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

Abstract

SUMMARYThe 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 1 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 1 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.

Immunoparalysis and Adverse Outcomes from Critical Illness

“…To date, the vast majority of data outlining the natural history of innate immune function in critical illness come from the adult literature. There are only a handful of studies which hint at the developmental aspects of acquired immune dysfunction in neonates, infants, and small children [100][101][102]. The challenge before the pediatric intensive care community, therefore, is to design and implement multi-center prospective observational and interventional studies to address these important issues and work toward promoting immunologic homeostasis in the pediatric ICU.…”

Phagocytosis of neonatal pathogens by peripheral blood neutrophils and monocytes from newborn preterm and term infants

“…Of note, although expression levels were significantly lower in preterm infants, as previously described (36,37), there was no correlation of expression with reduced phagocytosis, suggesting that the prognostic utility of HLA-DR expression is independent of phagocytosis functionality. The difference in HLA-DR expression and any effect on phagocytosis may be transient, as others have reported equivalent expression levels between preterm and term infants up to 7-9 d of age (38). Similarly, expression of CD64 on neutrophils and CD86 on monocytes was not correlated with phagocytic activity.…”

Antenatal Betamethasone Associates with Transient Immunodepression in Very Low Birth Weight Infants

“…In very low birth weight (VLBW) infants, signs include upregulation of monocyte surface markers (CD11b) [8,9], high proinflammatory cytokine levels [10], and monocyte deactivation (low HLA-DR expression) [11]. One mechanism for favorable glucocorticoid effects may be their effects on innate immunity and cytokine production [12].…”