Canis ISSN: 2398-2942

Thyroid: T4AA assay

Synonym(s): T4 autoantibody

Contributor(s): David Bruyette, Peter Graham, Roger Powell


  • Antibodies that cross react with thyroxine (T4) and tri-iodothyronine (T3) are collectively known as thyroid hormone antibodies (THAA).
  • THAA are subsets of thyroglobulin autoantibody (TgAA) Thyroid: TgAA assay.
  • During the process of inflammatory thyroid pathology, antibodies are generated against the largest protein mass within the thyroid gland which is thyroglobulin. Thyroglobulin molecule is the molecular protein location where T3 and T4 are manufactured and stored. When TgAA develops at an epitope within the thyroglobulin molecule that contains a T3 or T4 storage site, the resulting TgAA will have cross reactivity to T3 or T4 respectively. Consequently, only a proportion of TgAAs will cross-react with the thyroid hormones.
  • These antibodies have no functional significance in vivo but in a laboratory immunoassay designed to measure serum T3 or T4 they can interfere and cause false results to be generated.
  • In most of these assay systems THAA cause falsely high results to be generated, but in some others, a falsely low value could instead be the result. 
  • Measurement of Free T4 by equilibrium dialysis (fT4 Thyroxine assay (free) Thyroid: free T4 assay) is not subject to interference by these antibodies as the dialysis membrane does not allow the large antibody molecules to cross into the dialysate used for the measurement of fT4. 
  • Few veterinary studies have shown a varied prevalence of THAA in 6-30% of hypothyroid dogs and that T4AA are present in up to 10%. 


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  • The principle of the THAA tests is the identification of a factor in the patient’s serum that binds T3 or T4, despite the presence of a chemical (8-anilino-1-napthalene sulfonic acid; ANS) that is expected to displace thyroid hormones from their normal binding proteins. In simple terms, radiolabeled thyroid hormone is added to the patient sample and incubated. The proportion of radiolabeled hormone not then recovered out of the serum when in the presence of ANS gives an indication of the amount of THAA (ie inversely proportional to the radioactivity, so the more THAA there is, the less radioactivity is measured after incubation with ANS).  
  • The test is conducted separately for T3 and for T4.  
  • The chemical nature of this test means that it can be applied to any species (unlike TgAA which is species specific). 


  • A few specialist endocrine laboratories globally.



  • Not used in isolation as a diagnostic test, instead being used to determine whether T4 and T3 measurements are likely to have suffered analytical interference. 


  • Not used in isolation as a diagnostic test, instead being used to determine whether T4 and T3 measurements are likely to have suffered analytical interference. 
  • One large scale study of samples submitted to a commercial laboratory found 6% of samples from hypothyroid cases had circulating THAA (T4AA and/or T3AA), typically also having TgAA although the converse was not evident. Their presence can falsely elevate the measured T4 and/or T3 respectively, but clinically significant effects (ie altered clinical interpretation or misdiagnosis) are estimated at <1% of cases for T4. 

Result Data

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Further Reading


Refereed papers

  • Recent references from VetMed Resource and PubMed.
  • Graham P A, Refsal K R, Nachreiner R F (2007) Etiopathologic findings of canine hypothyroidism. Vet Clin North Am Small Anim Pract 37(4), 617-631, v (Review) PubMed.
  • Kemppainen R J, Behrend E N (2001) Diagnosis of canine hypothyroidism. Perspectives from a testing laboratory. Vet Clin North Am Small Anim Pract 31(5), 951-962, vii (Review) PubMed.
  • Gaschen F, Thompson J, Beale K, Keisling K (1993) Recognition of triiodothyronine-containing epitopes in canine thyroglobulin by circulating thyroglobulin autoantibodies. Am J Vet Res 54(2), 244-247 PubMed.
  • Thacker E L, Refsal K R, Bull R W (1992) Prevalence of autoantibodies to thyroglobulin, thyroxine, or triiodothyronine and relationship of autoantibodies and serum concentrations of iodothyronines in dogs. Am J Vet Res 53(4), 449-453 PubMed.