ISSN 2398-2950      

Thyroid: free T4 assay


Mark Peterson

Roger Powell

Synonym(s): Free thyroxine, free T4, fT4, fT4eq/fT4d


  • Thyroxine (total thyroxine/tT4/T4) is the main secretory product of thyroid gland, as both free and protein bound forms.
  • Over 99.9% of thyroxine is reversibly bound to carrier proteins.
  • The minimal free unbound fraction (free T4/fT4/fT4eq/fT4d) is metabolically active and also converted to tri-iodothyronine (T3) for cellular effects.
  • Diagnostic assays for thyroxine measure both protein-bound (inactive) and free forms.
  • Free T4 assays are designed to measure only the free unbound component but have varied accuracy, in part when using cross reacting human assays for veterinary species.
  • The addition of equilibrium dialysis to the testing (pre-incubation) improves the accuracy of free T4 measurement (fT4eq/fT4d) in veterinary species.


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  • fT4eq/fT4d Equilibrium dialysis (“modified”):
    • Free T4 isolated from serum by passive diffusion through a semi-permeable membrane, which allows only free T4 molecules to pass into the dialysate, but not larger plasma proteins and protein bound T4.
    • This diffusion is passive, taking hours and is typically set up and left overnight to run, the dialysate then tested the following day.
    • Control material should be tested to ensure the filtration system did not fail.
    • A slightly different “standard” dialysis technique using radioactive tracer T4 is also available and has similar performance.
    • Radioimmunoassay (RIA/IRMA) on the dialysate.
  • fT4 ‘Analogue’ competitive EIAs (eg CLIA) or RIAs:
    • Tubes or magnetized particles with coated T4 antibody, their final radioactivity inversely proportional to the sample’s free T4.
    • Have been designed to measure fT4 but in veterinary medicine (cf. human) appear to be less accurate, typically underestimating feline values.


  • Some commercial laboratories use analogue assays (fT4).
  • Fewer specialist laboratories use equilibrium dialysis (fTeq/fT4d).



  • Higher sensitivity for equilibrium dialysis (fTeq/fT4d) cf. ‘analogue’ assays (fT4).
  • Same/slightly higher than total T4 Thyroxine assaydepending on study and methodology, on average 98%.


  • Better specificity for equilibrium dialysis (fTeq/fT4d) cf. ‘analogue’ assays (fT4).
  • Same/slightly lower than total T4 depending on study and methodology, on average 86%.

Technique (intrinsic) limitations

  • fT4:
    • Rare anti T4 antibodies affect (falsely increase) only these analogue tests for free T4, the membrane in equilibrium dialysis preventing such antibodies entering the tested diasylate.
  • fT4eq/fT4d:
    • Samples are often run in duplicate to improve quality control, with repeat testing if the duplicate values are significantly different.
    • The semi-permeable membrane can fail, so the test has to be repeated (eg significantly lipemic or hemolyzed samples).
  • fT4eq/fT4d more expensive and time consuming, analogue fT4 assays more expensive (cf. standard (total) T4 measurement).

Technician (extrinsic) limitations

  • Equilibrium dialysis involves several practical steps in which technician factors may be important, eg filtration set up, sample loading.

Result Data

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Further Reading


Refereed papers

  • Recent references from VetMedResource and PubMed.
  • Peterson M E et al (2015) Evaluation of Serum Thyroid-Stimulating Hormone Concentration as a Diagnostic Test for Hyperthyroidism in Cats. JVIM 5, 1327-1334 PubMed.
  • Peterson M E, Melian C & Nichols C E (1998) Measurement of serum concentrations of total and free T4 in hyperthyroid cats and cats with nonthyroidal disease. J Vet Int Med 12, 211.
  • Mooney C T, Little C J L & MacRae A W (1996) Effect of illness not associated with the thyroid gland on serum total and free thyroxine concentrations in cats. JAVMA 208, 2004-2008.

Other sources of information

  • Specific commercial laboratory offering free T4 in regard to their methodology and reference data populations etc.
  • Feldman and Nelson's (2015) Canine and Feline Endocrinology and Reproduction. 4th edn. Elsevier Science, USA.


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