Thyroid Testing Best Practices: Reverse that Order for Reverse T3!

Nam K. Tran, PhD, HCLD (ABB), FACB, Director of Clinical Chemistry and POCT
Xenia Ivanova, BS, Clinical and Quality Research
Ky Van, BS, Clinical and Quality Research


Thyroid testing is one of the most common laboratory tests performed in the clinical chemistry section. The goal of thyroid testing is to evaluate thyroid function and to find the causes of problems such as hyperthyroidism or hypothyroidism.1 Unfortunately, there remains a trend towards inappropriate ordering of certain thyroid tests due to misinformation in the media or lack of understanding of thyroid function, and/or the analytical performance of these assays.

A simple web search for “thyroid testing” reveals a substantial number of non-medical sites with incorrect information on how to diagnose, monitor, and treat suspected thyroid disorders.1 These sites often encourage patients to order excessive number of thyroid tests without understanding concepts such as clinical sensitivity, specificity, and predictive values. There is also a lack of understanding about the limitations of many of these thyroid tests. The goal of this blog article is to highlight best practices for thyroid testing

Lab Best Practice

The UC Davis Health (UCDH) Clinical Laboratory has published a recommended thyroid testing algorithm developed in collaboration with the Division of Endocrinology and adapted from peer reviewed literature and guidelines.2,3

Thyroid Simulating Hormone: Thyroid stimulating hormone (TSH) levels serve as the primary screening test, and as needed, a free thyroxine, or free T4 (fT4) should be ordered if TSH values are abnormal.2,3 It must be noted that TSH assays may differ between manufacturers. The current state of the art TSH assay is considered a “3rd generation” test.4 Older generation tests lack the analytical sensitivity to measure lower concentrations of TSH levels. Fourth generation tests also exist, however, 3rd generation tests are the most common and provide sufficient sensitivity for the majority of clinical cases.

Free Thyroxine and Free Triiodothyronine Testing:  No thyroid test is perfect. For example, all  fT4 and free triiodothyronine (fT3) assays measure an analog due to relying on antibodies for detection and influencing the equilibrium conditions for free versus bound forms of thyroid hormone.5 Furthermore, these assays assume the protein binding capacity for T4 and T3 is known. When protein binding capacity changes, these assays become inaccurate. Protein binding abnormalities may be evaluated using total T4 and T3 assays in conjunction with fT4 and fT3 measurements respectively, as well as direct measurement of thyroid binding globulin, albumin, and transthyretin.

T3 Uptake Testing: T3 uptake testing is obsolete. This assay measures available thyroid hormone binding sites and to determine hormone levels.5 As noted above, with the availability of fT4, fT3, and binding protein assays, there is little need for T3 uptake testing. UCDH requires prior approval for send out T3 uptake testing and is highly restricted due to its obsolescence.

Reverse T3: Reverse T3 (rT3) is a byproduct of thyroid hormone metabolism.1,5 This test is not generally recommended for routine evaluation of thyroid disorders. Reverse T3 levels is elevated in sick euthyroid syndrome but has no other clinical value. Sick patients including those in intensive care units will have elevated rT3. Therefore, it can be assumed rT3 levels are elevated during sickness and its measurement should not change in clinical decision-making. To this end, UCDH highly restricts this test. In recent times, misinformation in the media has promoted the ordering of rT3 especially for naturopathic applications, however, there is little clinical evidence to support this practice.

Other Considerations

Discrepant thyroid testing results may arise for many reasons. These “confounding” factors are typically due to exogenous or endogenous interfering substances. It is highly encouraged that providers contact the clinical laboratory if they suspect an interfering substance. Interfering substances in thyroid testing is well documented in literature and includes:

Fibrin: Plasma specimens that were collected and inadequately mixed or with the presence of heparin degrading compounds may result the presence of fibrin.6 Certain thyroid assays may be susceptible to fibrin interference and cause an erroneously high value that does not match the clinical picture. Re-centrifuging the sample in the laboratory and visually checking specimens for fibrin may resolve this issue. Alternately, re-drawing the patient and ensuring good technique would be appropriate.

Biotin: Biotin interference has been documented in thyroid testing.7-10 At present, UCDH does not offer any thyroid tests affected by biotin. Typically, testing problems arise in patients taking extreme amounts of biotin as supplements. Patients with good renal function taking <5 mg/day of biotin should not result in any interferences. However, there has been a trend towards taking >100 mg of biotin per day for the treatment of thyroid disorders and/or multiple sclerosis. These biotin-based therapies are not approved by the United States Food and Drug Administration and there is presently insufficient clinical evidence to prove efficacy. It is recommended that patients needing thyroid testing be asked if they are actively taking biotin, and to refrain from taking these supplements at least 8 hours before laboratory testing. It must be noted that biotin may also be found in multivitamins and other compounds without patient knowledge.

Heterophile Antibodies: Heterophile antibody interference is a rare occurrence that can cause falsely high or falsely low thyroid testing results.1,6 In brief, antibodies produced by the patient directly bind to antibodies used by the assay. These heterophile antibodies may block the binding site for thyroid biomarkers or enhanced the detection to cause falsely low or falsely high results respectively. Being an immune system related interference intrinsic to the patient, heterophile antibody production will not go away, therefore, these patients should be “flagged” to avoid incorrect testing. Again, these are rare occurrences. If heterophile antibody interference is suspected, contact the clinical laboratory. Heterophile antibody treatment of samples is available as a send out test but must be approved by the Clinical Laboratory.

Iodine Containing Compounds: Iodine containing medications such as amiodarone may cause abnormal changes in thyroid hormone levels.11 TSH is found to increase early and significantly, starting from the first day of amiodarone therapy and reaching a value 2.7-fold higher than baseline values on the 10th day. After 1 to 4 months of therapy, serum T4 levels increase by an average of 40% compared to pre-treatment levels. Increases in T4 levels is an expected finding and in itself does not constitute evidence of hyperthyroidism. Likewise, TSH levels often return to normal during chronic amiodarone administration (>3 months).


  1. Koulouri O, Moran C, Halsall D, et al. Pitfalls in the measurement and interpretation of thyroid function tests. Best Pract Res Clin Endocrinol Metab 2013;27:745-762.
  2. Garber JR, Cobin RH, Gharib H, et al. Practice guidelines for hypothyroidism in adults: cosponsored by the American Association of Clinical Endocrinologists and the American Thyrid Association. Endocr Pract 2012;18:1-45.
  3. Bahn RS, Burch HB, Cooper DS, et al. Hyperthyroidism and other causes of thyrotoxicosis: management guidelines of the American Thyroid Association and American Association of Clinical Endocrinologists. Endocr Pract 2011;17:457-522.
  4. Saller B, Broda N, Heydarian R, et al. Utility of third generation thyrotropin assays in thyroid function esting. Exp Clin Endocrinol Diabetes 1998;106:S29-S33.
  5. Burtis CA, Ashwood ER. Tietz Fundamentals of Clinical Chemistry. 4th Edition. 1996.
  6. Tate J, and Ward G. Interferences in immunoassay. Clin Biochem Rev 2004;25:105-120.
  7. Chun KY. Biotin interference in diagnostics., Accessed on June 1, 2017
  8. Sedal F, Papeix C, Bellanger A, et al. High doses of biotin in chronic progressive multiple schlerosis: a pilot study. Mult Scler Relat Disord 2015;159.
  9. Kummer S. Biotin treatment mimicking Grave’s disease. N Engl J Med 2016;375:704
  10. Roche Diagnostics Product TSH Assay Product Insert, Accessed on June 1, 2017
  11. Loh KC. Amiodarone-induced thyroid disorders: a clinical review. Postgrad Med J 2000;76:133-140.
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