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Test code(s) 35167, 866

We currently offer two tests for free T4 measurement. One test uses a direct equilibrium dialysis method (T4, Free, Direct Dialysis, test code 35167). The other test measures free T4 in an automated immunoassay on the Siemens Centaur platform (T4, Free, test code 866).

In the direct equilibrium dialysis (ED) method, patient serum is dialyzed against a buffer for 16-18 hours. ED separates the free T4 molecules from the protein bound T4 molecules. The smaller free T4 molecules can cross the dialysis membrane, but the larger bound T4 molecules cannot. After dialysis, free T4 can be measured using either immunoassay or mass spectrometry. We currently use a sensitive radioimmunoassay that has an analytical sensitivity of 0.2 ng/dL. Results are independent of T4-binding protein concentrations and are unaffected by the presence of molecular variants of these proteins or by circulating thyroid autoantibodies.

Unexpected high free T4 results by ED may be seen in sera from heparin-treated patients. Free fatty acids (FFA), which can displace T4 from its binding proteins, are generated in these samples. The spuriously high free T4 result appears to be primarily an “in vitro” effect and does not reflect the true free T4 level in vivo.

Less commonly, impurities in the tracer may cause erroneously high results.

If you feel that the results are elevated beyond what was anticipated, please contact the lab to discuss your results.

The immunoassay is performed on the automated Siemens Centaur platform. This assay is a competitive immunoassay using a T4 analog. It assumes that the T4 binding capacity is normal; therefore, results can be impacted when the albumin or thyroid binding globulin (TBG) levels are abnormal. The analytical measurement range is 0.1 – 11 ng/dL. The advantage of this assay is that it provides a shorter turnaround time than does the direct equilibrium dialysis method.

The analog-based immunoassays perform poorly at extremes of thyroid hormone binding capacity. Thus, excess TBG, as seen in select genetic conditions, in pregnancy, and in other conditions with increased estrogen, can cause a slight positive bias for free T4 results. Genetically-related TBG deficiency can also cause a negative bias in the free T4 results for this method. Very low levels of albumin, as seen in critical illness, premature infants, etc., also alter free T4 measurements. Free T4 results tend to be low in patients with abnormally low albumin levels and high in patients with familiar dysalbuminemic hyperthyroxinemia or in those having thyroid hormone autoantibodies.

Heterophilic antibodies in patient serum may also cause erroneous results.

ED is considered by many as the “gold standard” method based on the consistent log-linear relationship seen between free T4 ED measurements and TSH in patients with stable thyroid status.1 Analog-based immunoassays performed on automated platforms provide a convenient estimation of free T4, but results do not always agree with the gold standard ED methods. We found that free T4 results from the Centaur immunoassay were generally 40% to 60% lower than those from ED.2 Linear regression analysis of our data gave a slope of 0.46 and an intercept of 0.05 (R2 =0.79).2 This is consistent with previous observations that immunoassays perform reasonably well for samples with normal free T4 but are less reliable in samples with abnormal free T4.3 We suggest free T4 measurements from the automated immunoassay be repeated using the ED method when they are inconsistent with the clinical picture.


  1. The National Academy of Clinical Biochemistry. Laboratory Medicine Practice Guidelines. Laboratory Support for the Diagnosis of Thyroid Disease. 2002. Accessed June 26, 2012.
  2. Song L, Furlanetto R, Lara M, et al. Comparison of two free T4 immunoassays with a direct dialysis method. Clin Chem. 2011;57(10):A189. Accessed June 26, 2012.
  3. Sapin R, d’Herbomez M. Free thyroxine measured by equilibrium dialysis and nine immunoassays in sera with various serum thyroxine-binding capacities. Clin Chem. 2003;49:1531-1535. Accessed June 26, 2012.


This FAQ is provided for informational purposes only and is not intended as medical advice. A clinician’s test selection and interpretation, diagnosis, and patient management decisions should be based on his/her education, clinical expertise, and assessment of the patient.

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