2016
Volume 16, Number 1, pp. 47-64
Revisiting red blood cell heterogeneity, membrane molecules and surface charge changes in normal, anaemic and thalassaemic red blood cells for blood microsample test development
J.N. Mehrishi1 and Angela Risso2
1The Cambridge Blood, Umbilical Cord Blood Stem Cells for Cell–Gene Thalassaemia, Sickle Cell Therapy and Cultivated RBC Research Initiative, 13 Macfarlane Close, Impington, Cambridge, CB24 9LZ, United Kingdom.
2Department of Agriculture and Environmental Sciences, Università di Udine, via delle Scienze 91, 33100 Udine, Italy
Mammalian cells are covered by a complex of glycoproteins, glycolipids, glycosphingolipids and proteoglycans, forming a dense 70-100 nm thin layer termed the glycocalyx. This is rich in sialoglycoproteins; the cell membrane sialic acid residues (SAs) are important components that play a rôle in determining cell structure, survival, chemical-electrical properties and functions such as recognition and homing. SAs contribute a substantial (~60%) electrical charge, which is one of the major physicochemical factors governing a wide variety of interactions. When these SA sites are compromised or poorly expressed, such as in aging red blood cells (RBCs) or in blood disorders, for instance thalassaemia, erythrophagocytosis is hastened and anaemia can ensue. Centrifugation of RBCs on a Percoll density-gradient provides a reasonably acceptable separation of fractions of RBCs. Analyses of biochemical and physical parameters of these RBC subsets have shown that each fraction is enriched successively in the lightest young (Y-RBC), middle aged (M-RBC), and the densest old red cells (O-RBC). Recently, Percoll density-gradient-fractionated RBCs from blood samples of β-thalassaemia intermedia patients were shown to have a very large number of high density RBCs. Furthermore, in β-thalassaemia intermedia not only do RBCs show physicochemical alterations of the membrane, but also changes in the levels of expression of the membrane proteins CD47 and CD55. In view of these observations, we propose that we have the basis for developing quantitative, rapid tests on microsamples of blood for monitoring the physiology and surface markers of RBCs to be applied to the clinical monitoring of blood disorders. At present, microsample blood tests using 0.1-1.0 mL (20-200 drops) for monitoring in clinics seems feasible at very low cost for a battery of several ligands, using fluorescence microscopy, imaging and flow cytometry.
Keywords:anaemias, CRISPRCas9, CRISPRPf1, electroporation, erythrocytes, erythropoietin, flow cytometry, gene therapy, hypoxia, membrane molecules, Percoll gradient separation, prolyl hydroxylase inhibitors, renal anaemia, spectrin, thalassaemia