Endocrinologists looking to quantify growth hormone in their patients frequently rely on laboratory tests for insulin-like growth factor-1 (IGF-I) and IGF-binding protein-3 (IGFBP-3). But getting accurate readings is tricky for a variety of reasons, including biological and both intra- and inter-assay variations. While efforts to push a universal standard for growth hormone and IGF-I immunoassays have made some progress, there is still room to improve, said laboratory experts. Labs also have a role to play in returning as robust results as possible and in better informing clinicians about the limitations of their methods and about interpreting test results.

IGF-I, a protein stimulated by growth hormone that plays an important role in growth during childhood, is primarily produced by the liver. It’s a useful marker for growth hormone deficiency or surplus because it is released fairly consistently, unlike growth hormone itself, which can fluctuate throughout the day, said Ravinder Singh, PhD, DABCC, director of the Mayo Clinic endocrine laboratory, in Rochester, Minnesota. Measuring serum IGF-I and IGFBP-3 concentrations helps assess short stature or gigantism and helps predict growth response in patients taking growth hormone therapy. Clinicians also monitor IGF-I levels to assess patients’ adherence to therapy and to ensure that doses remain in the therapeutic range.

But measuring IGF-I accurately has its challenges, said Martin Bidlingmaier, MD, head of the endocrine laboratory at the University of Munich, in Germany. For one, IGF-I binds not only to IGFBP-3 but also to acid labile subunit. One must separate out the IGF-I, he said, by adding an acid and then a factor like IGF-II to bind to the other proteins so as to measure free IGF-I. Measurement has to be done quickly, he added, as IGF-I will re-aggregate with its binding proteins within 20-30 minutes.

In addition, said Singh, the reference range for IGF-I has been evolving, now getting as specific as providing norms for patients at almost every year of age to reflect how dramatically IGF-I varies with age. Different assay manufacturers also may use different calibration materials, Singh added, and antibodies used for the assays may not be consistent, making it quite challenging to compare results from one assay to another. These nuances may not cause problems for one patient whose testing is performed consistently at one lab, but when patients move and their tests are run by different labs, the results could be vastly different.

In another twist, manufacturers should verify the reagents sent to clinical laboratories, but these agents may not be consistent from lot to lot. This happened at Mayo Clinic a few years back, when the lab was using a commercial IGF-I assay, said Alicia Algeciras-Schimnich, PhD, DABCC, chair of the medical center’s division of clinical biochemistry and immunology. She and her colleagues were noticing elevated IGF-I results in some patients that were considered false positives once they reviewed patients’ clinical data (Endocr Pract 2014;20:421-5). “The clinical picture was not consistent with growth hormone excess,” she said. “That created a challenge for us to try to figure out what was going on.”

Ultimately, they determined the problem to be related to inconsistent reagent lots. “It was an assay performance issue, not a patient or sample collection issue,” she said. Mayo Clinic subsequently switched to a lab-developed mass spectrometry assay to try to achieve better consistency over time (See Box).

These variances can go unnoticed by physicians, not all of whom are schooled in laboratory techniques, said David Clemmons, MD, Sarah Graham Kenan professor of medicine at the University of North Carolina at Chapel Hill. “They just get a number back, and if there’s interference or if there’s a lack of reproducibility, they really don’t know it,” he said. Inaccurate results could lead them to make errors in treatment, such as prescribing unnecessary therapies or improper doses.

Consensus on Assay Performance

Clemmons, along with Bidlingmaier and Algeciras-Schimnich, was part of a 2010 meeting of clinicians and clinical laboratory professionals convened by the Growth Hormone Research Society, the International Society for IGF Research, the International Federation of Clinical Chemistry and Lab Medicine, and the Pituitary Society to define criteria and strategies for harmonizing growth hormone and IGF-I assays. The group’s consensus statement recommended that manufacturers all adopt the same standard, and that all assays need appropriate, specific reference intervals (Clin Chem 2011;57:555-9).

Results are still a work in progress, said Bidlingmaier, although the proposal that all IGF-I assay manufacturers adopt a uniform, standard preparation has largely been achieved. Most assays are now calibrated against the most recent World Health Organization international reference preparation, 02/254. This in part led to better agreement among some assays, he noted.

In addition, Bidlingmaier and others, in 2014, published reference intervals for both IGF-I and IGFBP-3 based on a population of about 15,000 subjects for the IDS iSYS assay (J Clin Endocrinol Metab 99:1712-21 and 1675-86, respectively). “For the clinician, the key issue with IGF-I assays is to get appropriate reference intervals, or normative data to make a useful interpretation of the numbers you get. That’s a point where different assays have different quality to offer,” he observed.

Publication Requirements

In other updates, the Endocrine Society announced that it would no longer accept papers for publication in its journals unless growth hormone results were expressed in micrograms, said Catharine Sturgeon, PhD, FRCP, a clinical scientist at the Royal Infirmary of Edinburgh, in Scotland, and director of the U.K. National External Quality Assessment Service proficiency testing center. “That made a major difference because people had to state how their method had been calibrated, and to use those units, which meant that the manufacturers had to take notice. This changed things considerably in terms of bringing results closer together.”

This move has been important, she said, because when dealing with relatively small patient populations, “it’s highly desirable to be able to compare results across countries and studies.” Sturgeon also was a co-author of the consensus statement on growth hormone and IGF-I assays.

Detailed Reference Intervals

 As standardization efforts continue, clinical labs should take several steps to ensure the accuracy and solid interpretation of their IGF-I and IGFBP-3 test results. Bidlingmaier suggested providing clinicians with a very detailed, transparently established reference interval to help guide interpretation of test results. Some laboratory handbooks have reference intervals for IGF-I “which are absolutely insufficient in terms of the population investigated,” he noted—maybe testing just a few subjects with no appropriate age classification.

“You need a huge reference population to make these reference intervals reliable,” Bidlingmaier elaborated. “Many laboratories underestimate the importance of this point.” Labs should not simply rely on information provided in the manufacturer’s kit insert, he said. Ideally, there should be a publication providing detailed information on what population was investigated, including subjects’ ages, ethnicities, and genders.

In addition, laboratories should monitor long-term performance of their assays. “All assays come with quality control samples, but these in many cases are provided by the manufacturer so are not true serum samples,” Bidlingmaier said, and therefore do not ideally mimic clinical samples. Some companies provide quality control samples, but they have IGF-I spiked into an artificial matrix that does not contain all of the binding proteins, he added. Beyond these commercial controls, labs should measure patient serum samples daily.

“Prepare little aliquots from a large pool, which you put into the freezer, and every day you measure the same sample again and follow the results. This is the only way to get a better understanding of the performance of your method in real world samples,” said Bidlingmaier.

Laboratories also should participate in external quality assessment programs to compare their IGF-I results to those from other laboratories, ensure they calibrate their method against the most current reference standards, and evaluate the applicability of manufacturer-provided reference intervals to their patient populations. In addition, labs should inform physicians about the assay being used and be ready to confer with doctors about assay performance or other issues anytime a patient’s clinical presentation doesn’t jibe with test results.

“Reference laboratories are pretty well aware of these issues—it’s not like they’re mysterious,” Clemmons said. “But the extent to which they apply the principles on a daily basis can fluctuate. You have to keep readdressing the issue to make sure that there is reproducibility.”

Karen Blum is a freelance medical/science writer in Owings Mills, Maryland. +Email: karen_blum[at]verizon.net.