Re-establishing of stored RBC’s characteristics after plasma treatment studied by flow cytometry

DI TULIO; RELANCIO, M. F.; RIQUELME, B.; FORESTO, P.; VALVERDE, J.

Cape Town, South Africa

Congreso; ISBT 2006; 2006

It is well known that red blood cells show characteristic shape changes, especially discocyte - echinocyte transformation when they are stored. The normal discocyte represents an equilibrium state between two opposing shape changes: the echinocytic and the stomatocytic transformation. Transformation discocyte-echinocyte is influenced by many factors such as depletion of ATP, increase of intracellular calcium, changes in pH and alteration in the composition of the cell membrane. During storage, lyso-phosphatidylcholine is produced from phosphatidylcholine, which accumulates in cell membranes and is a potent echinocytogenic stimulant. Even though the RBC shape transformations have a potential reversibility, these alterations may alter transfusion effectiveness. Our previous studies in this field by flow cytometry have permitted to corroborate results of discocyte - echinocyte transformation by mean of this technique, which at difference of conventional microscopy is independent from operator subjectivity. The aim of this study was to observe the re-establishing of the stored erythrocyte characteristic after incubation in autologous plasma by flow cytometry. Rheological parameters were also studied by laser diffractometry technique for evaluate the alterations in the membrane of storage erythrocytes. Concentrated RBCs prepared from whole blood of a healthy donor collected with CPDA-1 solution was stored under blood bank conditions during 45 days. Each week after donation a sample was taken from the concentrated RBC storage bag under sterilized conditions to be analyzed. 500ml of RBCs and 500ml of autologous plasma were mixed and incubated during 60 minutes at room temperature. During this incubation time, aliquots of treated RBCs were re-suspended in PBS 300 mM pH 7,4 solution at six different times: 1, 5, 15, 30, 45 and 60 minutes to be analyzed.  The flow cytometric measurements were performed using a standard instrument (Coulter Epics XL-MCL), which measures the forward light scatter signal (FSC) and the 90º light scatter signal (SSC) at logarithmic amplification. In every sample run 100,000 events were acquired in list mode. The list mode files were processed by using a computer software WinMDI version 2.8.     We have observed that SS and FS values decrease with the storing period time. The SS values tend to recover the initial value with plasma treatment as the incubation time but the same behavior was not observed in the  FS values.   Data obtained with flow cytometry result useful to evaluate the RBC’s characteristics and their reversibility properties as the storage time increases, because it is possible to count a great number of cells in a few minutes and evaluate them in a multi-parametric way.