Simultaneous determination of membrane CD64 and HLA-DR expression by blood neutrophils and monocytes using the monoclonal antibody fluorescence capability of a routine haematology analyser

Abstract This study reports the design of an immunofluorescent method for the co-determination of neutrophil CD64 (PMN-CD64), monocyte CD64 (MON-CD64) and monocyte HLA-DR (MON-Ia) expression with the Cell-Dyn CD4000 haematology analyser. Normal PMN-CD64, MON-CD64 and MON-Ia expression, defined as the mean ± 2SD of 25 healthy adults after correction for isotype control staining, corresponded to 17–67, 515–1045 and 170–670 AFU respectively. Analytical reproducibility determined by duplicate analysis of 12 random samples revealed good assay consistency for all three analysed antigens, with day to day variation in normal subjects being relatively minor in significance. CD4000 PMN-CD64 and HLA-DR values showed good inter-method correlation with flow cytometry although short term (12 h) stability studies suggested an in vitro trend for increasing PMN-CD64 and variable HLA-DR antigen expression with progressive storage. Observed ranges of PMN-CD64, MON-CD64 and MON-Ia for 109 randomly-selected clinical samples were 31–1058, 307–2843 and 10–876 AFU. Abnormal PMN-CD64 and MON-CD64 shared the same trend (upregulation) while abnormal monocyte MON-Ia was characterised by declining expression. Normal PMN-CD64 was only seen with normal (45/52) or intermediate (7/52) MON-CD64, while high PMN-CD64 was mostly associated with intermediate (18/22) or high (3/22) MON-CD64. MON-Ia expression was largely independent ( p  = 0.04) of PMN-CD64 although marked decreases in MON-Ia were invariably associated with intermediate or high PMN-CD64. MON-Ia expression was inversely related ( p