Hormonal Assays and Interpretation

Assay Principles - Hormones Under Scope

• Immunoassays: Core of hormone measurement.
  • Competitive: Labeled & unlabeled hormone compete for limited antibody sites. Signal $\propto$ 1/[Analyte].
  • Non-competitive (Sandwich): Analyte captured between two antibodies. Signal $\propto$ [Analyte].
• Key Techniques:
  • ELISA: Enzyme-linked, colorimetric/fluorometric signal.
  • RIA: Radiolabeled antigen ($^{125}$I), highly sensitive.
  • CLIA: Chemiluminescent signal, high sensitivity & wide range.
  • LC-MS/MS: Gold standard for steroids; high specificity & multiplexing.
• Hormones Covered: Thyroid (TSH, T3, T4), PTH, Cortisol, Aldosterone, GH, ACTH, Prolactin, Insulin, Estrogen, Testosterone, hCG.

⭐ Free hormone fractions (e.g., fT4, fTestosterone) often better reflect biological activity than total hormone levels, as they are unbound to proteins.

Pituitary & Thyroid - Master Regulators

• Pituitary Gland:
  • Anterior Lobe: Secretes TSH, ACTH, GH, PRL, FSH, LH. 📌 Mnemonic: FLAT PiG.
    • TSH (Thyrotropin): Stimulates thyroid hormone (T3, T4) synthesis/release. Regulated by hypothalamic TRH (+) and thyroid hormone (-).
  • Posterior Lobe: Stores & releases ADH, Oxytocin (synthesized in hypothalamus).
• Thyroid Gland:
  • Hormones: Thyroxine (T4), Triiodothyronine (T3), Calcitonin.
  • T4 is prohormone; converted to T3 (more potent) in periphery by deiodinases.
  • Binding proteins: TBG (Thyroxine-binding globulin) carries most T4/T3.
• Hypothalamic-Pituitary-Thyroid (HPT) Axis:

• Thyroid Function Tests (TFTs) Interpretation:
  • TSH: Best initial screening. Normal: 0.4-4.0 mIU/L.
    • Primary Hypothyroidism: ↑ TSH, ↓ Free T4.
    • Primary Hyperthyroidism: ↓ TSH, ↑ Free T4.
    • Subclinical Hypothyroidism: ↑ TSH, Normal Free T4.
  • Anti-TPO Abs: Hashimoto's. TSH-Receptor Abs (TSI): Graves' disease.

⭐ TSH is the most sensitive indicator of primary thyroid dysfunction; changes in TSH often precede changes in T4/T3 levels.

Adrenal & Gonadal - Stress & Sex Hormones

• Adrenal Cortex:
  • Cortisol (Glucocorticoid): Stress response, ↑glucose. Reg: CRH-ACTH-Cortisol.
    • Tests: Dexamethasone suppression, ACTH stimulation, 24hr Urinary Free Cortisol (UFC).
    • Disorders: Cushing's syndrome (↑cortisol), Addison's disease (↓cortisol).
  • Aldosterone (Mineralocorticoid): Na⁺/H₂O reabsorption, K⁺ excretion, BP control. Reg: RAAS, K⁺.
    • Tests: Aldosterone:Renin Ratio (ARR). Conn's syndrome (↑aldosterone).
  • Adrenal Androgens (DHEA, DHEAS): Precursors to sex hormones.
    • Test: 17-hydroxyprogesterone (17-OHP) for Congenital Adrenal Hyperplasia (CAH).
• Adrenal Medulla:
  • Catecholamines (Epinephrine, Norepinephrine): Fight-or-flight.
    • Test: Plasma/Urinary Metanephrines, Vanillylmandelic Acid (VMA) for Pheochromocytoma.
• Gonadal Hormones:
  • Testosterone (Androgen): Male 2° sexual characteristics, spermatogenesis.
    • Tests: Total/Free Testosterone, LH, FSH.
  • Estrogen (Estradiol) & Progesterone: Female 2° sexual characteristics, menstrual cycle, pregnancy.
    • Tests: Estradiol, Progesterone, LH, FSH. Polycystic Ovary Syndrome (PCOS) common.

⭐ 21-Hydroxylase deficiency is the most common CAH cause: ↓cortisol, ↓aldosterone, ↑androgens. Presents with salt wasting & virilization (females).

Pancreas & Calcium - Metabolic Managers

• Pancreatic Hormones:
  • Insulin (β-cells): Anabolic. ↓ Blood glucose. C-peptide reflects endogenous insulin.
  • Glucagon (α-cells): Catabolic. ↑ Blood glucose, glycogenolysis, gluconeogenesis.
  • Somatostatin (δ-cells): Universal inhibitor (insulin, glucagon, GH).
• Calcium Homeostasis:
  • PTH: ↑ Serum $Ca^{2+}$, ↓ Serum $PO_4^{3-}$ (📌 Phosphate Trashing Hormone). Stimulates osteoclasts, renal $Ca^{2+}$ reabsorption, Vit D activation.
  • Vitamin D ($1,25(OH)_2D_3$): ↑ Intestinal $Ca^{2+}$ & $PO_4^{3-}$ absorption.
  • Calcitonin (Thyroid C-cells): Weakly ↓ Serum $Ca^{2+}$ by inhibiting osteoclasts.

⭐ Primary hyperparathyroidism classically presents with high PTH, high serum calcium, low serum phosphate, and increased urinary cAMP.

High‑Yield Points - ⚡ Biggest Takeaways

• Immunoassays (ELISA, RIA, CLIA) are mainstay for hormone quantification.
• Dynamic function tests (e.g., Dexamethasone suppression) assess endocrine reserve/autonomy.
• Free hormone levels (e.g., FT4) better reflect bioactivity than total hormone levels.
• Pulsatile secretion (LH) & diurnal variation (cortisol) demand timed/pooled sampling.
• Assay interferences (e.g., HAMA) can cause falsely high or low results.
• Interpret results with clinical context, considering age, sex, pregnancy, and medications.