Presented at the Neonatal Society 2008 Spring Meeting.
Ahmed I, Walker K, Thomas A, Midgley P
Simpson Centre for Reproductive Health, New Royal Infirmary, Edinburgh EH16 4SA, UK
Department of Haematology, Royal Hospital for Sick Children, Edinburgh EH9 1LW, UK
Background: Infection is a major cause of morbidity and mortality in preterm infants. Polymorphonuclear neutrophils (PMN) are the first line of defence against invasive organisms. In order to function effectively, neutrophils must adhere to vascular endothelium at sites of infection. The process of adhesion has been shown to be impaired in the neonatal neutrophil, partly due to reduced surface expression of binding antigens CD11b and CD18. Neutrophils must also generate toxic oxygen metabolites (a process known as oxidative burst) in order to fulfil their microbicidal function. However a combination of neutrophil oxidative burst and adhesion molecule expression, in the same preterm infant has not been previously reported.
Aim: To examine the function of oxygen-dependent intracellular killing mechanisms (oxidative burst) in neutrophils of newborn preterm infants and investigate the expression of neutrophil adhesion molecules CD11b and CD18.
Methods: Prospective observational study. Blood samples were collected on days 1, 3, 5 and 7 from preterm infants (n=58), median (range) gestation 29 (23-35) weeks, median (range) birth weight 1257 (635–2540) g. The neutrophils were stimulated with PMA (phorbol 12-myristate 13-acetate), a strong chemical stimulant, and with opsonised E.coli. A non-stimulated sample was included in each test as a control. Neutrophils exhibiting oxidative burst activity produce free radicals, which react with Dihydrorhodamine (DHR) to produce fluorescence. This is then detected by a flow cytometer and reported as mean fluorescence index (MnIX). The neutrophil oxidative index (NOI) is then calculated as the ratio of stimulated: non-stimulated MnIX. The density of CD11b and CD18 on neutrophils was determined using flow cytometry. Aliquots of blood were labelled with fluorescent monoclonal mouse anti-human CD11b and CD18 antibodies. Antigen quantitation was performed using a qualitative analysis based on the linear relationship observed between immunofluorescence intensity and antigen antibody binding capacity expressed as Mean channel of fluorescence or Mean index.
Ethics approval obtained from Lothian Research Ethics Committee.
Results: The median (interquartile range) neutrophil oxidative index for preterm infants following stimulation with PMA and E.coli with reference ranges for stimulated neutrophils from infants and children are shown in the table.
There was no effect of labour, pregnancy induced hypertension or being small for gestational age on the oxidative burst response. All preterm infants expressed both CD11b and CD18 antigens, however the percentage of neutrophils expressing binding antigens was variable.
Conclusion: This data show that the neutrophil oxidative burst response to stimulation with PMA or E.coli is reduced in preterm infants. This, in combination with variable expression of CD11b and CD18 binding antigens, may contribute to susceptibility to infection in this preterm population.