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 < 0.0001). Better results were obtained between EasyCounter BC and flow cytometer Guava, 0.998 for vCD45+ and 0.998 for vCD34+ (P < 0.0001).