Comparative study of monocyte enumeration by flow cytometry: improved detection by combining monocyte-related...

Monocyte-counting performance by hematological cell counters often lacks satisfactory accuracy and precision. Flow cytometry, the alternative, is a reliable reference method for monocyte enumeration in evaluation of the performance of automated analyzers. The present study was focused on evaluation and optimization of monocyte enumeration by flow cytometry. Fifty blood samples were stained with same-color CD163, CD64, and CD14 monoclonal antibodies (MoAbs), single or in mixtures, along with different colors of anti-CD45 and anti-CD16. The percentage positive monocytes and their median fluorescence intensity (MFI) were compared between the resulting MoAb combinations. CD163(+)14 increased monocyte detection by 5% compared with CD14 alone. The CD64(+)CD163 mixture resulted in a 6% detection increase relative to CD64 alone. CD64(+)CD14 labeled 2% more monocytes than CD14 and 0.03% more compared with CD64. Combining CD14 with CD163 or CD64 increased MFI 24% and 14% relative to CD14. CD163(+)CD64 increased monocyte MFI 115% compared with CD64. We found that combining same-color antibodies improved monocyte detection, especially when anti-CD163 was added to CD64 or CD14 antibodies. The use of CD163 antibody is the recommended flow cytometric reference method for monocyte counting.