Methods

The fundus of infants with ROP was examined with a slit lamp and contact lens (Volk Quad Pediatric Lens; Volk Optical Inc, Mentor, Ohio, USA) under general anesthesia. During the examinations, fundus photographs and fluorescein angiograms were taken with a RetCam 120 digital fundus camera (Clarity Medical Systems, Inc, Pleasanton, California, USA). The stage of the ROP was based on the International Classification of Retinopathy of Prematurity. The ROP eyes were also classified into 3 groups according to the vascular activity: highly vascular-active ROP, moderately vascular-active ROP, and mildly vascular-active ROP. The eyes with highly vascular-active ROP initially received 0.25 mg or 0.5 mg of intravitreal bevacizumab and underwent 23-gauge pars plicata vitrectomy without cannula system. The surgery was performed within 1 week after the injection when considered to be necessary.

Blood samples were collected before and 1 day, 1 week, and 2 weeks after the intravitreal bevacizumab. This schedule was based on the data from animal experiments demonstrating that the maximum blood level of bevacizumab is achieved about 1 to 2 weeks after the intravitreal bevacizumab. The blood samples were collected in sterile tubes by an anesthesiologist or a neonatologist and centrifuged at 5000 rpm for 10 minutes until a clear separation between serum and the cell components was seen. The serum was transferred to sterile tubes and stored at −80 C until the assay.

The serum concentration of bevacizumab was measured with an enzyme-linked immunosorbent assay (ELISA) kit (Protein Detector ELISA Kit; Kirkegaard & Perry Laboratories, Inc, Gaithersburg, Maryland, USA), according to the manufacturer’s protocol and also according to an earlier report with slight modifications. Briefly, microwell plates (Immuno 96 MicroCell solid plates; Nunc, Roskilde, Denmark) were coated with recombinant human VEGF ₁₆₅ (PeproTech, Rocky Hill, New Jersey, USA) at a concentration of 1.0 μg/mL for 1 hour at room temperature (100 μL/well). After blocking the wells to reduce nonspecific binding, 100 μL of each sample and different concentrations of the standard were added to the plates. A standard curve was prepared with bevacizumab ranging from 1 ng/mL to 5000 ng/mL. The bound bevacizumab was made visible with 0.1 μg/mL of horseradish peroxidase–goat anti-human IgG (H+L) conjugate prepared by the ELISA kit. The optical density was determined at 405 nm with an absorption spectrophotometer (ARVO _MX ; PerkinElmer Japan, Kanagawa, Japan). The background absorbance was subtracted from all values. This assay measures the free bevacizumab, and all measurements were performed twice according to the manufacturer’s recommendation.

The serum concentration of VEGF was measured with an ELISA kit for human anti-VEGF (R & D Systems, Minneapolis, Minnesota, USA) according to the manufacturer’s protocol. The anti-VEGF kit can detect the 121 and 165 isoforms of VEGF. The minimum detectable level of the test was 9.0 pg/mL for VEGF. The optical density was determined at 450 nm with the absorption spectrophotometer with the correction wavelength set at 540 nm. The assay was also performed in duplicate.

Statistical analyses were performed using the SPSS software (Sigma Stat; Systat Software, Inc, San Jose, California, USA). Data are presented as the means and standard deviations. If the data were normally and equally distributed, 1-way repeated-measures analysis of variance was used to compare 3 or more matched groups, followed by the Holm-Sidak method to detect significant differences between each set of data. If the data were not normally or equally distributed, Friedman repeated-measures analysis of variance on ranks was performed to compare 3 or more matched groups, followed by Dunn’s method to detect significant differences between each set of data. The significance of differences between 2 groups was determined by t tests if the data were normally and equally distributed and by the Mann-Whitney rank sum test if not normally distributed. The correlation between 2 parameters was determined by the Spearman rank order correlation because the residuals were not normally distributed with constant variance. A P value less than .05 was considered to be statistically significant.

Results

Eleven infants (4 girls and 7 boys) with highly vascular-active ROP were studied. The demographics of the patients are summarized in Table 1 . Three patients received intravitreal bevacizumab in 1 eye and the other 8 received intravitreal bevacizumab in both eyes. The mean gestational age of the infants was 25 weeks (range, 23–27 weeks), and the mean body weight at birth was 660 grams (range, 332–1042 grams). All of the infants had received laser photocoagulation of the peripheral avascular retina before the intravitreal bevacizumab. The mean postmenstrual age of the infants at the time of intravitreal bevacizumab was 38 weeks (range, 32–51 weeks), and the mean body weight at the time of the intravitreal bevacizumab was 1720 grams (range, 940–2600 grams). In Patients 6, 8, 9, and 11 with stage 3 ROP, vitrectomy was not performed after the intravitreal bevacizumab because of the reduction of vascular activities. In the remaining patients, vitrectomy was performed 0 to 6 days after the intravitreal bevacizumab.

The average serum levels of bevacizumab before and 1 day, 1 week, and 2 weeks after a total of 0.5 mg of intravitreal bevacizumab were 0 ng/mL, 195 ± 324 ng/mL, 946 ± 680 ng/mL, and 1214 ± 351 ng/mL, respectively ( Figure 1 , Table 2 ). In Patients 2, 8, 9, 10, and 11, the serum bevacizumab levels were significantly different ( P = .008) before and 1 day and 1week after the intravitreal bevacizumab, and the serum bevacizumab level at 1 week after the intravitreal bevacizumab was significantly higher ( P < .05) than that before the intravitreal bevacizumab.

Time course of serum level of bevacizumab in infants with retinopathy of prematurity who received intravitreal bevacizumab. The abscissa represents the time before and after intravitreal bevacizumab and the ordinate represents the serum level of bevacizumab.

TABLE 2

Serum Levels of Bevacizumab (Avastin) and Vascular Endothelial Growth Factor in Infants With Retinopathy of Prematurity

Patient/Sex	Eye/Stage	Total Dosage of Intravitreal Bevacizumab (mg)	Serum Level of Bevacizumab (ng/mL)				Serum Level of Vascular Endothelial Growth Factor (pg/mL)
			Before Intravitreal Bevacizumab	1 Day After Intravitreal Bevacizumab	1 Week After Intravitreal Bevacizumab	2 Weeks After Intravitreal Bevacizumab	Before Intravitreal Bevacizumab	1 Day After Intravitreal Bevacizumab	1 Week After Intravitreal Bevacizumab	2 Weeks After Intravitreal Bevacizumab
1/Male	Right/5	0.5	0	23	NA	NA	NA	NA	NA	NA
	Left/4A
2/Male	Right/4B	0.5	0	31	81	NA	NA	NA	NA	NA
	Left/4A
3/Female	Right/4A	1.0	0	206	665	NA	NA	NA	NA	NA
	Left/4A
4/Male	Right/5	1.0	0	396	513	NA	NA	NA	NA	NA
	Left/4A
5/Male	Right/4A	1.0	0	19	560	NA	NA	NA	NA	NA
	Left/3
6/Female	Right/3	1.0	0	372	453	NA	NA	NA	NA	NA
	Left/3
7/Female	Right/3	0.25	0	11	113	NA	418	303	301	NA
	Left/4B
8/Male	Right/3	0.5	0	33	1204	NA	603	227	106	NA
	Left/3
9/Male	Right/3	0.5	0	36	610	844	1140	515	208	129
	Left/3
10/Female	Right/3	0.5	0	841	1905	1255	2110	433	331	439
	Left/5
11/Male	Right/3	0.5	0	209	928	1542	2660	533	337	239
	Left/3

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