Review article

Interferences in hormone immunoassays

The accurate interpretation of the cosyntropin (adrenocorticotropic hormone [ACTH]) stimulation test requires method- and assay-specific cutoffs of the level of cortisol. Compared with a historical cutoff (18 μg/dL) for polyclonal antibody-based immunoassays, lower thresholds were proposed for the Roche Elecsys II assay, which uses a monoclonal antibody. However, cutoffs for other commonly adopted, monoclonal antibody-based cortisol assays were not yet available. Here, we established the thresholds for the level of cortisol specific to the Abbott Architect immunoassay by comparing the measurements of the level of cortisol using 3 immunoassays.

The ACTH stimulation test was performed in patients with suspected adrenal insufficiency (n = 50). The serum cortisol level was measured using the Abbott Architect, Roche Elecsys II, and Siemens Centaur assays. The results of the Abbott assay were also compared with those of liquid chromatography-tandem mass spectrometry. The receiver operating characteristic analysis was performed to derive new diagnostic thresholds for the Abbott assay using the polyclonal antibody-based Siemens assay as the reference method.

The concentrations of cortisol measured using the Abbott assay were similar to those measured using liquid chromatography-tandem mass spectrometry and the Roche Elecsys II assay but significantly lower than those measured using the Siemens assay. The optimized threshold for cortisol using the Abbott assay was 14.6 μg/dL at 60 minutes after stimulation (sensitivity, 92%; specificity, 96%) and 13.2 μg/dL at 30 minutes after stimulation (sensitivity, 100%; specificity, 89%).

We recommend a threshold of 14.6 μg/dL for the level of cortisol at 60 minutes after ACTH stimulation for the Abbott assay. In comparison with the historical threshold of 18 μg/dL, the application of the new cutoff may significantly decrease false-positive results due to ACTH stimulation testing. The use of assay-specific cutoffs will be essential for reducing misclassification and overtreatment in patients with suspected adrenal insufficiency.

Progesterone is one of the female steroid hormones and plays an important role in the menstrual cycle and during pregnancy. It is especially important in preparing the uterus for the implantation of the blastocyst and maintaining pregnancy. The concentration in human serum is measured to determine the ovarian function retroactively and the cause of abortion in early pregnancy.

A quantification assay based on isotope dilution mass spectrometry to determine the concentration of progesterone in human serum is reported. Incorporated with ¹³C₃-progesterone, serum samples were subjected to progesterone extraction and clean-up by C4 solid-phase-extraction columns and hexane-based liquid/liquid extraction, respectively. The cleaned-up serum samples were then subjected to MALDI-TOF mass spectrometry for the quantification of progesterone.

Progesterone and the internal standard, ¹³C₃-progesterone, were measured in the selected reaction monitoring mode for the transitions m/z 315.4 to 108.9 and m/z 318.4 to 111.9, respectively. We calculated the peak area ratio of progesterone to ¹³C₃-progesterone. The progesterone concentration in human serum was calculated by substituting the peak area ratio into an isotope dilution calibration curve, and then compared with the radioimmunoassay.

In the study, the concentrations of serum progesterone were measured, and the recovered progesterone concentration determined by the assay showed good robustness and consistency in comparison to the conventional radioimmunologic assay. We concluded that the ¹³C₃-progesterone-based quantification assay is a robust method for the measurement of serum progesterone.

This chapter discusses the basic knowledge of laboratory medicine that helps a pediatric endocrinologist make better test-ordering decisions, avoid interpretation errors, and make the best decisions for their patients. Knowing some of the “language of the lab” will allow for better discussions with laboratory experts and makes it easier to understand relevant publications from the clinical chemistry community. The statistics behind diagnostic decision-making, understanding how tests are validated for quality purposes, and understanding the various methods available (including the limitations and possible sources of error) are also discussed. An additional goal of this chapter is to reveal some of the uncertainties involved with laboratory testing, to assuage some of the frustration experienced when one sees widely different results for the same test done at different laboratories, or to realize the current gaps in reference interval information.

Sources of error in laboratory tests include preanalytical, analytical, and postanalytical factors that potentially influence the integrity or interpretation of a test result. Immunoassays that are used to measure many of the endocrine biomarkers such as hormones, binding proteins, and metabolic products, are particularly susceptible to analytical interferences due to the limited specificity of antibody–antigen interactions. Endogenous substances such as lipids and bilirubin may interfere with laboratory tests when present at unusually high concentrations. Hemoglobin is a common interferent that leaks into the noncellular fraction of blood when red blood cells are disrupted during collection or storage of a blood specimen. Dietary supplements such as biotin can sometimes interfere with laboratory tests when present at high enough concentrations. Finally, the proper interpretation of a laboratory test result requires some knowledge of the sensitivity and specificity of the test, the predictive value of positive results, and the inherent biological variability of the biomarker the test measures. This chapter discusses the most common sources of error in laboratory tests used in diagnostic endocrinology. Avoidance of these sources of error maximizes the value of laboratory tests.

The concentrations of a variety of hormones are measured in the clinical laboratory typically by using immunoassays. These techniques are known to be vulnerable to a number of interferences, for example, heterophilic antibodies and macro-complexes, to name a few. In addition, there are other factors that make these measurements challenging, such as the existence of multiple isoforms of a single hormone and supplemental dietary ingestion of biotin which is a component of many immunoassay systems. This chapter reviews the interferences and analytical difficulties in measuring hormones using immunoassays.

Insulin glargine 300 U/mL and insulin detemir are synthetic long-acting insulin analogs associated with minimal day-to-day variability or episodes of hypoglycemia in people. Here, 8 healthy purpose-bred dogs each received 2.4 nmol/kg subcutaneous injections of insulin detemir (0.1 U/kg) and insulin glargine 300 U/mL (0.4 U/kg) on 2 different days, >1 wk apart, in random order. Blood glucose (BG) was measured every 5 min, and glucose was administered intravenously at a variable rate with the goal of maintaining BG within 10% of baseline BG (“isoglycemic clamp”). Endogenous and exogenous insulin were measured for up to 24 h after insulin injection. The effect of exogenous insulin was defined by glucose infusion rate or a decline in endogenous insulin. Isoglycemic clamps were generated in all 8 dogs after detemir but only in 4 dogs after glargine. Median time to onset of action was delayed with glargine compared to detemir (4.0 h [3.3–5.8 h] vs 0.6 h [0.6–1.2 h], P = 0.002). There was no difference in time to peak (median [range] = 6.3 h [5.0–21.3 h] vs 4.3 h [2.9–7.4 h], P = 0.15) or duration of action (16.3 h [6.1–20.1 h] vs 10.8 h [8.8–14.8 h], P = 0.21) between glargine and detemir, respectively. Glargine demonstrated a peakless time-action profile in 4/8 dogs. The total metabolic effect and peak action of detemir was significantly greater than glargine. Significant concentrations of glargine were detected in all but 1 dog following administration. Glargine might be better suited than detemir as a once-daily insulin formulation in some dogs based on its long duration of action and peakless time-action profile. Day-to-day variability in insulin action should be further assessed for both formulations.