Assessment of erythrocyte shape by flow cytometry techniques

Background: Red blood cell (RBC) rheology is altered in different diseases, including acute conditions such as patients in intensive care units (ICU) with sepsis or with an inflammatory reaction due to postoperative states or intracerebral haemorrhage, or chronic conditions such as diabetes mellitus or terminal renal failure. Several techniques are available to assess alterations in RBC rheology, especially deformability, but they are too cumbersome to be used on a large number of cells. Objective: To develop a new, rapid flow cytometry technique for easy assessment of RBC shape in patients. Methods: In flow cytometry, healthy human RBC shape shows a bimodal distribution related to the biconcave form. On this histogram, the second Pearson coefficient of dissymmetry (PCD) representing the asymmetry of this histogram and the spherical index (M2:M1) were calculated, both representing the spherical shape. This technique was used in healthy volunteers (n = 17) and in diseases characterised by abnormalities in RBC rheology, including terminal renal failure requiring haemodialysis (n = 28), diabetes mellitus (n = 18), sepsis (n = 19) and acute inflammatory states (postoperative, intracerebral haemorrhage, chronic obstructive pulmonary disease, epilepsy or severe drug intoxication; n = 21). Multivariate analysis was performed to determine the factors influencing RBC shape. Results: Measurement of RBC shape was highly reproducible. A good correlation was observed between the PCD and the spherical index, except in the critically ill patients without sepsis. RBCs were more spherical in patients with terminal renal failure (PCD −0.56 (0.14), p<0.05), diabetes mellitus (PCD −0.59 (0.23), p<0.05), sepsis (PCD −0.58 (0.22), p<0.05) or an acute inflammatory state (PCD −0.65 (0.29), p<0.05) than in healthy volunteers (PCD −0.89 (0.12)). The spherical index was also increased in all populations compared with healthy volunteers (terminal renal failure 2.30 (0.20); diabetes mellitus 2.27 (0.38); sepsis 2.28 (0.37); acute inflammatory state 2.35 (0.42) vs healthy volunteers 2.72 (0.47); all p<0.05). Multivariate analysis demonstrated that the underlying pathology (sepsis, acute inflammatory state, diabetes mellitus, terminal renal failure) was the principal cause of these RBC shape abnormalities. Conclusion: 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.

[1]  C. Rice-Evans,et al.  Red blood cell biomembrane structure and deformability , 1981 .

[2]  S J Singer,et al.  On the mechanism of ATP-induced shape changes in human erythrocyte membranes. I. The role of the spectrin complex , 1977, The Journal of cell biology.

[3]  T. Kamada,et al.  Erythrocyte membrane fluidity decreased in uremic hemodialyzed patients. , 1985, Nephron.

[4]  G. Gambaro,et al.  Abnormal Erythrocyte Charge in Diabetes Mellitus: Link with Microalbuminuria , 1988, Diabetes.

[5]  G. Staal,et al.  Alterations in size, shape and osmotic behaviour of red cells after splenectomy: a study of their age dependence , 1988, British journal of haematology.

[6]  J. Hudson,et al.  Flow cytometric analysis of human erythrocytes: I. Probed with lectins and immunoglobulins , 1991, Experimental Gerontology.

[7]  J. L. Gall,et al.  A simplified acute physiology score for ICU patients , 1984, Critical care medicine.

[8]  S B Shohet,et al.  The influence of membrane skeleton on red cell deformability, membrane material properties, and shape. , 1983, Seminars in hematology.

[9]  Jean-Charles Preiser,et al.  Microvascular blood flow is altered in patients with sepsis. , 2002, American journal of respiratory and critical care medicine.

[10]  G. Machiedo,et al.  Decreased red blood cell deformability and impaired oxygen utilization during human sepsis. , 1993, The American surgeon.

[11]  I. Kushner,et al.  Acute-phase proteins and other systemic responses to inflammation. , 1999, The New England journal of medicine.

[12]  N. Mohandas,et al.  Red blood cell deformability, membrane material properties and shape: regulation by transmembrane, skeletal and cytosolic proteins and lipids. , 1993, Seminars in hematology.

[13]  M. Cazzola,et al.  Flow cytometry evaluation of erythroid dysplasia in patients with myelodysplastic syndrome , 2006, Leukemia.

[14]  C A Grimbergen,et al.  Measurement of the distribution of red blood cell deformability using an automated rheoscope. , 2002, Cytometry.

[15]  E. Maher,et al.  Increased free-radical activity during haemodialysis? , 1987, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[16]  J. Vincent,et al.  Effects of sample temperature on red blood cell shape in septic patients. , 2004, Clinical hemorheology and microcirculation.

[17]  Samuel E. Lux,et al.  Dissecting the red cell membrane skeleton , 1979, Nature.

[18]  M. Bitbol.  Red blood cell orientation in orbit C = 0. , 1986, Biophysical journal.

[19]  W H Reinhart,et al.  Echinocytosis induced by haemodialysis. , 1998, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[20]  M. McMullin.  The molecular basis of disorders of the red cell membrane. , 1999, Journal of clinical pathology.

[21]  O. Baskurt,et al.  Erythropoietin‐induced rheological changes of rat erythrocytes , 2000, British journal of haematology.

[22]  O. Baskurt,et al.  Importance of measurement temperature in detecting the alterations of red blood cell aggregation and deformability studied by ektacytometry: a study on experimental sepsis in rats. , 2000, Clinical hemorheology and microcirculation.

[23]  L. Mazzanti,et al.  Sialic acid, diabetes, and aging: a study on the erythrocyte membrane. , 1997, Metabolism: clinical and experimental.

[24]  F. Leoncini,et al.  Variations of erythrocyte morphology in different pathologies. , 1997, Clinical hemorheology and microcirculation.

[25]  T. Kamada,et al.  Erythrocyte membrane microviscosity in diabetes. , 1981, Hormone and metabolic research. Supplement series.

[26]  C. Watala,et al.  Changes in fluidity and composition of erythrocyte membranes and in composition of plasma lipids in Type I diabetes , 1986, British journal of haematology.

[27]  G. Cokelet,et al.  Decreased Hydrodynamic Resistance in the Two‐Phase Flow of Blood Through Small Vertical Tubes at Low Flow Rates , 1991, Circulation research.

[28]  J. Vincent,et al.  Alterations of red blood cell shape and sialic acid membrane content in septic patients , 2003, Critical care medicine.

[29]  N. Mohandas,et al.  Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking. , 1985, The Journal of clinical investigation.

[30]  E. Abadie,et al.  Increased adhesion of erythrocytes to endothelial cells in diabetes mellitus and its relation to vascular complications. , 1981, The New England journal of medicine.

[31]  C A Grimbergen,et al.  Analyzing red blood cell-deformability distributions. , 2002, Blood cells, molecules & diseases.

[32]  F. Turci,et al.  Osmotic fragility of erythrocytes, cell deformability and secondary hyperparathyroidism in uremic patients on maintenance hemodialysis. , 1985, Clinical nephrology.

[33]  B. Deuticke.  Membrane Lipids and Proteins as a Basis of Red Cell Shape and its Alterations , 2003 .

[34]  J. Dobbe,et al.  Laser-assisted optical rotational cell analyser (L.O.R.C.A.); I. A new instrument for measurement of various structural hemorheological parameters , 1994 .

[35]  O. Linderkamp,et al.  Haemorheological changes in uraemic children in response to erythropoietin treatment , 1993, Pediatric Nephrology.

[36]  W. Birchmeier,et al.  On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP , 1977, The Journal of cell biology.

[37]  A. Arduini,et al.  Increased erythrocyte phosphatidylserine exposure in chronic renal failure. , 1999, Journal of the American Society of Nephrology : JASN.

[38]  H J Meiselman,et al.  Red blood cell deformability in sepsis. , 1998, American journal of respiratory and critical care medicine.

[39]  Mitchell M. Levy,et al.  2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference , 2003, Intensive Care Medicine.

[40]  M. Välimäki,et al.  Alcohol and sex hormones. , 1981, Scandinavian journal of clinical and laboratory investigation.

[41]  H. J. Barnes.  Blood rheology in diabetes mellitus. , 1986, Acta medica portuguesa.

[42]  G. Omann,et al.  Flow cytometric analysis of human erythrocytes: II. Possible identification of senescent RBC with fluorescently labelled wheat germ agglutinin , 1991, Experimental Gerontology.

[43]  A. Goldberg,et al.  Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes. , 1987, The Journal of biological chemistry.