrocytes, with a large proportion of PC and almost no SM, hot-cold hemolysis induced by PlcHR 2 is not observed. Fluorescence microscopy observations confirm the formation of ceramide-enriched domains as a result of PlcHR 2 activity. After cooling down to 4 °C, the erythrocyte ghost membranes arising from hemolysis contain large, ceramide-rich domains. We suggest that formation of these rigid domains in the originally flexible cell makes it fragile, thus highly susceptible to hemolysis. We also interpret the slow hemolysis observed at 37 °C as a phenomenon of gradual release of aqueous contents, induced by the sphingomyelinase activity, as described by Ruiz-Argüello et al. [(1996) J. Biol. Chem. 271, 26616]. These hypotheses are supported by the fact that ceramidase, which is known to facilitate slow hemolysis at 37 °C, actually hinders hot-cold hemolysis. Differential scanning calorimetry of erytrocyte membranes treated with PlcHR 2 demonstrates the presence of ceramide-rich domains that are rigid at 4 °C but fluid at 37 °C . Ceramidase treatment causes the disapperance of the calorimetric signal assigned to ceramiderich domains. Finally, in liposomes composed of SM, PC, and cholesterol, which exhibit slow release of aqueous contents at 37 °C, addition of 10 mol % ceramide and transfer to 4 °C cause a large increase in the rate of solute efflux.Hemolysis consists of the release of hemoglobin from red blood cells (RBC) 1 to the surrounding medium. In vivo hemolysis is a pathologic event that occurs in various forms of anemia, as well as in response to intoxications and infections. In addition, in vitro hemolysis is often used as a sensitive and reliable method to assay the lytic action on cell membranes of any physical or chemical agent. In this context RBC membranes are used as a membrane model. It was found long ago that erythrocytes become more sensitive to hemolysis induced by certain drugs or toxins when, after incubation at 37 °C, the cell suspension is cooled to 4 °C (1-3). This is known as "hot-cold hemolysis". The molecular basis for this phenomenon is not known. Different explanations have been proposed, but none has gained wide acceptance.PlcHR 2 toxin from Pseudomonas aeruginosa is an enzyme with both phospholipase C and sphingomyelinase activities. Stonehouse et al. (4) found that PlcHR 2 was highly hemolytic when the RB...

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Section: Resultsmentioning

confidence: 99%

Ceramide-Enriched Membrane Domains in Red Blood Cells and the Mechanism of Sphingomyelinase-Induced Hot−Cold Hemolysis

et al. 2008

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“…Thus, it seems that both senescent and acutely damaged red blood cells are eliminated through the same pathway, and we speculate that K Ca 3.1 and TMEM16A activation allow early recognition and removal of erythrocytes attacked by HlyA. Previous studies have shown that erythrocytes from septic patients show severe signs of volume reduction and crenation (40,41) and PS exposure (42). Patients that survive fulminate sepsis usually present with some degree of anemia, which is partially a consequence of intravascular hemolysis and partially the result of selective removal of damaged red blood cells (42).…”

Section: Discussionmentioning

confidence: 99%

Escherichia coli α-Hemolysin Triggers Shrinkage of Erythrocytes via KCa3.1 and TMEM16A Channels with Subsequent Phosphatidylserine Exposure

Skals

Jensen

Ousingsawat

et al. 2010

Journal of Biological Chemistry

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“…Shape is also an indicator of cell state useful in clinical diagnostics. For example, changes of red blood cells from their normal biconcave shapes to other shapes accompany many diseases, such as sickle-cell disease, anamia, or malaria [3,4]. Thus, shape can be a specific marker in bioparticle separation and may serve as a useful new basis for passive particle fractionation.…”

Section: Introductionmentioning

confidence: 99%

Continuous Inertial Focusing and Separation of Particles by Shape

et al. 2012

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An effective approach to separating shaped particles is needed to isolate disease-causing cells for diagnostics or to aid in purifying nonspherical particles in applications ranging from food science to drug delivery. However, the separation of shaped particles is generally challenging, since nonspherical particles can freely rotate and present different faces while being sorted. We experimentally and numerically show that inertial fluid-dynamic effects allow for shape-dependent separation of flowing particles. (Spheres and rods with aspect ratios of 3:1 and 5:1 have all been separable.) Particle rotation around a conserved axis following Jeffery orbits is found to be a necessary component in producing different equilibrium positions across the channel that depend on particle rotational diameter. These differences are large enough to enable passive, continuous, high-purity, high-throughput, and shape-based separation downstream. Furthermore, we show that this shape-based separation can be applied to a large range of particle sizes and types, including small, artificially made 3-m particles as well as bioparticles such as yeast. This practical approach for sorting particles by a previously inaccessible geometric parameter opens up a new capability that should find use in a range of fields.

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Assessment of erythrocyte shape by flow cytometry techniques

Abstract: RBCs are characterised by an increased spherical shape in many disease states. The measure of the second PCD in flow cytometry is a new, easy method to investigate RBC shape in various diseases. This technique could facilitate the investigation of abnormalities of RBC rheology.

Cited by 68 publications

References 34 publications

Ceramide-Enriched Membrane Domains in Red Blood Cells and the Mechanism of Sphingomyelinase-Induced Hot−Cold Hemolysis

Ceramide-Enriched Membrane Domains in Red Blood Cells and the Mechanism of Sphingomyelinase-Induced Hot−Cold Hemolysis

Escherichia coli α-Hemolysin Triggers Shrinkage of Erythrocytes via KCa3.1 and TMEM16A Channels with Subsequent Phosphatidylserine Exposure

Continuous Inertial Focusing and Separation of Particles by Shape

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