Investigation of canine thyroid function

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Hypothyroidism is a common endocrine disorder in dogs. Approximately 90% of cases are due to acquired, primary disease (lymphocytic thyroiditis or idiopathic thyroid atrophy). Middle-aged dogs are most commonly affected and Golden Retrievers, setters, Doberman Pinschers, Great Danes, Schnauzers, spaniel breeds and terriers appear predisposed. The clinical findings include skin changes (alopecia, seborrhoea, pyoderma, puppy coat, hyperpigmentation, hyperkeratosis, myxoedema and slow hair growth), lethargy, obesity, intolerance to cold and reproductive abnormalities. When thyroid disease is suspected routine haematology and biochemistry screens are recommended to eliminate other conditions which may produce similar clinical signs. Nonspecific laboratory findings in hypothyroid dogs include hypercholesterolaemia, mild non-regenerative anaemia, high serum alkaline phosphatase activity and high serum creatinine kinase activity. None of these changes are pathognomonic. Skin biopsy may reveal non-specific histological findings for example, orthokeratotic hyperkeratosis, epidermal atrophy and epidermal melanosis or findings suggestive of hypothyroidism for example, vacuolated and/or hypertrophied arrector pili muscles, thickened dermis and increased dermal mucin. There are a great many factors affecting thyroid hormone levels in dogs and confirmation of hypothyroidism can be difficult. It is essential that a thorough clinical examination is performed and history taken prior to any diagnostic testing. The following factors are also very important to consider when carrying out thyroid function tests:

Concurrent therapy

Certain therapies can affect thyroid hormone results and complicate their interpretation. Glucocorticoid, barbiturate medications and sulphonamides often cause low total T4 (TT4) concentrations. Where possible, it helps to discontinue these therapies prior to sampling (for 1 month for glucocorticoids and 3 weeks for sulphonamides). However, these guidelines are not rigid and there is a high degree of confidence in ‘normal’ results obtained at any time. Where therapy cannot be withdrawn or where there is a possible false positive result, a diagnostic panel including FT4D (see below) is recommended. If a previous diagnosis of hypothyroidism is in question, levothyroxine therapy should be withdrawn for 4-6 weeks prior to testing, but as noted above a valid ‘normal’ result could be obtained at any point prior to this.

Breed specific ranges

Sight hounds are known to have lower TT4 levels compared to other breeds. The diagnosis of hypothyroidism in these breeds should be made cautiously and more reliance should be placed on other measures of thyroid function that are less influenced by breed such as TSH (or total T3).

Concurrent illness

Dogs with non-thyroidal illnesses will often have low serum TT4 (sick euthyroid syndrome) as part of their physiological response to that illness. This is not a T4 deficient state and thyroid supplementation is not appropriate. Instead, it is a reflection of the mechanism used to control metabolic rate during illness that is believed to improve the chances of survival. The effect of non-thyroidal illness on FT4D values is less common and less dramatic. Ill dogs will often have depressed TT4 levels so the use of additional or alternative thyroid function tests along with careful evaluation of clinical signs and assessment of the likelihood of nonthyroidal illness are important in distinguishing the true hypothyroid dogs from those that are just responding to a non-thyroidal illness.

We recommend the investigation of possible thyroid disease or dysfunction is performed using panels of several tests rather than individual tests. The diagnostic power of a group of thyroid tests is much greater than that of any available single test. Our panels are made up of selections of the following tests: total T4, free T4 by equilibrium dialysis, thyroid stimulating hormone, thyroglobulin antibody and thyroid hormone antibodies.

Individual diagnostic tests

Total T4 (TT4)

Most dogs with hypothyroidism would be expected to have a low TT4 (~95% of them) making this a test with high, but slightly less than perfect, diagnostic sensitivity. On the other hand, the effects of non-thyroidal illness on TT4 means that many dogs with normal thyroid function will also have a low TT4 making the test poorly specific. Depending on the type of population sampled, up to 25% of dogs with normal thyroid function will yield a low TT4. This poor diagnostic specificity and less than perfect sensitivity means that TT4 has limited value as a stand-alone test for hypothyroidism. Diagnostic power is improved by combining it with TSH measurement. Total T4 also has a role to play in monitoring thyroid therapy where it can be used alone (if hours post-pill and dose frequency are recorded) or ideally in combination with TSH (see below).

Free T4 by Equilibrium Dialysis (FT4D)

Almost all (>99.9%) of circulating T4 is bound to carrier proteins leaving only a tiny fraction available to interact with tissues. This free fraction can be measured in an ultra sensitive radioimmunoassay following an equilibrium dialysis step. The analysis of FT4D is the most accurate way of assessing the physiologically important thyroid status of an animal. Samples are dialysed, separating FT4 from serum proteins and protein bound T4. In most cases, TT4 and FT4D will be highly correlated, but this is not always so and in the following circumstances it is advisable to measure FT4D instead of, or in addition to, TT4:

Non-thyroidal illness

One of the contributing mechanisms to the low TT4 we see in nonthyroidal illness is an alteration in thyroid hormone-protein binding. Although TT4 concentrations may be greatly reduced, the lower protein affinity for T4 means a higher fraction is available as free hormone and the FT4D concentration usually remains within the reference range. This makes FT4D a good test for distinguishing non-thyroidal illness from true hypothyroidism as the cause of a low TT4. Less than 10% of dogs with non-thyroidal illness have a FT4D below the reference interval.

Concurrent therapies

Part of the effect on certain therapies on TT4 is mediated through thyroid hormone-protein binding meaning that FT4D is less commonly and less dramatically affected by concurrent therapy. FT4D is the analysis of choice when glucocorticoid or barbiturate therapies cannot be withdrawn prior to embarking on a thyroid diagnostic investigation.

T4 autoantibodies

The presence of T4 cross-reacting antibodies (T4AA) in the patient’s serum will interfere with TT4 measurement causing false high values. The FT4D procedure is unaffected by these antibodies because they are removed by the dialysis step. For an accurate estimation of thyroid status in a dog with T4AA, FT4D is required. T4AA are present in the serum of approximately 10% of hypothyroid dogs as part of the thyroid pathologic process.

Canine TSH (cTSH)

We expect serum concentrations of cTSH to be high in animals with primary hypothyroidism because the negative feedback effect of thyroid hormones on pituitary production of TSH is lost. Indeed, this is the case most of the time but the diagnostic sensitivity is less than ideal. About 80- 85% of hypothyroid dogs will have the expected high cTSH, unfortunately, leaving a proportion that will not. Conversely, cTSH measurement has good diagnostic specificity (up to 100%) meaning that false positives are rare. The combination of thyroid hormone analysis with cTSH measurement makes the most of the advantages of the individual tests while minimising their deficiencies. Canine TSH may be measured at 30 minutes as part of a TRH stimulation test in the diagnosis of secondary hypothyroidism. Normal dogs should increase cTSH by at least 0.4ng/ml.

Total T3 (TT3)

The analysis of TT3 is of little value in the diagnosis of hypothyroidism principally because of the high prevalence of cross-reacting T3 autoantibodies (T3AA) in hypothyroid dogs. These antibodies cause false results to be generated in T3 assays.

Thyroglobulin autoantibody (TgAA)

The presence of TgAA in serum is strongly suggestive of immune mediated lymphocytic thyroiditis which is responsible for more than half of the cases of canine hypothyroidism. In the remainder there is no serological or histological evidence of inflammation. This test is used to document the presence and type of thyroid pathology. It does not provide information on the functional status of the thyroid glands. Evidence of thyroid dysfunction does not occur until lymphocytic thyroiditis has destroyed more than 50-60% of thyroid functional mass. Therefore TgAA evidence for thyroid pathology can be seen in animals before dysfunction occurs and while serum TT4 and cTSH concentrations are still normal. Some breeds of dog have a much higher prevalence of serum TgAA indicating a genetic predisposition to immune mediated thyroiditis. The screening of breeding lines or families for TgAA can be helpful for people wishing to breed away from a thyroiditis and hypothyroidism pre-disposition. A study which monitored the progress of dogs with positive TgAA results revealed that after one year, 1 in 20 dogs have laboratory evidence of overt hypothyroidism and 1 in 5 have laboratory evidence of early/partial thyroid failure.

T4 and T3 antibodies

Subsets of thyroglobulin antibody exist in certain hypothyroid dogs which cross-react with T4 or T3 assays. These antibodies lead to falsely high thyroid hormone levels due to interference in the respective assay systems. FT4D is not subject to this interference.

Thyroid diagnostic panels

  • Canine Thyroid Profile Full-Standard (CTP5): canine TSH and TT4 are measured in addition to TgAA. Recommended initial test, which provides information regarding thyroid function and thyroid gland pathology (lymphocytic thyroiditis)
  • Canine Thyroid Diagnostic Profile Full-Premium (CTP6): TT4, Canine TSH, Free T4D and TgAA. This panel provides the most comprehensive information available from a single sample. The TgAA identifies thyroid pathology and the TT4, FT4D and TSH provide a complete picture of thyroid function. Recommended where there is non-thyroidal illness and concurrent therapy
  • Canine Thyroid Diagnostic Profile Mini Standard (CTP1): canine TSH is measured in combination with TT4 and this increases diagnostic accuracy over TT4 alone
  • Canine Thyroid Diagnostic Profile Mini-Premium (CTP4): canine TSH is measured with Free T4D

Dynamic testing

TRH stimulation test (TRHS)

The TRH stimulation test is used to detect primary and secondary hypothyroidism in dogs and is the alternative to the thyrotropin (TSH) stimulation test. The main limitation of the test is that little information has been published regarding the effects of drugs or non-thyroidal illness. The response, seen as increases in T4, is less than seen with the TSH stimulation test using bovine TSH.


  • Take 2ml of clotted or heparinised blood
  • Inject TRH slowly i/v over 1 minute using the dose rates below. Please note: TRH can cause mild side effects such as defecation, salivation, tachypnoea. If these cause a problem, atropine is an effective antidote. Thyrotropin Releasing Hormone is available on prescription as TRH Cambridge Laboratories*.
1-5kg 100μg TRH
5-30kg 200μg TRH
>30kg 300μg TRH
  • Take a second 2ml of clotted or heparinised blood 4 hours after TRH injection
  • If possible separate the serum or plasma before dispatch to the laboratory and label the tubes ‘pre’ and ‘post’ TRH


  • If the pre-stimulation T4 is >25nmol/l, regardless of the post-stimulation concentration, the dog is normal
  • With a pre-stimulation T4 <25nmol/l, the post-stimulation values can be interpreted as follows:
0-20nmol/l Hypothyroid or sick euthyroid
20-30nmol/l Status equivocal
>30nmol/l Normal
  • Dogs with equivocal values should be retested after 1 month
  • In dogs with repeated equivocal results it may prove helpful to assess the degree of stimulation
  • The post TRH stimulation T4 concentration in a normal dog should increase to >1.2 x the basal T4 concentration, and rise to >30nmol/l

TRH Stimulation of TSH

The TRH stimulation of TSH is essentially a `differentiation test’ and should not be used in an attempt to diagnose canine hypothyroidism. When the results of a TRH stimulation test indicate hypothyroidism this test may be useful in identifying those dogs suffering from secondary disease as a result of inadequate pituitary thyrotropin secretion (TSH). Decreased secretion of thyrotropin releasing hormone (TRH) from the hypothalamus - tertiary hypothyroidism - has not been confirmed in the dog. Secondary hypothyroidism is comparatively rare, such cases representing <5% of the true hypothyroid population. The TSH response is measured on samples taken 30 minutes after i/v injection of TRH, the protocol is as recommended for a standard TRH stimulation test (see earlier text).


  • In normal dogs the pre stimulation TSH is between 0-0.41ng/ml and this should increase by at least 0.4ng/ml
  • Basal concentrations of TSH >0.6ng/ml are consistent with primary hypothyroidism and effectively exclude a diagnosis of secondary disease
  • In cases of secondary hypothyroidism the pre and post stimulation TSH concentrations ought to be low
  • Dogs with hyperadrenocorticism, a common differential diagnosis, may elicit a similar pattern to secondary hypothyroid cases, with a reduced response to TRH
  • Concentrations of TSH measured 30 minutes post TRH stimulation:
0-0.3ng/ml Reduced TSH response to TRH. Likely secondary hypothyroid (possibly HAC)
0.3-1.1ng/ml Normal TSH response to TRH

Therapeutic monitoring

TT4 levels may be monitored after two weeks of therapy to determine whether a dose change would be appropriate. It is assumed that maintaining T4 concentrations above the lower limit of the reference interval will correct the clinical signs of hypothyroidism. Samples for total TT4, collected at 3-4 hours post treatment (peak) and just before drug administration (trough) provide the most information regarding drug metabolism and the likely therapeutic success in any individual. However, where this approach is not practicable, it is possible to collect one sample, at least 2 hours after administration of medication, and to extrapolate the expected T4 concentrations for the remainder of the day. Extrapolation of the T4 concentrations from a single result is based on the range of half-lives reported for the drug (6-10 hours). To draw and interpret a therapeutic graph for your patient, we will require the timing of the sample in relation to the last medication (at least two hours) and the frequency of dosing. A trough sample may be more helpful to assess once daily therapy.

The measurement of both TT4 and cTSH is recommended in monitoring thyroid therapy in dogs. Canine TSH reflects the adequacy of thyroid replacement therapy in the preceding days, not just the day of the test. This can help identify inconsistencies in dosing and also prevent an inappropriate management decision being made based on a non-representative single day TT4 result.

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