Simultaneous enumeration of CD34+ and CD45+ cells using EasyCounter image cytometer.

Abstract Accurate counting of CD34-positive cells is important for successful hematopoietic stem cell transplantation that is applied to various diseases. The aim of this study was simultaneous counting of viable CD34+ (vCD34+) and CD45+ (vCD45+) cells in apheresis samples by automatic immunofluorescence counter – EasyCounter BC. CD34+ and CD45+ cells were counted using two conjugates anti-CD34 antibody – dR110 and anti-CD45 antibody – ATTO620, respectively. The conjugates were prepared by carbodiimide method. Dead nuclear cells were counted by using monomethine cyanine dye PO-TEDM 1. The linearity and reproducibility of EasyCounter BC for CD34+ cell counting were determined (R2 = 0.99; CV values for vCD34+ cells were 6.8 ÷ 8.5% and for vCD45+ cells 4.1 ÷ 7.2%). The obtained results by EasyCounter BC were compared with those by other two standard methods – flow cytometry (Guava easyCyte 8HT) and fluorescence microscopic method (Olympus BX51) with the same conjugates. Passing–Bablok regression was performed to determine the relationship between the results of the three methods, analyzing 43 apheresis samples. Correlation coefficients for vCD45+ and vCD34+ between EasyCounter BC and Olympus microscope were 0.987 and 0.982, respectively (P