Subproject 4 – Study of electrical and mechanical propertis of erithrocyte membrane

Participants: : Carlos Lenz César (IFGW-UNICAMP), Sara T. O. Saad (Hemocentro-Unicamp)

This collaboration is very well stablished and already resulted in a PhD thesis, part of another PhD thesis, many published papers and congress communications. There is a collaborative supervision of a MSc thesis.
The erithrocytes exhibit unusual mechanical properties that allow the cell to perfuse capillary bed and spleenic sinusoids half their diameter (7-9 mm). The erithrocyte deformability results from a combination of diverse factor such as the surface volume ratio, internal viscosity, and membrane elasticity and viscosity. The evaluation of blood red cell elasticity was first done by researchers from Unicamp, who studied normal cells (Blood 15: 2975, 1998), S hemoglobin donors
(AS) (Vox Sang 85: 213, 2003), irradiated cells (Transfusion 42: 1196, 2002), falciform cells (SS), S heteroziygotes S (AS), and hydroxiurea treated cells (Eur J Hematol 70: 207, 2003). Currently, a manuscript on ferropriva anemia has been submitted. The erythrocyte has negative electrical charges of sialic acid bound to membrane glycoproteins. So, the cell atracts positive counterions that establish a ionic cloud around the cell surface, with a gradient from the membrane surface out. This property establish a potential difference, named zeta potential that maintain the red blood cell apart from each other and from the endothelial surface. The aggregability of red blood cells is defined as the hability of these cells to form multi-aggregates or rouleaux (pilled coins) in the presence of proteins and other macromolecules such as dextrans, used as a plasma expansor. In normal circunstances, blood flow is albe to disperse such aggregates. However, pathological situations such as slow fluxes and alterations of the mechanical propertis of the membrane (elasticity/deformability), resistant aggregates may form and cause clinical complications. Preliminary results of the methodological characterization of zeta potential and erithrocyte aggregation were presented at the Brazilian Congress of Hematology and Hemotherapy (2007), at the SPIE 2007/2008 and submitted to the American Society of Hematology Congress (2008). Erithrocyte concentrates used in therapy are kept under low temperatures (4-6°C) for 35-42 days, depending on the preservative solution employed. The
Storage of concentrates is related to numerous biochemical alterations, between them the loss of anti-oxidant capacity with consequent oxidative damage of the membrane, lipid loss and reduction of sialic acid. Oxidative damage and changes in electrical charges of the membrane reduce the capacity of the membrane to deform and increase the tendency to aggregate. Few studies have been conducted to determine the erithrocyte damage related to storage. A better understanding of these phenomena could contribute to the development of new preservative solutions in addition to the use of antioxidant drugs, with benefits for the transfusion. Hemotherapy services rely on transfusional immunehematological testsem based on antibody-antigen interactions. The antierithrocyte IgG antibodies are monomers and can not agglutinate cells without the addition of substances that favors cell adhesion. In this sense, substances that reduce the zeta potential are required and diminish the distance between the cells. The specificity and sensibility of these tests depend on the correct use of these substances, and a quantitative valuation of the efficiency of different substances or even new drugs could improve the quality of the tests used before transfusion and more importantly, contribute to a better automatization of routines. Objectives: (1) Measure zeta potencial and erithrocyte aggregation in fresh and stored blood samples; (2) Measure the aggregation force using double optical tweezers after different periods of storage, associated with the use of substances that potentiate the agglutination such as low ionic force buffers (LISS), polyethyleneglycol (PEG), dextran, bromelin and papain.