Additional Laboratory Tests for Thyroid Function Assessment

When TSH is abnormal, free T4 (FT4) is the essential next test to distinguish between subclinical and overt thyroid dysfunction. 1

Core Additional Tests Beyond TSH

Free T4 measurement is necessary when TSH is abnormal to differentiate subclinical thyroid dysfunction (normal FT4) from overt dysfunction (abnormal FT4). 1
FT4 represents a more useful index of thyroid status than total T4 because it is not influenced by variations in thyroid hormone-binding proteins, particularly T4-binding globulin (TBG). 2
In patients with suspected central hypothyroidism, free T4 must be measured alongside TSH, as TSH may be inappropriately normal or only mildly elevated despite low FT4. 1
Free T4 has the highest diagnostic performance when considered alone, with superior sensitivity and specificity compared to total hormone measurements. 3

Free T3 should be measured when FT4 is normal but hyperthyroidism is still suspected, as approximately 5% of hyperthyroid patients have selective T3 elevation (T3 thyrotoxicosis). 4
FT3 measurement is particularly useful in difficult diagnostic cases when combined with FT4, though it is not routinely needed for initial thyroid assessment. 3
In hyperthyroidism diagnosis, FT3 (along with TSH) may be more reliable than FT4, since FT4 can occasionally be elevated in euthyroid subjects taking chronic amiodarone or levothyroxine. 2

Anti-thyroid peroxidase (anti-TPO) antibodies should be measured to confirm autoimmune etiology when hypothyroidism is detected, as positive antibodies predict 4.3% annual progression to overt hypothyroidism versus 2.6% in antibody-negative patients. 1
Measuring anti-TPO antibodies is particularly important when TSH is between 4.5-10 mIU/L, as positive antibodies may influence the decision to initiate treatment. 1

Anti-TG antibodies should be measured simultaneously with TPO antibodies when screening for autoimmune thyroid disease, as approximately 10-20% of patients with Hashimoto's thyroiditis have isolated anti-TG positivity without TPO antibodies. 1

Thyroglobulin (Tg) must be measured at follow-up visits in thyroid cancer patients, as it serves as a tumor marker for recurrence. 5, 1
Thyroglobulin antibodies must be measured simultaneously with Tg, as their presence interferes with Tg measurement and can produce falsely low or undetectable results. 1
Both basal and rhTSH-stimulated serum Tg measurements should be obtained at 6-12 months post-initial treatment to assess disease status. 5

Early morning ACTH and cortisol must be checked before starting thyroid hormone replacement, as initiating levothyroxine before corticosteroids can precipitate adrenal crisis in patients with concurrent adrenal insufficiency. 1
Free T4 is the critical test in central hypothyroidism, as TSH cannot be relied upon and may appear inappropriately normal despite low thyroid hormone levels. 1

Measuring lipid panel is reasonable, as subclinical hypothyroidism may affect cholesterol levels and treatment with levothyroxine may lower LDL cholesterol. 1
Glucose and HbA1c should be measured in patients on immunotherapy, as new-onset diabetes can occur with checkpoint inhibitor therapy. 1

Both TSH and free T4 should be measured together during levothyroxine dose titration every 6-8 weeks, as FT4 helps interpret ongoing abnormal TSH levels. 1
Free T4 measurement is particularly important when TSH remains abnormal during therapy, as TSH may take longer to normalize than FT4. 1

TSH and free T4 must be measured together during pregnancy, as inadequate treatment increases risk of preeclampsia, low birth weight, and neurodevelopmental effects. 1
Target TSH should be maintained in the low-normal range (0.5-2.5 mIU/L) during pregnancy, requiring more frequent monitoring than in non-pregnant patients. 1

Avoid ordering free thyroid hormones (FT4, FT3) when TSH is normal in asymptomatic patients, as this represents inappropriate testing that rarely contributes to diagnosis or management. 6
Do not rely on total T4 or total T3 measurements in routine clinical practice, as these are influenced by binding protein variations and have been superseded by free hormone measurements. 7, 2
Never treat based on a single abnormal TSH value—repeat TSH in 3-6 weeks before initiating treatment, as 30-60% of elevated TSH values normalize spontaneously. 1
Recognize that free hormone measurements remain technically demanding in sera from severely ill patients with low serum thyroxine binding capacity, potentially leading to artifactual results. 7