108-P: BONE MARROW NUCLEATED ERYTHROCYTES SELECTION AND ENGRAFTMENT EVALUATION

Aim Bone Marrow transplantation is used to treat a spectrum of diseases including hematologic disorders. Chimerism studies using short tandem repeat (STR) analysis of white blood cell lineages are routinely performed after bone marrow transplantation. However red cell chimerism is not routinely performed due to the difficulty to isolate nucleated red blood cells (NRBC). It is clinically significant to evaluate red cell chimerism after transplantation for thalassemia major, sickle cell disease, and other red cell disorders. Several methods have been employed to evaluate erythroid chimerism, including STR analysis of picked burst forming unit-erythroid colonies (BFU-E), detection of mixed red blood cell population by agglutination, and flow cytometry analysis with antibodies to ABO, Rhesus, Kell, Duffy or MNSs antigens. Here we report a simple and reliable molecular method to isolate NRBC for chimerism study from bone marrow samples. Methods Ficoll-Paque™ Plus density gradient (1.077 g/ml) (GE Healthcare, Uppsala, Sweden) was used to fractionate nucleated erythrocytes from bone marrow samples. After 30 minutes centrifugation at 400 g at room temperature, NRBCs banded together with mononuclear white cells. The NRBCs were then selected from the fraction by removing mononuclear white cells with CD45 conjugated magnetic beads. Results The purity of NRBC fraction was evaluated by histological method. After concentrating the cell preparation onto a glass slide by cytospin, the cells were stained with H&E and manually counted under microscope by an experienced hematology technologist. Out of a total of 703 cells counted from a randomly selected sample, 695 (98.9%) were NRBCs and 8 (1.1%) were contaminating white cells. Conclusions Ficoll-Paque fraction followed by CD45 magnetic beads negative selection of bone marrow sample is a simple and reliable method to isolate NRBC for erythroid lineage chimerism study after bone marrow transplantation.