Flow cytometry methodology applied to the study of RBC characteristics and its re-establishment after storing

DI TULIO, LILIANA; FORESTO, PATRICIA GLADYS; DELANNOY, M.; VALVERDE, JUANA ROSA; RIQUELME, BIBIANA DORIS

Punta del Este, Uruguay

Congreso; XXXI World Congress of the International Society of Hematology; 2007

ISH

Introduction: It is well known that red blood cells (RBCs) 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 ATP depletion, intracellular calcium increase, pH changes and alterations 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. Objectives: The aims of this study were to analyses the discocyte-echinocyte transformation and evaluate the re-establishment of the stored erythrocyte characteristic after incubation in autologous plasma by means of flow cytometry. Materials and Methods: Concentrated RBCs prepared from a healthy donor’s whole blood and collected with CPDA-1 solution were stored under blood bank conditions during 45 days at 4°C. A sample of these concentrated RBCs was taken every week after donation in order to be analyzed. Then, 500µl of RBCs and 500µl 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 cytometry measurements were performed using a standard instrument (Coulter Epics XL-MCL), which measures the forward light scattering intensity (FSC) and the 90º side light scattering intensity (SSC) at logarithmic amplification. 100,000 events were acquired in list mode at every sample run. The list mode files were processed by using a computer software Win MDI version 2.8. Results: We present the FS and SS dot plot and histogram in which can observe that during RBCs storage the SS and FS values decrease according to the discocyte-echinocyte transformation. All over this storage time, the SS value tends to recover the initial value with fresh frozen plasma treatment in function of the incubation time but the same behavior was not observed in the FS value, where all values tend to declined. Conclusions: Data obtained with flow cytometry turned out to be useful to evaluate the RBCs characteristics and their reversibility properties as 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. This method is a non-conventional tool to evaluate accurate RBCs behavior during storage under blood bank conditions, but unlike conventional microscopy, flow cytometry is not affected by the operator’s subjectivity. Therefore, flow cytometry could also be applied to quality control protocols in transfusion medicine.