CD34+ stem cell counting using labeled immobilized anti-CD34 antibody onto magnetic nanoparticles and EasyCounter BC image cytometer.

ABSTRACT

The ability of immobilized conjugate anti-CD34+ monoclonal antibody-dR110 and free conjugate anti-CD45+ monoclonal antibody-ATTO620 to precisely enumerate CD34+ stem cells and CD45+ cells in apheresis samples were evaluated. The conjugates anti-CD34+ antibody-dR110 and anti-CD45+- antibody-ATTO620 were prepared. Functionalized magnetic nanoparticles (MNPs) were synthesized. The anti-CD34+ antibody-dR110 conjugate was immobilized on the modified MNPs using a carbodiimide method. The stem cell count in thawed apheresis samples was determined using the free and the immobilized conjugate anti-CD34+ antibody-dR110 on MNPs and an image cell counter EasyCounter BC. A higher stem cell count and more accurate results were obtained with the immobilized conjugate, because a separation and concentration of the stem cells bound to antibody-dR110 on MNPs by external magnet were performed. Coefficients of variation of CD34+ cell count in apheresis samples, determined by EasyCounter BC, were ranged from 5.5 to 6.9% and those of CD45+ cell count from 3.8 to 4.7%. The viability of CD34+ cells was high from 98.5 to 99.6%. It was found that correlation coefficient between the flow cytometer and automatic cell counter, using free anti-CD34+ antibody-dR110 was 0.94, and when using immobilized anti-CD34+antibody-dR110 on MNPs, the correlation coefficient was 0.97.