Subjects
This study is part of a large prospective study on interleukin levels conducted at the NICU of Harapan-Kita Women and Children Hospital, Jakarta, Indonesia from October 2007 till October 2009. For the present study patients were selected by consecutive sampling when they were inborn and showed no clinical signs compatible with an infection during the first seven days of life other than mild respiratory problems for which N-CPAP was given with 21% of FiO2. As CPAP is used very frequent in our unit for mild respiratory problems in infants without other symptoms, the use of CPAP was allowed. The blood culture, taken soon after birth, had to be negative. As is practice in our NICU, all infants admitted with -mild- respiratory problem, were treated with broad spectrum antibiotics (Ampicillin-sulbactam and an aminoglycoside). The study was approved by the Research Ethical Committee, Harapan Kita Women and Children Hospital, Jakarta. Informed consent was obtained from the parents when infants were included.
Routine laboratory and cytokine measurement
Blood was taken on admission for clinical purposes, including a blood culture. An additional 0.3 ml was taken for cytokine levels. Thereafter the same amount of blood was taken at 4, 12, 24 hrs and at day 2, 3, 4, 5, 6 and 7 together with blood taking for clinical purposes. In infants with a gestational age of 32 weeks and less the blood sampling for this study was stopped after day three, as it is was considered not ethical to take blood in these tiny infants for study purposes only. The blood sample was centrifuged immediately, 50 μl of serum was sampled and stored at -80°C. Samples were shipped on dry ice to the University Medical Center Groningen, The Netherlands, where they were analyzed. Sera were thawed and analysed using Invitrogen’s Multiplex Bead Immunoassay. In a 96 well plate samples were prepared by adding beads of defined spectral properties which were conjugated to protein-specific capture antibodies, incubation buffer to bind cytokines to the protein-specific capture antibodies and biotinylated detector antibodies. Finally streptavidin conjugated to the light-sensitive fluorescent protein R-Phycoerythrin was added and cytokine concentrations were analyzed with the Luminex detection system (Luminex Corp., Austin, Texas) using the programme StarStation 2.3. By monitoring the spectral properties of the beads and the amount of associated R-Phycoerythrin (RPE) fluorescence, the concentration of proteins was determined. The cytokines/chemokines of IL-1b, IL-1Ra, IL-2, IL-2r, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL-8), IL-10, IL-12, IL-13, IL-15, IL-17, TNFα, IFNa, IFNγ, IP-10 (CXCL-10), MIP-1a (CCL-3), MIP-1b (CCL-4), Eotaxin, Rantes (CCL-5), GM-CSF, MIG, MCP-1 (CCL-2) were measured.
Statistical analysis
To study the effect of gestational age the infants were divided in three groups, 30-32 wks, 33-35 wks and ≥36 wks. Differences between groups were analyzed by Non Parametric statistic (Mann–Whitney U test). The trend over time was evaluated using the Rank- Spearman test. To correct for multiple comparisons, the significance for all analysis was set at p < 0.01.