In this study, sE-selectin was measured prospectively in 50 preterm infants at high risk of ROP (gestational age [GA] between 23 and 32 weeks) admitted to the neonatal intensive care unit of the University of Freiburg. One child died before eye examination, two were discharged without a complete ophthalmic screening, and five had no measurements because of hemolytic samples; thus, 42 infants were analyzed. ROP staging was performed by two experienced pediatric ophthalmologists (CP, FB) by dilated indirect ophthalmoscopy. Ophthalmic examinations started 5 weeks after birth and were repeated after 2 to 14 days, depending on the stage of ROP, until retinal maturation was completed. Infants with no or with stage I ROP are referred to as the no-ROP group, and infants who had stage II (independent of the presence of plus disease) or stage III ROP formed the ROP group. To avoid excessive blood loss in this vulnerable cohort, we used only residues of blood samples withdrawn for other clinical purposes in the analyses. Scheduled blood withdrawals for study purposes were not authorized by the local ethics committee. Sample collection started as early as 2 days after birth and continued up to 15 weeks, depending on length of hospitalization. A total of 85 plasma samples were collected. One to five samples were available per infant: 20 infants had one, 10 had two, 6 had three, 3 had four, and 3 had five. A minimal volume of 50 μL of plasma was necessary for repeated ELISAs. Samples were gathered in serum tubes (Vacuette; Greiner Bio-One, Kremsmuenster, Austria) containing EDTA and were centrifuged at 23,000
g at room temperature for 10 minutes. The clear supernatant was immediately separated and frozen at −20°C until analyzed. A sandwich enzyme immunoassay was performed with factor-specific monoclonal mouse antibodies (R&D Systems, Wiesbaden-Nordenstadt, Germany). sE-selectin levels were determined in 100 μL 1:20 diluted plasma per data point. The minimum detectable concentration of sE-selectin was 2.12 ng/mL. Information regarding clinical parameters that have been associated with ROP, such as GA, birth weight, oxygen supplementation, respiratory distress syndrome,
20 BPD, sepsis, and cerebral hemorrhage,
21,22 and other parameters was obtained from the medical records of all the children and analyzed. Our study adhered to the tenets of the Declaration of Helsinki and was approved by the ethics committee of the Albert-Ludwigs-University Freiburg, Germany. Written informed consent was obtained from the parents or guardians of all the children.
To examine the dependence of sE-selectin plasma levels on GA kernel, we applied smoothing to fit a nonlinear curve to all sE-selectin measurements obtained by using a Gaussian kernel with a bandwidth (SD) of approximately 2.1 days (2σ
2 = 9).
23 For statistical evaluation of differences between infants with and those without ROP, only the first plasma sample collected in each child was used.
For group comparisons, we evaluated median plasma concentrations for each group by univariate analysis and tested for significance with the Mann-Whitney test for nonparametric data. The following clinical data were assessed in a univariate analysis with respect to the ROP groups: GA, birth weight, arterial umbilical cord pH, days of mechanical ventilation, need for surfactant therapy, days of FiO
2 > 0.4 per month, occurrence of BPD (by definition of Bancalari et al.
24 ), vasopressor treatment, sepsis (defined as clinical signs and interleukin-6 >100 pg/mL or CRP >20 mg/L), mode of ductus botalli closure, necrotizing enterocolitis (NEC, if surgery was performed), and intraventricular hemorrhage (any grade by Papile's definition). Differences were considered significant when
P < 0.05. Univariate analysis was performed (SPSS ver. 15.0; SPSS, Chicago, IL), and multiple regression analysis and the receiver operating characteristic (ROC) curve were calculated (PROC logistic procedure, SAS; SAS Institute, Cary, NC). The models were tested in forward, backward, and stepwise selections. Correlation coefficients were calculated by Spearman rank order correlation.