Thyroid Function Tests (TSH, Free T4, and Free T3)

Thyroid Function Tests (TFTs) are a standardized battery of biochemical immunoassays that evaluate the functional integrity of the Hypothalamic-Pituitary-Thyroid (HPT) axis and measure circulating concentrations of the active metabolic hormones governing cellular oxygen consumption, thermogenesis, and basal metabolic rate throughout the body.

1. Hypothalamus: Synthesizes and secretes Thyrotropin-Releasing Hormone (TRH) into the hypophyseal portal circulation.
2. Anterior Pituitary Thyrotrophs: In response to TRH, the pituitary synthesizes and releases Thyroid-Stimulating Hormone (TSH / Thyrotropin), a 28 kDa glycoprotein heterodimer sharing an alpha subunit with hCG, LH, and FSH, but possessing a unique beta subunit conferring receptor specificity.
3. Thyroid Gland: TSH binds to the G-protein-coupled TSH receptor on thyroid follicular cell basolateral membranes, stimulating iodine organification, coupling of iodotyrosines into thyroglobulin, and secretion of Thyroxine ($\text{T}_4$, ~80%) and Triiodothyronine ($\text{T}_3$, ~20%).
4. Peripheral Deiodination: T₄ is essentially a prohormone. In peripheral target tissues (liver, kidneys, muscle), the selenium-dependent enzyme 5'-deiodinase (Types 1 and 2) cleaves a 5'-iodine atom to convert T₄ into active T₃, which has a 10-fold higher affinity for nuclear thyroid hormone receptors (TRα and TRβ). Inactivating deiodinase (Type 3) converts T₄ into metabolically inactive reverse $\text{T}_3$ ($\text{rT}_3$).

Pituitary TSH secretion responds with extraordinary sensitivity to tiny fluctuations in circulating free thyroid hormones. A mere 50% decrease in serum Free T₄ stimulates a 100-fold increase in serum TSH! Consequently, serum TSH is the most sensitive and cost-effective first-line test for evaluating suspected primary thyroid pathology.

Over 99.97% of circulating T₄ and 99.7% of circulating T₃ are bound to plasma carrier proteins, predominantly Thyroxine-Binding Globulin (TBG, ~75%), transthyretin (prealbumin, ~15%), and albumin (~10%). Only the tiny unbound fraction (Free $\text{T}_4$ and Free $\text{T}_3$) is biologically active and accessible to cellular receptors.

• Elevated TBG (Pregnancy, oral estrogen contraceptives): Increases Total T₄ while Free T₄ remains completely normal.
• Decreased TBG (Cirrhosis, nephrotic syndrome, androgens): Lowers Total T₄ while Free T₄ remains completely normal.

Clinical Rule: Always measure Free $\text{T}_4$ rather than Total T₄ to avoid protein-binding confounding.

• Diurnal Variation: Serum TSH exhibits a modest circadian rhythm, peaking in the early hours of the morning (02:00–04:00) and reaching its lowest nadir in the late afternoon. Routine testing should ideally occur consistently in the morning.
• Timing of Levothyroxine Dose: In patients established on daily oral levothyroxine (L-T₄), taking the daily tablet within 2 to 4 hours prior to blood collection produces a transient post-absorptive peak in Free T₄ (up to 15% to 30% above steady-state trough levels). Blood draws should occur in the morning BEFORE taking the daily levothyroxine tablet.
• Biotin Interference (Megavitamin Warning): Patients consuming > 5–10 mg/day of biotin for cosmetic hair and nail enhancement experience profound analytical immunoassay interference. Biotin produces falsely suppressed TSH combined with falsely elevated Free $\text{T}_4$ and Free $\text{T}_3$, creating a perfect biochemical facsimile of acute Graves' thyrotoxicosis in a clinically euthyroid patient! Discontinue biotin for at least 48 to 72 hours prior to testing.
• Critical Illness: Non-thyroidal illness syndrome disrupts HPT physiology; avoid routine thyroid screening in intensive care units.

1. Suspected Hypothyroidism: Fatigue, weight gain, cold intolerance, constipation, dry coarse skin, alopecia, menorrhagia, bradycardia, delayed deep tendon reflex relaxation, or carpal tunnel syndrome.
2. Suspected Hyperthyroidism / Thyrotoxicosis: Weight loss despite increased appetite, heat intolerance, palpitation/tachycardia, fine hand tremor, frequent bowel movements, oligomenorrhea, anxiety, or exophthalmos/goiter.
3. Monitoring Thyroid Hormone Replacement: Titrating levothyroxine in treated hypothyroidism (recheck TSH every 6 to 8 weeks after dose changes until stable).
4. Monitoring Antithyroid Pharmacotherapy: Titrating methimazole or propylthiouracil in Graves' disease or toxic nodular goiter (monitor Free T₄ and Free T₃, as TSH may remain suppressed for months after normalization of peripheral hormones).
5. New-Onset Atrial Fibrillation or Unexplained Dyslipidemia: Routine screening for occult thyrotoxicosis (atrial fibrillation) or hypothyroidism (refractory hypercholesterolemia).
6. Pre-conception and Antenatal Surveillance: Screening women with infertility, recurrent miscarriage, or known thyroid autoimmunity.

┌─────────────────────────────────────────────────────────────┐
│          STEP-BY-STEP THYROID FUNCTION ALGORITHM            │
├─────────────────────────────────────────────────────────────┤
│ Step 1: Evaluate Serum TSH First                            │
│         ├─ High TSH (> 4.0 mIU/L): Go to Step 2             │
│         ├─ Suppressed TSH (< 0.4 mIU/L): Go to Step 3       │
│         └─ Normal TSH (0.4 - 4.0 mIU/L): Euthyroid          │
│            (Unless pituitary disease is suspected!)         │
├─────────────────────────────────────────────────────────────┤
│ Step 2: If TSH is ELEVATED, Inspect Free T4:                │
│         ├─ Low Free T4: Overt Primary Hypothyroidism        │
│         │  (Check anti-TPO antibodies; start Levothyroxine) │
│         ├─ Normal Free T4: Subclinical Hypothyroidism       │
│         │  ├─ TSH >= 10 mIU/L: Treat with Levothyroxine     │
│         │  └─ TSH < 10 mIU/L: Treat if pregnant, young,     │
│         │     symptomatic, or anti-TPO positive             │
│         └─ High Free T4: Discordant! TSH-secreting pituitary│
│            adenoma or Thyroid Hormone Resistance            │
├─────────────────────────────────────────────────────────────┤
│ Step 3: If TSH is SUPPRESSED (< 0.4), Inspect FT4 and FT3:  │
│         ├─ High FT4 and/or High FT3: Overt Hyperthyroidism  │
│         │  (Check TRAb for Graves; uptake scan for nodules) │
│         ├─ Normal FT4 and Normal FT3: Subclinical Hyper-    │
│         │  thyroidism (Risk of AF and osteoporosis)         │
│         └─ Low FT4: Discordant! Central Hypothyroidism or   │
│            Non-Thyroidal Illness Syndrome (NTIS)            │
├─────────────────────────────────────────────────────────────┤
│ Step 4: Adjudicate Suspected Central Hypothyroidism         │
│         Low FT4 + Low/Normal TSH = Pituitary Failure        │
│         Order brain MRI; check morning cortisol/ACTH        │
│         (CRITICAL: Always replete cortisol BEFORE levothyr- │
│         oxine to prevent fatal adrenal crisis!)             │
└─────────────────────────────────────────────────────────────┘

• Amiodarone: Contains 37% iodine by weight. Induces two distinct forms of thyrotoxicosis:
• Type 1 Amiodarone-Induced Thyrotoxicosis (AIT): True hyperthyroidism driven by excessive iodine substrate (Jod-Basedow effect) in patients with underlying autonomous nodular goiter. Treated with antithyroid drugs (methimazole).
• Type 2 AIT: Destructive drug-induced subacute thyroiditis with release of preformed hormone. Treated with high-dose corticosteroids.
• Amiodarone-Induced Hypothyroidism (AIH): Failure of the gland to escape the acute Wolff-Chaikoff effect. Treated with levothyroxine.
• Lithium: Actively concentrates within thyroid follicular cells and inhibits thyroid hormone release. Up to 20% to 30% of patients on chronic lithium develop primary hypothyroidism or goiter.
• Immune Checkpoint Inhibitors (Nivolumab, Pembrolizumab, Ipilimumab): Frequently trigger acute autoimmune destructive thyroiditis, presenting with a transient hyperthyroid phase that transitions into permanent lifelong primary hypothyroidism within 6 to 12 weeks.

1. The "Cortisol Before Thyroxine" Golden Rule: In patients with Central Hypothyroidism (low Free T₄ with low/normal TSH due to pituitary dysfunction), initiating levothyroxine before ruling out and replacing co-existing adrenal insufficiency can precipitate fatal acute adrenal crisis! Levothyroxine accelerates hepatic clearance of cortisol. Always test morning cortisol or synacthen stimulation first!
2. Treating Non-Thyroidal Illness Syndrome with Thyroid Hormones: In sick hospitalized patients with low T₃, clinical trials have demonstrated that administering triiodothyronine or levothyroxine does NOT improve clinical outcomes and may be harmful. Withhold hormone replacement and treat the underlying medical illness.
3. The "Transient Postpartum Phase" Misstep: Postpartum thyroiditis classically exhibits a triphasic course: hyperthyroidism (1–3 months postpartum), followed by hypothyroidism (4–8 months postpartum), followed by spontaneous resolution. Initiating radioactive iodine ablation during the hyperthyroid phase is strictly contraindicated, as the gland has low radioiodine uptake.

• The Subclinical Hypothyroidism Cutoff (10 mIU/L): In asymptomatic subclinical hypothyroidism (elevated TSH with normal Free T₄), the consensus threshold where treatment is universally indicated is $\text{TSH} \ge 10\ \text{mIU/L}$ (due to elevated risk of progression and ischemic heart disease). If TSH is 4.5–9.9 mIU/L, treat only if patient is pregnant, symptomatic, young, or has positive anti-TPO antibodies.
• The Reverse T3 Secret: When struggling to differentiate central hypothyroidism from euthyroid sick syndrome in the hospital, measure reverse $\text{T}_3$ ($\text{rT}_3$):
• Central hypothyroidism = Low or normal rT₃.
• Euthyroid sick syndrome = High rT₃ (shunting of T₄ into inactive rT₃ by Type 3 deiodinase).
• The First-Trimester hCG Mimic: Because hCG stimulates the TSH receptor, a mildly suppressed TSH in early pregnancy is physiological and expected. Never diagnose hyperthyroidism based on TSH alone in pregnant women.

• Complete Blood Count (CBC): Screens for normocytic or macrocytic anemia associated with hypothyroidism (Complete Blood Count).
• Serum Electrolytes: Evaluates for hyponatremia seen in severe primary hypothyroidism (Serum Electrolytes).
• Inflammatory Markers (ESR/CRP): Markedly elevated in subacute painful granulomatous (de Quervain) thyroiditis (Inflammatory Markers).
• Thyroid Autoantibodies (Anti-TPO, Anti-Tg, TRAb): Confirms Hashimoto thyroiditis (anti-TPO) or Graves' disease (TRAb / TSI).
• Radioactive Iodine Uptake and Scan (RAIU): Differentiates Graves' disease (diffuse high uptake) from toxic nodules (focal high uptake) and thyroiditis (absent / zero uptake).