Filter Paper Blood Spot Assay of Human Insulin-Like Growth Factor I (IGF-I) and IGF-Binding Protein-3 and Preliminary Application in the Evaluation of Growth Hormone Status

Abstract

To facilitate broader applications of insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) analysis, we developed procedures for their measurements in extracts of whole blood dried on filter paper. A single 8-mm diameter filter paper disc containing about 13 μL blood was used. IGFBP-3 was efficiently extracted in a buffer within 1 h of incubation. IGF-I extraction involved incubation in buffer followed by acidification and neutralization steps. Blood spot assays showed intra- and interassay coefficients of variation (including interspot variations) of 5.4–16.7% for IGF-I and 6.6–11.7% for IGFBP-3; recoveries were 97 ± 7.1% and 101± 8.7%, respectively. Recoveries of IGF-I and IGFBP-3 in response to 4- to 8-fold variations in extraction buffer volume were 97 ± 8.2% and 107 ± 6.1%, respectively. Dried blood spot IGF-I and IGFBP-3 showed greater than 1-month stability at −20 C, 4 C, and room temperature and retained more than 65% of the immunoreactivity after approximately 1 month at 37 C. Both IGF-I and IGFBP-3 were contained within the plasma fraction of whole blood, and variations (mean ± sd) in IGF-I (204 ± 29 μg/L) and IGFBP-3 (4.4± 0.48 mg/L) measured in extracts of dried blood spot with adjusted hematocrit of 0.2–0.62 were acceptable. IGF-I and IGFBP-3 in paired plasma and dried blood spot extracts of random samples (n = 46) showed excellent correlation (r > 0.94) with slopes of near unity. Compared to conventional methods, the filter paper procedures were equally effective in distinguishing IGF-I and IGFBP-3 levels in untreated GH receptor-deficient (n = 11) and age-matched normal controls (n = 16). We conclude that blood collected on filter paper is ideal for IGF-I and IGFBP-3 analysis and may find applications in pediatric and large scale infant screening programs.