Flow cytometric measurement of HLA-DR expression on circulating monocytes in healthy and sick neonates using monocyte negative selection

Abstract

Summary The aim of this study was to investigate the effect of prematurity, neonatal sepsis, respiratory distress syndrome (RDS) and perinatal asphyxia on monocyte HLA-DR expression of neonates using a flow cytometric method based on monocyte negative selection. The subjects were one hundred and thirty-one neonates (59 healthy, 44 septicaemic, 20 with RDS and eight with perinatal asphyxia) and 20 healthy adults. Monocyte HLA-DR expression was measured using one-colour HLA-DR labelling in a gate for monocytes obtained using the combination of CD3-CD19–PE/CD15–FITC MoAbs. In addition, the common dual staining method using MoAbs against two CD14 epitopes (TUK4, MO2) was evaluated. With the one-colour HLA-DR labelling higher purity and recovery values of monocytes were achieved than with the dual labelling method. Healthy neonates had significantly lower percentages of HLA-DR+ monocytes than adults (69 ± 13% versus 91·5 ± 2·5%) and comparable mean fluorescence intensity (MFI) (119 ± 25 versus 131 ± 26). Values did not differ significantly between healthy term and preterm neonates. Preterm neonates with RDS had a significantly lower percentage of HLA-DR+ monocytes than the healthy preterm neonates. In neonates with asphyxia both parameters were comparable to those of the healthy ones. Septicaemic neonates presented significantly lower values of both parameters than the healthy, RDS and asphyxiated neonates. Monocyte negative selection provides a reliable estimation of HLA-DR expression on monocytes. Expression of monocyte HLA-DR is lower in healthy neonates in comparison with adults and is further decreased in neonates with sepsis and RDS, but it is not influenced by prematurity and perinatal asphyxia.