# Endocrine Laboratory Testing: Thyroid, Adrenal, and Pituitary

## Introduction

Endocrine laboratory testing requires understanding of feedback loops, dynamic testing, and the physiologic context of hormone measurements. The pathologist and laboratory director must ensure appropriate test selection, recognize assay interferences, and guide clinicians in interpreting results within the correct clinical framework.

## Thyroid Function Testing

### Thyroid-Stimulating Hormone (TSH)

TSH is the **first-line screening test** for thyroid dysfunction. Its reference range is approximately 0.4-4.0 mIU/L, though this varies by assay and population. Third-generation assays can detect levels down to 0.01-0.02 mIU/L. TSH is inversely related to free thyroid hormones via the **hypothalamic-pituitary-thyroid axis**.

### Free T4 and Free T3

The free (unbound) hormone is the biologically active fraction. **Free T4** is the primary confirmatory test when TSH is abnormal. Free T3 is useful in **T3 thyrotoxicosis**, where T4 is normal but TSH is suppressed. Immunoassay methods estimate free hormone levels and can be affected by binding protein abnormalities.

### Common Patterns

| TSH    | Free T4 | Free T3 | Interpretation                    |
|--------|---------|---------|-----------------------------------|
| Low    | High    | High    | Overt hyperthyroidism             |
| High   | Low     | Low     | Overt hypothyroidism              |
| Low    | Normal  | Normal  | Subclinical hyperthyroidism       |
| High   | Normal  | Normal  | Subclinical hypothyroidism        |
| Normal | Low     | Low     | Central hypothyroidism or NTI     |

### Thyroid Antibodies

**Anti-TPO** antibodies are present in approximately 90% of Hashimoto thyroiditis cases. **TSH receptor antibodies (TRAb)** are characteristic of Graves disease. **Thyroglobulin antibodies** interfere with thyroglobulin measurement, which is used as a tumor marker for differentiated thyroid carcinoma.

![Thyroid function test algorithm for abnormal TSH](images/thyroid-test-algorithm.jpg)

## Adrenal Function Testing

### Cortisol Assessment

Morning serum cortisol is highest at 6-8 AM due to the **diurnal rhythm**. **24-hour urine free cortisol** integrates cortisol production over the entire day. **Late-night salivary cortisol** is a convenient screening test for Cushing syndrome because it captures the loss of the normal diurnal nadir.

### Dynamic Tests for Cushing Syndrome

The **1 mg overnight dexamethasone suppression test** excludes Cushing syndrome if cortisol suppresses below 1.8 mcg/dL. The **low-dose dexamethasone suppression** test (2-day protocol, 0.5 mg every 6 hours) serves as confirmation. The **ACTH level** distinguishes ACTH-dependent causes (pituitary or ectopic) from ACTH-independent causes (adrenal).

### Adrenal Insufficiency

The **cosyntropin (ACTH) stimulation test** uses 250 mcg IV, with a cortisol level above 18 mcg/dL at 30 or 60 minutes considered normal. The low-dose (1 mcg) ACTH stimulation test may be more sensitive for secondary adrenal insufficiency. **Morning ACTH** is elevated in primary adrenal insufficiency (Addison disease) and low or normal in secondary or tertiary disease.

### Aldosterone and Renin

The **aldosterone-to-renin ratio (ARR)** is the screening test for primary aldosteronism (Conn syndrome). An ARR above 30 with aldosterone above 15 ng/dL is suspicious and requires confirmatory testing. Medications such as spironolactone, ACE inhibitors, and beta-blockers must be managed before testing to avoid misleading results.

![Cushing syndrome diagnostic algorithm](images/cushing-algorithm.jpg)

## Pituitary Function Testing

### Prolactin

Prolactin is elevated in **prolactinoma**, with medications (dopamine antagonists), and due to stalk effect from other sellar masses. The **hook effect** is an important pitfall: very high prolactin levels may give falsely normal results, so serial dilutions should be requested when a large pituitary macroadenoma is present.

### Growth Hormone (GH) and IGF-1

GH is secreted in pulses, making random levels unreliable. **IGF-1** is the screening test for acromegaly and must be interpreted with age- and sex-adjusted reference ranges. The **oral glucose tolerance test (OGTT)** confirms acromegaly when GH fails to suppress below 1 ng/mL after a 75g glucose load.

### Gonadotropins (FSH, LH)

FSH and LH are elevated in primary gonadal failure such as menopause or Klinefelter syndrome, and low in hypothalamic-pituitary dysfunction. An **LH/FSH ratio greater than 2** is suggestive of polycystic ovarian syndrome (PCOS).

### ADH and Water Balance

The **water deprivation test** differentiates central diabetes insipidus, nephrogenic DI, and primary polydipsia. **Copeptin** is a surrogate marker for ADH that is increasingly used because it is a more stable analyte in clinical testing.

## Assay Interferences and Pitfalls

**Biotin interference** is a growing problem: high-dose biotin supplementation causes false results in streptavidin-biotin immunoassays, producing falsely low TSH and falsely high free T4 in competitive assay formats. **Heterophilic antibodies** can cause false elevations or decreases and should be suspected when results are discordant with the clinical picture. **Macro-analytes** such as macro-prolactin and macro-TSH are large complexes that are immunoreactive but biologically inactive, and they can be detected by PEG precipitation.

![Biotin interference patterns in immunoassays](images/biotin-interference.jpg)

## Clinical Pearls

TSH is the single best screening test for thyroid dysfunction in outpatients with an intact hypothalamic-pituitary axis. The hook effect should always be considered when prolactin is unexpectedly normal in a patient with a large pituitary adenoma. Biotin interference is a growing clinical problem, and laboratories should have policies for managing biotin-containing supplements. Dynamic testing through stimulation or suppression protocols is often needed to confirm endocrine diagnoses, as static levels alone can be misleading.

## References

1. Garber JR, et al. Clinical practice guidelines for hypothyroidism in adults. *Thyroid*. 2012;22(12):1200-1235.
2. Nieman LK, et al. The diagnosis of Cushing's syndrome: an Endocrine Society clinical practice guideline. *J Clin Endocrinol Metab*. 2008;93(5):1526-1540.
3. Fleseriu M, et al. Hormonal replacement in hypopituitarism in adults: an Endocrine Society clinical practice guideline. *J Clin Endocrinol Metab*. 2016;101(11):3888-3921.
4. Favresse J, et al. Interferences with thyroid function immunoassays: clinical implications and detection algorithm. *Endocr Rev*. 2018;39(5):830-850.