Thyroid testing
Total Thyroxine (Total T4 or TT4)
Total T4 refers to both protein-bound and free T4 in the circulation. It is typically measured in serum by immunoassay, although it can also be measured with other methods, including liquid chromatography–mass spectrometry.
Comments:
• Measurement of total T4 requires the release of T4 from its binding proteins to facilitate its interaction with the measurement system, usually through the use of an agent such as 8-anilino-1-naphthalene-sulphonic acid (ANS).
• With the exception of the uncommonly used liquid chromatography–mass spectrometry (LC-MS/MS) method, all veterinary total T4 analyses are competitive immunoassays reliant on antibodies to capture thyroid hormone and facilitate its detection.
• Assays designed to measure total T4 in humans are optimised for higher concentrations than are useful for the diagnosis of hypothyroidism in dogs and cats, requiring adaption or canine/feline optimised assays.
• Although the underlying immunoassay principle is the same for all commonly used total T4 assays, there are many signal detection technologies and platforms.· Detection systems include radiation (radioimmunoassay, RIA), chemiluminescence (chemiluminescence immunoassay, CLIA), fluorescence (immunofluorescence assay, IFA), and colorimetric (enzyme-linked immunosorbent assay, ELISA, lateral flow).
• Most methods require a step to separate the antibody bound from unbound T4 usually by performing a “wash” step (heterogeneous assays). There are technologies now in use in laboratories (cloned enzyme donor immunoassay [CEDIA], Diagnostic Reagents Inc. [DRI]) and clinics (DRI, surface plasmon enhanced fluorescence [SPEF]) that avoid the wash step (homogeneous assay).
• RIA has long been considered the gold standard method, but no detection system is uniformly superior to the others, as each individual assay method, its particular capture antibodies, its design, and optimised measurement range will determine its usefulness for veterinary total T4 measurement. Each method used by laboratories or clinics should be validated or verified as fit for purpose with appropriate and ongoing quality control.
• Different assay methods used in veterinary laboratories and clinics yield different results, emphasising the importance of local validation and method specific reference intervals and diagnostic cut-offs.
• In dogs, subsets of thyroglobulin autoantibodies (TgAA), such as T3 autoantibodies (T3AA) and T4 autoantibodies (T4AA), can interfere with the immunoassay measurement of their respective hormones. Depending on individual assay design, they can cause falsely higher (results that can potentially increase into or above the reference interval) or lower total T4 results.
• It is important to recognise the likely direction of change caused by T4AA in the chosen assay method.
• For use in diagnostic decision-making, total T4 results generated in clinics should be subject to method verification, performance evaluation, internal quality control, and external quality assessment in the same way as they would in reference laboratories. Results should be interpreted with caution and repeated in a commercial laboratory particularly if equivocal or unexpected.
Free Thyroxine (Free T4 or fT4)
Free T4 refers to the small amount (approximately 0.1%) of T4 in the circulation that is not protein-bound. It is typically measured in serum by immunoassay either with or without a step to separate free from bound T4 in the sample. It can also be measured with other methods, including liquid chromatography–mass spectrometry.
Comments:
- • Immunoassays for free T4 do not include 8-anilino-1-naphthalene-sulphonic acid (ANS), and theoretically only unbound T4 is able to interact with detection antibodies.
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There are two distinct methods commonly used for free T4 measurement.• One method involves equilibrium dialysis to separate the protein-bound from the free T4, followed by an ultrasensitive radioimmunoassay for measurement of T4 in the dialysate. This method is not widely available, subject to stringent sample handling requirements, and is more difficult for routine quality control. Nevertheless, it is the only method currently commercially available that measures true free T4 concentrations capable of providing more diagnostic information than total T4 alone.• The second method does not include a separation step and is made possible through the use of a labelled T4 analogue that theoretically does not react with thyroid hormone binding proteins and is thus available to compete with free T4 for antibody binding sites.• Because the protein binding kinetic assumptions underlying analogue assays can be violated in certain physiological and pathological states, analogue free T4 methods may be subject to interference that generates results that do not provide more information than obtained by total T4 measurement. Circumstances that promote the release of T4 from binding proteins such as increased free fatty acid concentrations and furosemide therapy will cause increases in the measured free T4.• In dogs, subsets of thyroid hormone antibodies (T3AA and T4AA) or anti-thyroid antibodies can interfere with the immunoassay measurement of their respective hormones. Interference is more likely with analogue methods compared with those that utilise equilibrium dialysis for separation of free from bound thyroid hormones. For the analogue methodologies commonly used for free T4 measurement, T4 AAs can cause falsely higher (results that can potentially increase into or above the reference interval) free T4 results. Free T4 results may be affected, with or without a similar effect on total T4 results.
Thyrotropin (TSH)
Hormone secreted from the pituitary gland that regulates thyroid activity. Its measurement can aid the diagnosis of thyroid disorders. The pituitary gland constantly monitors circulating concentrations of T4 and T3 and decreases or increases the secretion of TSH, respectively, in response to even the slightest increase or decrease in thyroid hormone concentrations. Therefore, finding a low serum TSH concentration is consistent with hyperthyroidism, even if total or free T4 concentrations remain in the high-normal range (a situation defined as subclinical hyperthyroidism). In contrast, finding a high serum TSH concentration is considered diagnostic for hypothyroidism, even if serum total or free T4 concentrations remain low-normal (a situation defined as subclinical hypothyroidism).
Minimum Canine Hypothyroidism Panel
A minimum thyroid diagnostic panel for canine hypothyroidism would include a measure of thyroid gland output (looking for decreased total T4 or free T4 in case of hypothyroidism) and a measure of feedback on thyrotrophs (looking for elevation of TSH in case of primary hypothyroidism).
Comments:
- • The additional inclusion of TgAA or T4AA in a profile, if negative, reassures that there is no potential antibody interference in thyroid hormone results.
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• The inclusion of free T4 by equilibrium dialysis may assist in correctly identifying the effects of non-thyroidal illness or medications.
Minimum Feline Hyperthyroidism Panel
In cats suspected of hyperthyroidism, mostly only TT4 is measured; however, in cats with emerging or mild disease, vague clinical signs, or a thyroid nodule cannot be palpated, the use of free T4 and TSH concentration can be helpful.
Comments:
- • Emerging, more sensitive TSH tests might help in the future. A low TSH concentration (<0.01 ng/mL) detected by such sensitive tests supports the diagnosis of hyperthyroidism (example test: pubmed.ncbi.nlm.nih.gov/38382201 [accessed on 4 April 2024]).
T3 and T4 Autoantibodies
In dogs, T3AA and T4AA, respectively, are circulating markers for inflammatory pathology of the canine thyroid glands. They are subsets of TgAA, such that if a dog is TgAA negative, it will not have T3AA or T4AA. However, the converse is not true; it is possible to be TgAA positive and T3AA and T4AA negative.
Comments:
• T3AA can be expected in a higher proportion of hypothyroid dogs than T4AA.
Currently, there are no commercially available assays for their measurement, and those that are available in laboratories will be in-house methods and may provide numerical or titre results that are not directly comparable.
• Autoantibodies do not provide an indication of thyroid function, and their sole presence does not provide an indication for treatment.
Thyroglobulin Autoantibodies (TgAA)
Thyroglobulin autoantibodies are circulating markers for inflammatory pathology of the canine thyroid gland.
Comments:
• In human thyroiditis, the more commonly detected antibodies are anti-thyroperoxidase, but these are inconsistently found in the canine disease.
• A commercial canine TgAA ELISA is available and widely used.
In-house-derived methods are also in use.
• Whichever method is used, it is important that steps are taken in the method to mitigate the possibility of misleading results from non-specific binding of non-TgAA antibodies.
• Autoantibodies do not provide an indication of thyroid function, and their sole presence do not provide an indication for treatment.
• A commercial canine TgAA ELISA is available and widely used.
In-house-derived methods are also in use.
• Whichever method is used, it is important that steps are taken in the method to mitigate the possibility of misleading results from non-specific binding of non-TgAA antibodies.
• Autoantibodies do not provide an indication of thyroid function, and their sole presence do not provide an indication for treatment.
Total Triiodothyronine (Total T3 or TT3)
Total T3 refers to both protein-bound and free T3 in the circulation. It is not the main product of the canine or feline thyroid gland and is mostly generated peripherally. As is the case for total T4, total T3 measurement relies on immunoassay. Its measurement for clinical purposes is not recommended by the ALIVE panel. Total T3 has a much lower diagnostic performance than total T4 for both canine hypothyroidism and feline hyperthyroidism, as it is a less direct measure of thyroid gland function than T4. In addition, a large proportion of hypothyroid dogs (approximately 30%) have TgAA subsets that cross react with T3 (T3AA) that could interfere in total T3 immunoassays and generate misleading results.
An example circumstance in which total T3 has been suggested to be more useful than total T4 is in the attempted diagnosis of hypothyroidism in sighthounds. The total T4 and free T4 reference intervals for sighthounds often extend below reportable range of assays, whereas sighthound reference intervals for total T3 may be more similar to other breeds and be within assay reportable ranges. This is, however, not a universally accepted diagnostic practice, given the lack of evidence in its favour.
Free Triiodothyronine (Free T3 or fT3)
Free T3 it is the bioactive form of thyroid hormone, and it is free to interact with tissues. For reasons similar to those for total T3, free T3 is seldomly included in thyroid diagnostic panels. There is a theoretical rationale as to why free T3 would be an ideal assessment of thyroid function. However, in practice, it does not appear to be a good indicator of thyroid gland hormone output.
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