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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

Diagnostic Systems Laboratories (Canada), Inc. (A.D., M.J.K.), and the Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto (M.J.K.), Toronto, Ontario, Canada; Diagnostic Systems Laboratories, Inc. (J.M.), Webster, Texas 77598; and the Institute of Endocrinology, Metabolism, and Reproduction (V.M., J.G.-A.), Quito, Ecuador

Address all correspondence and requests for reprints to: M. J. Khosravi, Ph.D., Diagnostic Systems Laboratories (Canada), Inc., Mount Sinai Hospital, Room 653, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5.

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.

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