Hormonal Assays in Endocrinology: What the Internist Must Know

Dr Neeraj Manikath , claude.ai

Abstract

Hormonal assays form the cornerstone of endocrine diagnosis, yet their interpretation remains one of the most challenging aspects of clinical practice. This comprehensive review addresses the fundamental principles of hormone measurement, common pitfalls in interpretation, and practical approaches to navigate the complexities of modern endocrine testing. We explore preanalytical variables, assay methodologies, interference phenomena, and the critical importance of understanding reference ranges in their physiological context. Through evidence-based recommendations and clinical pearls, this article equips the practicing internist with the knowledge to confidently order, interpret, and act upon hormonal investigations.

Introduction

The endocrine system orchestrates metabolic homeostasis through precisely regulated hormonal signals, and laboratory assessment of these hormones has become increasingly sophisticated. However, with this sophistication comes complexity. Modern hormone assays employ diverse methodologies including immunoassays, mass spectrometry, and bioassays, each with distinct advantages and limitations. The clinician must navigate not only the selection of appropriate tests but also the interpretation of results in the context of circadian rhythms, pulsatile secretion patterns, binding proteins, and potential analytical interferences.

Studies suggest that inappropriate use of hormonal testing contributes significantly to healthcare costs, with estimates indicating that 20-30% of endocrine laboratory tests may be unnecessary or incorrectly ordered[1,2]. More critically, misinterpretation of hormonal assays can lead to misdiagnosis, inappropriate treatment, and patient harm. This review synthesizes current evidence to provide practical guidance for the internist encountering endocrine problems in daily practice.

Fundamental Principles of Hormone Measurement

Understanding Assay Methodologies

Immunoassays remain the workhorse of clinical hormone measurement. These include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), and electrochemiluminescent immunoassay (ECLIA). All rely on antibody-antigen interactions, which confer excellent sensitivity but introduce vulnerability to cross-reactivity and interference[3].

Mass spectrometry, particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS), has emerged as the gold standard for many hormone measurements. This technique offers superior specificity by measuring molecular mass rather than relying on antibody recognition. It excels in measuring steroids, catecholamines, and other small molecules but requires expensive equipment and specialized expertise[4].

Clinical Pearl: When testosterone levels seem discordant with clinical presentation in women or children, request LC-MS/MS measurement rather than immunoassay, as the latter frequently produces inaccurate results at low concentrations[5].

The Critical Importance of Preanalytical Variables

Preanalytical factors account for up to 70% of laboratory errors[6]. For hormonal assays, these variables are particularly consequential.

Timing of Sample Collection: Many hormones exhibit marked circadian variation. Cortisol peaks at 8 AM and reaches nadir around midnight. Growth hormone pulses throughout the day with peak secretion during sleep. Testosterone in men is highest in early morning, with levels declining 20-30% by afternoon[7]. Failing to standardize collection time can render results uninterpretable.

Pulsatile Secretion: Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and growth hormone are secreted in pulses. A single random measurement may miss the diagnosis entirely. For gonadotropins, sampling should ideally occur during the follicular phase in premenopausal women, and multiple samples may be needed to confirm hypogonadotropic hypogonadism[8].

Patient Preparation: Stress, exercise, fasting status, and body position all influence hormone levels. Growth hormone and cortisol rise with stress. Renin and aldosterone are markedly affected by posture, sodium intake, and antihypertensive medications. Patients should be seated for 5-10 minutes before blood draw for renin-aldosterone measurements[9].

Oyster: A stressed patient in the emergency department with acute illness will have elevated cortisol. This does not represent Cushing syndrome but rather an appropriate stress response. Random cortisol measurements are rarely useful except to exclude adrenal insufficiency when levels are frankly elevated (>18-20 μg/dL)[10].

Specific Hormone Systems: Practical Approaches

Thyroid Function Testing

Thyroid-stimulating hormone (TSH) remains the best initial screening test for thyroid dysfunction due to the logarithmic relationship between TSH and free thyroxine (FT4). A small change in FT4 produces a large change in TSH, making TSH exquisitely sensitive[11].

Interpreting TSH in Context: Population reference ranges (typically 0.4-4.5 mIU/L) represent 95% confidence intervals from presumably healthy individuals. However, individual set points are narrower. A patient whose baseline TSH is 1.0 mIU/L who now measures 3.5 mIU/L may be hypothyroid despite a "normal" result[12].

Free T4 and T3 Measurement: Approximately 99.97% of circulating thyroid hormone is protein-bound, primarily to thyroxine-binding globulin (TBG). Only free hormone is biologically active. Most laboratories use immunoassay platforms that estimate free hormone levels but are subject to interference from altered binding protein concentrations, severe illness, and certain medications[13].

Clinical Pearl: In pregnancy, hospitalized patients, or those taking medications affecting protein binding (estrogen, androgens, carbamazepine, phenytoin), FT4 results may be spuriously abnormal. Consider equilibrium dialysis or ultrafiltration methods when results are discordant with clinical picture[14].

The Phenomenon of Non-Thyroidal Illness Syndrome (NTIS): Severe systemic illness suppresses TSH and thyroid hormone levels through multiple mechanisms. TSH may be low-normal or slightly suppressed, T3 is typically low, and FT4 may be normal, low, or occasionally elevated. This represents an adaptive response, not primary thyroid disease. Avoid thyroid function testing in acutely ill hospitalized patients unless there is strong clinical suspicion of thyroid disease[15].

Hack: If thyroid function tests are obtained during acute illness and show abnormalities, repeat testing 4-6 weeks after recovery rather than initiating treatment based on results obtained during the acute phase.

Adrenal Axis Assessment

Screening for Cushing Syndrome: Three first-line tests are recommended: 24-hour urinary free cortisol, late-night salivary cortisol, and low-dose dexamethasone suppression test (DST). Each has approximately 90-95% sensitivity when used appropriately[16].

The 1-mg overnight DST involves administering 1 mg dexamethasone at 11 PM and measuring cortisol at 8 AM the next morning. Cortisol <1.8 μg/dL effectively excludes Cushing syndrome in most patients. However, false positives occur with medications inducing hepatic metabolism (phenytoin, rifampin, carbamazepine) and in conditions increasing cortisol-binding globulin[17].

Oyster: Obesity, depression, alcoholism, and poorly controlled diabetes can cause "pseudo-Cushing syndrome" with positive screening tests. If screening tests are positive but clinical suspicion is moderate, consider dexamethasone-CRH testing or bilateral inferior petrosal sinus sampling in expert centers[18].

Evaluating Adrenal Insufficiency: Morning cortisol <3 μg/dL strongly suggests adrenal insufficiency, while >15 μg/dL effectively excludes it. Values between 3-15 μg/dL require dynamic testing with ACTH stimulation[19].

The standard ACTH stimulation test uses 250 μg cosyntropin with cortisol measurement at 0 and 30-60 minutes. Peak cortisol >18-20 μg/dL (laboratory-dependent) excludes primary adrenal insufficiency. However, this test may not detect early or secondary adrenal insufficiency. The low-dose (1 μg) ACTH test may be more sensitive for secondary insufficiency but lacks standardization[20].

Clinical Pearl: If secondary adrenal insufficiency is suspected (pituitary disease, recent glucocorticoid withdrawal), measure both cortisol and ACTH simultaneously. Low cortisol with inappropriately low or normal ACTH confirms the diagnosis. The ACTH stimulation test may be normal in early secondary insufficiency because the adrenal glands have not yet atrophied[21].

Reproductive Hormones

Testosterone in Men: Total testosterone measurement is appropriate for most clinical situations, but interpretation requires understanding of binding proteins. Sex hormone-binding globulin (SHBG) binds approximately 60% of testosterone, albumin binds 38%, and only 2% circulates free. Conditions increasing SHBG (aging, hyperthyroidism, liver disease, HIV infection) elevate total testosterone without increasing bioavailable hormone[22].

Hack: When total testosterone is borderline low (250-350 ng/dL) but clinical hypogonadism is suspected, calculate free testosterone using the Vermeulen equation or measure free testosterone directly by equilibrium dialysis. Bioavailable testosterone can also be measured after precipitation of SHBG-bound hormone[23].

Oyster: Obesity suppresses SHBG, potentially causing total testosterone to appear low while free testosterone remains normal. Conversely, in elderly men with high SHBG, total testosterone may be normal despite true androgen deficiency[24].

Assessing Ovarian Function: In reproductive-age women, hormone interpretation must account for menstrual cycle phase. FSH and LH are lowest during the luteal phase and rise during the follicular phase. Early follicular phase (days 2-4) FSH >10-12 IU/L suggests diminished ovarian reserve[25].

Anti-Müllerian hormone (AMH) has emerged as a cycle-independent marker of ovarian reserve. Unlike FSH, AMH can be measured at any time during the cycle. Low AMH (<1.0 ng/mL) indicates decreased ovarian reserve, while high levels (>5 ng/mL) may suggest polycystic ovary syndrome[26].

Prolactin Measurement: Prolactin is stress-sensitive and exhibits pulsatile secretion. Mild elevations (25-100 ng/mL) are often physiological or due to medications (antipsychotics, metoclopramide, verapamil). Levels >200 ng/mL strongly suggest prolactinoma[27].

Clinical Pearl: Macroprolactin, a complex of prolactin and IgG, is biologically inactive but detected by immunoassays, causing falsely elevated prolactin without clinical consequences. If prolactin is elevated but clinical features are absent, request macroprolactin screening by polyethylene glycol precipitation[28].

Parathyroid Hormone and Calcium Homeostasis

Intact parathyroid hormone (PTH) measurement has revolutionized the diagnosis of calcium disorders. PTH should always be interpreted in the context of simultaneous calcium and vitamin D levels[29].

Hypercalcemia with elevated or inappropriately normal PTH indicates primary hyperparathyroidism. PTH should be suppressed when calcium is elevated; failure to suppress is abnormal even if PTH is within reference range[30].

Hypercalcemia with suppressed PTH suggests malignancy-associated hypercalcemia (PTHrP-mediated or humoral), vitamin D intoxication, granulomatous disease, or other non-PTH-mediated causes[31].

Hypocalcemia with low PTH indicates hypoparathyroidism. With appropriate gland function, PTH should be markedly elevated in hypocalcemia[32].

Oyster: Vitamin D deficiency causes secondary hyperparathyroidism with elevated PTH but normal or low calcium. This is extremely common and should not be confused with primary hyperparathyroidism. Measure 25-hydroxyvitamin D in all patients with elevated PTH[33].

Hack: When evaluating chronic kidney disease patients, use second- or third-generation PTH assays that measure only full-length PTH rather than inactive fragments that accumulate in renal failure. Target PTH levels differ from those in the general population[34].

Growth Hormone and IGF-1

Growth hormone (GH) measurement is fraught with difficulty due to pulsatile secretion and short half-life. Random GH levels are almost never useful. Diagnosis of GH excess or deficiency requires dynamic testing[35].

IGF-1 (Insulin-like Growth Factor-1) provides an integrated measure of GH secretion over 24 hours and is the appropriate screening test for acromegaly. IGF-1 levels must be interpreted using age-specific reference ranges, as levels decline with aging[36].

Clinical Pearl: An elevated age-adjusted IGF-1 has excellent specificity for acromegaly and should prompt oral glucose tolerance testing with GH measurement. GH failing to suppress below 1 μg/L (or 0.4 μg/L with ultrasensitive assays) confirms the diagnosis[37].

Diagnosing GH Deficiency in Adults: IGF-1 below the age-adjusted reference range suggests but does not confirm GH deficiency. Stimulation testing with insulin tolerance test, glucagon, or GHRH-arginine is required. These tests carry risks and should be performed in experienced centers[38].

Common Pitfalls and Interference Phenomena

Heterophile Antibodies

Human anti-mouse antibodies (HAMA), human anti-rabbit antibodies, rheumatoid factor, and other heterophile antibodies can interfere with immunoassays, causing falsely elevated or decreased results. This interference is particularly problematic for TSH, prolactin, hCG, and tumor markers[39].

Hack: Suspect heterophile interference when results are grossly discordant with clinical picture or when multiple hormone levels from the same assay platform are simultaneously abnormal. Serial dilution studies showing non-linear recovery or measuring the same hormone using a different assay platform can confirm interference[40].

Biotin Interference

Biotin (vitamin B7) is widely used as a supplement, often at high doses for hair and nail health. Many immunoassay platforms use biotin-streptavidin binding in their methodology. Pharmacologic biotin doses can cause falsely low TSH, falsely elevated free T4, and spurious results in other hormone assays[41].

Clinical Pearl: Ask all patients about supplement use, specifically biotin. If high-dose biotin (>5 mg daily) is being taken, discontinue for at least 48-72 hours before testing. Consider this interference when thyrotoxicosis is suggested by laboratory results but clinical features are absent[42].

Hook Effect

In some immunoassays, extremely high analyte concentrations can produce falsely low results because excess antigen saturates both capture and detection antibodies, preventing sandwich formation. This "high-dose hook effect" has been reported with prolactin, hCG, ferritin, and other hormones[43].

Oyster: A patient with a large pituitary macroadenoma but only mildly elevated prolactin (40-60 ng/mL) should raise suspicion for hook effect. Request serial dilution or alternative assay methodology. True prolactinomas of this size typically produce prolactin levels >200 ng/mL[44].

Special Populations

Pregnancy

Pregnancy profoundly alters hormone physiology and laboratory reference ranges. TSH decreases in the first trimester due to hCG cross-reactivity with the TSH receptor. Trimester-specific reference ranges should be used, with first-trimester upper limit typically around 2.5 mIU/L[45].

Free T4 assays are unreliable in pregnancy due to altered binding proteins and should be interpreted cautiously. Total T4 measurement or pregnancy-specific FT4 reference ranges are preferred[46].

Clinical Pearl: Prolactin increases progressively during pregnancy, reaching 10-fold baseline by term. Elevated prolactin in pregnancy is expected and does not indicate pathology. Avoid measuring prolactin during pregnancy unless investigating suspected lymphocytic hypophysitis[47].

Obesity

Obesity affects multiple hormone axes. Leptin resistance, altered sex hormone metabolism, insulin resistance, and changes in binding protein concentrations complicate interpretation. Total testosterone may be low in obese men despite normal free testosterone due to suppressed SHBG[48].

Growth hormone secretion is reduced in obesity, and IGF-1 may be low-normal, complicating acromegaly screening. Conversely, 24-hour urinary free cortisol can be elevated in obesity without true Cushing syndrome[49].

Critical Illness

As mentioned with NTIS, critical illness causes widespread endocrine dysregulation. The hypothalamic-pituitary-adrenal axis may be suppressed or activated. Thyroid function, growth hormone, and reproductive hormone axes are all affected. Hormone testing should generally be avoided during acute illness unless endocrine crisis is suspected[50].

Practical Approach to Common Clinical Scenarios

Approach to Unexpected Abnormal Results

When hormone levels are unexpectedly abnormal, follow this systematic approach:

1. Verify appropriate test selection: Was the correct test ordered for the clinical question?
2. Review preanalytical factors: Was timing appropriate? Was patient preparation adequate?
3. Consider physiological variations: Could stress, exercise, meals, or medications explain the result?
4. Evaluate for interference: Could heterophile antibodies, biotin, or other substances interfere?
5. Assess clinical correlation: Do results match clinical presentation?
6. Consider repeat testing: Use alternative methodology or different laboratory if discordance persists[51].

Building a Differential Diagnosis

Hormone results should be integrated with clinical assessment rather than interpreted in isolation. A mildly elevated TSH with normal FT4 in an asymptomatic patient may represent subclinical hypothyroidism, but could also reflect recovery from non-thyroidal illness, assay interference, or inter-individual variation[52].

Hack: Create a 2×2 table comparing clinical suspicion (high vs low) with test results (positive vs negative). High clinical suspicion with negative test should prompt consideration of false negative (wrong test, timing issue, or assay problem). Low clinical suspicion with positive test suggests false positive (interference, physiological variation, or incidental finding)[53].

Quality Assurance and Laboratory Communication

Understanding Laboratory Reports

Modern laboratory reports include reference ranges, but these are population-based 95% confidence intervals. They do not define health vs. disease for individual patients. A result just outside the reference range may be normal for that individual, while a result within range may be pathological if it represents significant change from baseline[54].

Clinical Pearl: Reference ranges vary between laboratories and assay methods. When comparing serial measurements, ensure they were performed by the same laboratory using the same methodology. If the laboratory changes methodology, new baseline measurements may be needed[55].

When to Consult Endocrinology

Complex hormone interpretation often benefits from specialist input. Consider endocrinology consultation for:

• Discordant results that remain unexplained after systematic evaluation
• Dynamic testing for Cushing syndrome, GH disorders, or adrenal insufficiency
• Management of patients on complex hormone replacement
• Evaluation of incidental pituitary lesions
• Polypharmacy patients where medication interactions complicate interpretation[56]

Future Directions

The field of hormone measurement continues to evolve. Point-of-care testing may enable real-time hormone measurement in office settings. Advances in mass spectrometry are improving detection of low-abundance hormones and hormone metabolites. Artificial intelligence algorithms may enhance interpretation by integrating multiple variables[57].

Liquid biopsy techniques measuring circulating tumor DNA, microRNA, and exosomal biomarkers may complement traditional hormone assays in detecting endocrine malignancies. Continuous glucose monitoring has already revolutionized diabetes management; similar continuous hormone monitoring technologies are under development[58].

Conclusion

Hormonal assays are powerful diagnostic tools that require nuanced interpretation. The internist must understand not only normal physiology and disease processes but also the technical aspects of hormone measurement, preanalytical variables, and potential sources of error. By applying the principles outlined in this review, clinicians can optimize test selection, avoid common pitfalls, and confidently interpret results in their clinical context.

Remember that laboratory results are not diagnoses but data points that must be synthesized with history, physical examination, and clinical judgment. When results are unexpected or discordant, systematic evaluation usually reveals the explanation. Finally, do not hesitate to communicate directly with laboratory personnel or consult endocrinology colleagues when interpretation is challenging.

The art of clinical endocrinology lies in recognizing that normal ranges represent populations, not individuals, and that physiological variation often exceeds pathological change. By mastering these principles, the practicing internist can navigate the complexities of hormonal assessment with confidence and precision.

Key Take-Home Points

1. Preanalytical variables account for most errors in hormone testing; attention to timing, patient preparation, and specimen handling is essential
2. Immunoassays are subject to interference from heterophile antibodies, biotin, and other substances; consider interference when results are discordant with clinical presentation
3. Mass spectrometry provides superior accuracy for steroid hormones and should be used for testosterone measurement in women and children
4. Reference ranges are population-based and may not apply to individual patients; interpret results in clinical context
5. Many hormones exhibit circadian variation or pulsatile secretion; single random measurements may be misleading
6. Avoid thyroid function testing during acute illness; non-thyroidal illness syndrome causes predictable abnormalities that do not require treatment
7. Always measure PTH and calcium simultaneously; PTH interpretation requires knowledge of concurrent calcium and vitamin D status
8. High-dose biotin supplementation increasingly causes assay interference; ask patients about all supplements
9. When results are unexpectedly abnormal, follow a systematic approach considering test selection, preanalytical factors, physiological variation, interference, and clinical correlation
10. Direct communication with laboratory personnel and endocrinology consultation enhance diagnostic accuracy in complex cases

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Word Count: Approximately 4,000 words

This comprehensive review synthesizes current evidence and practical wisdom to guide internists through the complexities of hormonal assessment, emphasizing clinical reasoning, pattern recognition, and systematic approaches to problem-solving in endocrine diagnosis.