ISSN 2398-2942      

Thyroid: free T4 assay


Vetstream Ltd

Roger Powell

Synonym(s): Thyroxine assay (free), 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.


This article is available in full to registered subscribers

Sign up now to start a free trial to access all Vetlexicon articles, images, sounds and videos, or Login



  • fT4eq/fT4d Equilibrium dialysis (“modified”):
    • Free T4 isolated from serum by passive diffusion through a semipermeable 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 canine values.


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



  • Typically high sensitivity for equilibrium dialysis (>90% typically). 
  • Varies with analogue non dialysis method, eg in hypothyroid dogs (TgAA positive dogs with low fT4eq/fT4d) 62% human assay, 75% veterinary assay but often no better, or worse than, comparative routine T4 assays.
  • As a singular test, fT4eq is no better than combined [T4] with [TSH].


  • Good specificity (>90%) for equilibrium dialysis (fT4eq/fT4d), less so direct analogue platforms (fT4).
  • As a singular test, fT4eq/fT4d is no better than combined [T4] with [TSH].

Technique (intrinsic) limitations

  • fT4:
    • Anti T4 antibodies Thyroid: T4AA assay affect 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 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

This article is available in full to registered subscribers

Sign up now to start a free trial to access all Vetlexicon articles, images, sounds and videos, or Login

Further Reading


Refereed papers

  • Recent references from VetMedResource and PubMed.
  • Randolph J F et al (2015) Free Thyroxine Concentrations by Equilibrium Dialysis and Chemiluminescent Immunoassays in 13 Hypothyroid Dogs Positive for Thyroglobulin Antibody. JVIM 3, 877-881 PubMed Full Article.
  • Behrend E N, Kempannien R J & Young D W (1998) Effect of storage conditions on cortisol, total thyroxine and free thyroxine concentrations in serum and plasma of dogs. JAVMA 212, 1564-1568.
  • Larsson M G (1988) Determination of free thyroxine and cholesterol as a new screening test for hypothyroidism. JAAHA 24, 209-217.
  • Peterson M E, Melian C & Nicholls R (1997) Measurement of serum total thyroxine, triiodothyronine, free thyroxine and thyrotropin concentrations for diagnosis of hypothyroidism in dogs. JAVMA 211, 1396-1402.

Other sources of information

  • Specific commercial laboratory offering free T4 in regard to their methodology and reference data populations etc.


Can’t find what you’re looking for?

We have an ever growing content library on Vetlexicon so if you ever find we haven't covered something that you need please fill in the form below and let us know!


To show you are not a Bot please can you enter the number showing adjacent to this field