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CD64 is a high-affinity Fc(gamma)RI receptor expressed by activated neutrophils that has been recently evaluated as a potential sepsis parameter. In the present study, the kinetics of neutrophil membrane CD64 expression were examined during a standardized inflammatory response, using a human endotoxemia model, and compared with hematological indices, CRP, cytokines and interleukins. Ten healthy subjects received 2 ng/kg intravenous Escherichia coli lipopolysaccharide (LPS). After administration of LPS, neutrophil CD64 showed a biphasic response, characterized by a first increase from 108.5 +/- 7.5 to 133 +/- 6 AFU after 1 h (P = 0.047) and a second increase that started at 6 h and reached its maximum of 167 +/- 13 AFU at 22 h (P < 0.0001). CRP concentrations increased to 40 +/- 5 mg/dl 22 h after the administration of LPS. The cytokines and interleukins reached their maximum response within 1-2 h. The maximum values of pro-inflammatory cytokines (TNF-alpha, IFN-gamma and IL-6) correlated with the CD64 expression at 22 h after LPS administration (r(2) = 0.76, r(2) = 0.78, r(2) = 0.81, respectively, all P < 0.05), whereas this correlation was not found for the anti-inflammatory IL-10 (r(2) = 0.058, P = 0.54), suggesting that neutrophil CD64 expression might be a quantitative marker for innate immunity that could easily be used in the clinical setting.

A patient with abdominal sepsis, had both intra and extracellular bacteria in a blood smear, and high levels of neutrophil membrane CD64 and HLA-Dr. Intracellular bacteria are only observed in the terminal phase of a sepsis. Our patient recovered, suggesting that a high expression of neutrophil CD64 is indicative for a good prognosis.

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 CD4,000 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. CD4,000 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<0.0001) to absolute granulocyte counts, and patients with high PMN-CD64 were more likely (8/25) to have in excess of 10% Band Cells compared to samples with normal/intermediate PMN-CD64 (0/84). When compared to C-reactive protein (CRP), high PMN-CD64 and MON-CD64 were always associated with an increased CRP concentration, but minor proportions of samples with normal PMN-CD64 (11/52) or normal MON-CD64 (11/65) could also have an increased CRP. The procedures described in this communication overcome a number of limitations associated with flow cytometry, and co-determination of CD64 and HLA-DR antigen expression may provide complimentary insights into patient heterogeneity in the assessment of suspected sepsis compared to CD64 analysis alone.