Low or increased serum insulin-like growth factor 1 (IGF-1)1 concentrations might indicate growth hormone (GH) deficiency or overproduction, respectively. The rate of false positives is also a big concern by the clinicians with the current reference ranges. Currently, IGF-1 is mostly measured by automated immunometric assays. However, reagent availability issues prompted us to evaluate LC-MS with high-resolution accurate mass measurement (HRAM) as an alternative method.
Objectives: Validation of mass spec assay for IGF-1 and expanding its use in detecting pathological and non pathological mutations.
For all samples and QCs, we added 400 μL of 7:1 ethanol/1 N HCl to 100 μL of human serum and 25 μL of internal standard to precipitate excess proteins and dissociate IGF-1 from binding proteins. Following 30 min incubation at room temperature, 90 μL of 1.5 M Tris was added to neutralize the solution, which was then centrifuged at 3000 rpm for 10 min. This was followed by 30 min incubation at −20 °C and centrifugation for 10 min at 3000 rpm. The resulting supernatant then underwent LC-MS analysis. Data collection, clinical correlation, and interpretation of thousands of samples have been performed in the lab.
We have identified common variants of IGF-1, A70T-IGF1 and A67T-IGF1, of unknown clinical significance by our new LC-MS HRAM assay, which cannot be distinguished from wild-type by the current market-leading immunoassay. Our method has also identified V44M pathogenic mutations in a few patients. One reason for doing this is that a “missed” pathogenic variant would give the clinically correct result for the wrong reason, which might result in a failed opportunity for definitive genetic family testing. The other reason is that several of the variants are either nonpathogenic or of uncertain significance, leading to a potentially falsely low IGF-1 result being reported.
When known pathogenic mutations exist or discordant results with established immunoassays are discovered, variant protein sequences should be considered and evaluated. When mass spectrometric assays give unexpectedly low results, one should contemplate the possibility of the presence of variants and scan for them.