Concentrations of thyroid hormone triiodothyronine (T3) and thyroxine (T4) is tightly regulated by thyroid stimulating hormone (TSH) secreted by pituitary. T3 and T4 in turn provide negative feedback to the pituitary to control the TSH secretion, and TSH is sensitive to small thyroid hormone increases. Consequently, TSH testing has high sensitivity and specificity for detecting hyperthyroidism. The American Thyroid Association recommends TSH as the first-line test for evaluating thyroid function. Free T4 (fT4) should be ordered when TSH is abnormal or TSH-mediated hyperthyroidism is suspected. Measurement of T3 is most valuable for detecting hyperthyroidism when the fT4 is normal or suppressed at the context of low TSH [1].

The majority of T3 circulates as the protein-bound form, and the biologically active free T3 accounts for ~0.3% of total T3 in circulation. Accordingly, T3 can be measured and reported as total T3 (tT3) or free T3 (fT3). fT3 is measured by direct or indirect method. Direct method requires equilibrium dialysis or ultrafiltration to separate the free form from the protein-bound fraction, and the concentration of the free form is detected by immunoassay or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Even though the direct method is considered the gold standard for fT3 measurement, its complexity and expense render it from wide adaptation in the clinical laboratory setting. The widely used assay for routine fT3 analysis is the indirect immunoassay method, which measures the free T3 in the presence of the protein-bound form and assumes the equilibrium between free T3 and bound T3 remains undisrupted during the analysis process. On the other hand, the total T3 measurement relies on the displacement of T3 from binding proteins. The total concentration of free and the released T3 is subsequently assessed by LC-MS/MS or immunoassay [3].

fT3 measurement is more analytically challenging than tT3 measurement due to the presence of the small quantity of free T3 in circulation. fT3 measurement used to suffer from imprecision but it has now become less of a concern with the improved free T3 assay. At our institution, we use the fT3 assay primarily based on the assay imprecision. According to instructions for use from the manufacturer, the imprecision for fT3 and tT3 are ~2.0% and ~3.3% respectively at comparable concentrations, which is consistent with our experience. To the best of our knowledge, there is no literature describing the comparison of accuracy of the modern generation tT3 and fT3 assays. Future studies are needed to assess which assay is most appropriate for routine clinical use. As evidence of this, the 2022 College of American Pathology survey reported that tT3 and fT3 are adopted in ~44% and ~56% of the laboratories respectively, implying that there is not a preferred method adopted by laboratories for T3 testing [4]. Consistent with this, a multicenter study found that T3 testing is almost equally distributed among tT3 and fT3 [5]. Therefore, institutions should make decision on adopting tT3 vs fT3 based on their available choices from the manufacturer. However, fT3 may be the preferred assay in the future because the concentration of active form fT3 is a more accurate representation of the physiological state.

High T3 in the context of low TSH and low/normal fT4 in a symptomatic patient is referred to as T3 thyrotoxicosis, a form of hyperthyroidism. We recently investigated the frequency of biochemical T3 thyrotoxicosis in our institution, a 1,400-bed tertiary care inner city hospital that tests both in-patients and outpatients routinely for potential thyroid disease. We found that T3 thyrotoxicosis was relatively uncommon, with a frequency of 1.6% (70 out of 4,366 grouped TSH-fT4-fT3 results). Among these, 20 (0.5%) results revealed newly diagnosed T3 thyrotoxicosis. We also found that the likelihood of detecting high fT3 increased with a lower TSH cutoff (TSH <0.3 μIU/mL, 10.3% vs TSH <0.01μIU/mL, 27.6%). Notably, the concentrations of TSH in the newly diagnosed T3 thyrotoxicosis cases were all lower than 0.01 μIU/mL. These results suggested that fT3 testing has limited clinical utility for hyperthyroidism diagnosis in our hospital setting and that a fT3 testing reflex triggered by TSH < 0.01 μIU/mL and low/normal fT4 may improve the fT3 test clinical utility and reduce unnecessary testing for patients [2].

In a nutshell, T3 can be measured using direct fT3, indirect fT3 and tT3 assay (summarized in Table 1). Direct fT3 method is used as the reference method. Routine laboratory analysis uses indirect fT3 or tT3 measurement, and the choice of fT3 vs tT3 should be based on institution’s available options. Overall, the clinical utility of T3 testing is limited and the T3 assessment is most useful for capturing T3 thyrotoxicosis when the fT4 is low or normal in the context of low TSH.

Table 1 Summary of T3 measurements

Assay Requirement/Assumption Utility  Distribution among laboratories 
Direct fT3 Equilibrium dialysis/ultrafiltration to separate the free form from the protein-bound fraction. Reference method  NA 
Indirect fT3 Assumes the equilibrium remains undisrupted during the analysis process.  Routine analysis  ~50% [4,5]
tT3  Displacement of T3 from binding proteins. Routine analysis  ~50% [4,5] 



1. Ladenson, P.W., et al., American Thyroid Association Guidelines for Detection of Thyroid Dysfunction. Archives of Internal Medicine, 2000. 160(11): p. 1573.

2. Lin, Y., et al., Limited Utility of Free Triiodothyronine Testing. J Appl Lab Med, 2023. 8(5): p. 847-855.

3. Freedman D.B., et al., In: Rifai N, Horvath AR, Witter CT, editors. Tietz textbook of clinical chemistry and molecular diagnostics. 6th Ed.

4. College of American Pathologists. 2022 Surveys and anatomic pathology education programs.

5. Lin, D.C., et al., Multi-Center Benchmark Study Reveals Significant Variation in Thyroid Testing in United States. Thyroid, 2017. 27(10).