What methodologies are available for insulin-like growth factor 1 (IGF-1) quantitation?

Most clinical laboratories use commercially available immunoassays for IGF-1 quantitation, but some large reference laboratories now also offer high-resolution accurate-mass mass spectrometry (HRAM-MS) assays for this biomarker, which is widely used to diagnose growth disorders.

What are the drawbacks of IGF-1 immunoassays?

IGF-1 circulates as part of a ternary complex with insulin-like growth factor binding protein 3 (IGFBP3) or other members of the IGFBP family and acid-labile subunit. Immunoassays for IGF-1 involve an extraction using an acidified ethanol buffer followed by centrifugation wherein the acidic solution facilitates dissociation of IGF-1 from IGFBPs. The ethanol then acts as a precipitating agent, enabling unbound IGF-1 to be quantified in the supernatant. However, interference from residual IGFBPs can yield falsely low or high results depending on whether the assay format is competitive or immunometric. Another challenge with IGF-1 immunoassays is the poor correlation among various methods, which is likely due to differential cross-reactivity of the proprietary reagent antibodies with IGF-1.

What are the advantages of quantitating IGF-1 using HRAM-MS compared to immunoassays?

The HRAM-MS assay for IGF-1 uses the same precipitation and acidification method used in immunoassays followed by separation on a liquid chromatography column and analysis on a HRAM-MS platform. The method quantifies the intact, mature IGF-1 (7,649 Da) protein using a specific mass-to-charge ratio (m/z) at high resolution and low mass error, enabling separation from interfering peaks and a narrow peak extraction, respectively. The combination of high resolution (approximately 40,000 FWHM) and a narrow mass extraction window (approximately 5–10 ppm) afforded by these platforms accounts for the high specificity of this assay. As a result, the HRAM-MS assay for IGF-1 is devoid of the endogenous interferences commonly seen in IGF-1 immunoassays and can be standardized across clinical laboratories.

This is especially important for patients with mutations in the IGF1 gene. Studies have shown that IGF-1 immunoassay results can differ depending on the cross reactivity of the IGF-1 polymorphic/mutant form with the reagent antibodies. Additionally, a research team I was part of at the Mayo Clinic in Rochester, Minnesota observed that certain immunoassays fail to discriminate between wild type and a polymorphic form of IGF-1 (Clin Chem 2015;61:990–1). Using the change in mass-to-charge ratio (∆m/z) of the polymorphic IGF-1 compared with the wild type IGF-1 on our HRAM-MS assay along with confirmatory molecular evidence, we demonstrated the presence of an IGF-1 single nucleotide polymorphism (SNP) causing an amino acid change from alanine to threonine (Δm/z ~4.2 Da) at the 70th position in the mature IGF-1 protein. Due to the presence of this heterozygous SNP, in 0.5% of clinical samples, the value of the HRAM-MS IGF-1 result was half that of the immunoassay result for the same sample. While the clinical impact of this SNP is currently unknown, this observation opens up the possibility and technical feasibility of identifying patients with deleterious IGF-1 mutations using HRAM-MS.

Do you think investing in mass spectrometry is a good strategy?

The sensitivity offered by mass spectrometers is constantly improving, circumventing the need for expensive consumables and reagents (like antibodies) for quantitation of a variety of analytes. HRAM-MS is useful for qualitative and quantitative multi-drug analysis, steroid profiling, and in proteomic applications, which most hospitals want to implement in-house to reduce financial burden on their clinical laboratories. While implementing technical innovations like HRAM-MS presents a daunting initial capital investment burden, in the long run it offers what we really need in today’s environment—sustainable, long-term clinical benefit and value. What we learned from the IGF-1 HRAM-MS story is obviously just the beginning. We are moving towards an integrated biochemical-proteomic-genomic clinical practice and from a technical and analytical standpoint, mass spectrometry is on the leading edge.

Hemamalini KethaHemamalini Ketha, PhD, is director of toxicology and drug analysis and associate director of clinical chemistry at the University of Michigan, Ann Arbor. +Email: [email protected]