Cd45 clone 2d1

Heparin (20 IU/ml) was added to the blood sample collected in citrate or EDTA coated tubes within 20 min of collection. Whole blood (500 µl) was plated in a 24-well plate and placed in the incubator at 37⁰C for 20 min. Polystyrene particles (500 nm) were sonicated for 5 min and then added to the cells at an approximate ratio of 1:200 (cells to particles) according to the combined theoretical neutrophil (5 million/ml) and PBMC (2 million/ml) counts in the blood. Whole blood and particle suspension was subjected to flow in the peristaltic pump at the desired flow rate for 2 hours. The cells were then collected and subjected to RBC lysis for 8 min. The cells were spun down and resuspended in flow cytometry buffer (PBS containing 1% BSA and 4mM EDTA), and were stained with antibodies against CD45 (clone 2D1, BD Biosciences, USA), CD14 (clone M5E2, BD Biosciences) and CD15 (clone HI98, BD Biosciences) for 20 min at 4⁰C. Finally, the cell suspension was stained with propidium iodide (2 µg/ml) and run on the flow cytometer. All flow cytometry data analysis was performed using FlowJo (Treestar, USA).

CD34 positive cell counts were determined preprocedurally in the autologous donor's peripheral blood and in the leukapheresis product by flow cytometry (FACS Calibur; Becton Dickinson, Heidelberg, Germany). In 48 cases, counting was also done in peripheral blood 1 h after the leukapheresis procedure. The flow cytometric analysis followed the accepted protocol provided by the International Society of Haematology and Graft Engineering. Total Leukocyte Count (TLC) was performed in a calibrated automated cell counter (Sysmex XE 2100; Sysmex Corporation, Japan) using monoclonal CD34 antibody (clone 8G12; BD Biosciences, San Jose,, CA, USA) and CD45 (clone 2D1; BD Biosciences).