A Review on Challenges of Insulin-Like Growth Factor-1 Testing

Rongrong Huang, PhD, DABCC, Director of Clinical Chemistry, Harris Health Ben Taub Hospital; Assistant Professor, Department of Pathology and Immunology, Baylor College of Medicine

Junyan Shi, PhD, DABCC, FCACB, Clinical Chemist, Vancouver General Hospital, Vancouver Coastal Health; Clinical Assistant Professor, Department of Pathology and Laboratory Medicine, University of British Columbia, Canada

Ruhan Wei, PhD, DABCC, Director of Duke Health Clinical Laboratories Clinical Chemistry and Duke Central Automated Laboratory; Assistant Professor of Duke University School of Medicine

Jieli Shirley Li, MD, PhD, DABCC, NRCC, FCACB, Co-Director of Clinical Chemistry and Toxicology Laboratory, Wexner Medical Center; Associate Professor of Pathology, the Ohio State University

In a recent article published in Critical Reviews in Clinical Laboratory Sciences, Huang et al. provide a comprehensive overview of the current status of insulin-like growth factor 1 (IGF-1) testing, focusing on three key areas: pre-analytical variability, disparities among different analytical methods, and variations in reference range determination.

IGF-1, primarily synthesized in the liver, was initially discovered for its ability to mimic the metabolic effects of insulin. It later emerged as a key regulator of growth hormone (GH) actions, influencing critical processes such as cell proliferation, differentiation, and apoptosis. With a longer half-life than GH, IGF-1 is less affected by factors that influence GH levels, making its measurement more specific and sensitive for diagnosing conditions like acromegaly and GH deficiency.

Pre-analytical factors, such as sample handling, timing of blood collection, and storage conditions, significantly impact IGF-1 measurements. Huang et al. emphasize the importance of consistent phlebotomy practices, including fasting blood collection and prompt processing, to reduce variability. Improper handling, such as delayed processing or repeated freeze-thaw cycles, can lead to altered IGF-1 levels, compromising the accuracy of results.

Immunoassays have traditionally been the predominant method for IGF-1 testing but face challenges related to interference from IGF-binding proteins (IGFBPs). These proteins can hinder the accurate measurement of IGF-1 by immunoassays, leading to variability in test results. To address this issue, techniques such as acid-ethanol extraction and the use of IGF-2 have been introduced to dissociate IGFBPs, but these methods are not always completely effective.

To overcome the limitations of immunoassays, modern mass spectrometry-based methods have been developed. These methods offer more robust, reliable, and accurate IGF-1 quantification by minimizing IGFBP interference and improving precision. Two approaches are used: the "bottom-up" method, which involves enzymatic digestion of IGF-1 followed by peptide detection using a triple quadrupole mass spectrometer, and the "top-down" approach, which detects intact IGF-1 using high-resolution mass spectrometry. The latter also allows for the detection of pathogenic mutations through protein sequence analysis. In a study of 243,808 patient samples, 1,099 samples containing 14 IGF-1 variants were identified, demonstrating the value of mass spectrometric techniques.

Despite advances in analytical methods, discrepancies in IGF-1 reference intervals remain a significant challenge. Establishing accurate reference intervals for each assay is complex, requiring collaboration among laboratorians, clinicians, and manufacturers. A nationwide multicenter effort is needed to standardize IGF-1 assays in a cost-effective and resource-efficient manner.

This review provides an in-depth examination of the ongoing challenges related to IGF-1 testing, including the need for improved standardization of measurement methods, mitigation of pre-analytical variability, and harmonization of reference intervals. (1–4)

胰岛素样生长因子-1 检测全面综述

近期在《临床实验室科学评论》期刊上发表的一篇文章中，黄博士等人对胰岛素样生长因子1（IGF-1）检测的现状进行了全面的综述，重点关注了三个关键领域：分析前误差多样性、不同的分析方法之间以及参考区间建立上导致的差异。

IGF-1 主要在肝脏合成，最初发现其能够模拟胰岛素的代谢作用。后来发现IGF-1 作为生长激素（GH）的关键调节因子，能够调控细胞增殖、分化和凋亡等关键过程。值得注意的是，与生长激素相比，IGF-1 的半衰期更长，因此不易受到干扰生长激素水平的因素的影响。因此，在诊断肢端肥大症或生长激素缺乏症等疾病时，IGF-1 的检测更具特异性和敏感性。

分析前因素如样本处理、采血时间和样本存储条件都会显著影响 IGF-1检测。黄博士等人强调，采血操作一致性，包括空腹采血和样本及时处理，对减少测量变异性至关重要。样本不当处理（如处理延迟或多次解冻）会影响 IGF-1 结果的准确性。

免疫测定法一直是 IGF-1 检测的主要方法，但这种方法可能会被胰岛素样生长因子结合蛋白（IGFBP）干扰。这些蛋白可能会妨碍免疫测定法对 IGF-1 的准确测量，会影响 IGF-1 结果的准确性。要想解决这个问题，可以用酸乙醇提取和使用 IGF-2 等技术来分离 IGFBP，但这些方法并非总是完全有用。

为了克服免疫测定法的局限性，现代质谱法应运而生。这些方法通过减少 IGFBP 干扰并提高其精确度，提供了更稳定、可靠和准确的 IGF-1 定量分析。质谱法有两种方法：一种是 “自下而上” 的方法，通过酶切 IGF-1 并用三重四极杆质谱仪检测肽段；另一种是 “自上而下” 的方法，使用高分辨率质谱仪检测完整的 IGF-1 分子。后者还可以通过蛋白质序列分析检测致病突变。在对243,808个患者样本的研究中，质谱技术可以识别出1,099个包含14种 IGF-1 变异的样本，显示出其临床应用价值。

尽管分析方法上取得了进展，但 IGF-1 参考范围的不一致仍然是一个重大难题，可能导致对结果的不同解释。每个检测方法建立准确的参考范围非常复杂，需要实验室、临床医生和检测方法制造商在全国范围内开展多中心合作，以经济高效和节约资源的方式实现这一共同目标。该综述深入探讨了 IGF-1 检测中面临的持续挑战，包括需要把 IGF-1 测量方法标准化、减少分析前差错以及规范参考区间的设定。

References

1.         Huang R, Shi J, Wei R, Li J. Challenges of insulin-like growth factor-1 testing. Critical Reviews in Clinical Laboratory Sciences. 2024;1–16.

2.         Bystrom CE, Sheng S, Clarke NJ. Narrow Mass Extraction of Time-of-Flight Data for Quantitative Analysis of Proteins: Determination of Insulin-Like Growth Factor-1. Anal Chem. 2011;83:9005–10.

3.         Motorykin I, Li H, Clarke NJ, McPhaul MJ, Wu Z. Isotopic Peak Index, Relative Retention Time, and Tandem MS for Automated High Throughput IGF-1 Variants Identification in a Clinical Laboratory. Anal Chem. 2021;93:11836–42.

4.         Motorykin I, Mu J, Miller BS, Li A, Clarke NJ, McPhaul MJ, et al. Detection rate of IGF-1 variants and their implication to protein binding: study of over 240,000 patients. Clinical Chemistry and Laboratory Medicine (CCLM). 2024;62:484–92.