Diagnostic Evaluation of The Hypothalamic-Pituitary-Thyroid Axis

• Canine Hypothyroidism

• Feline Hyperthyroidism

Hypothyroidism is a multisystemic disorder associated with reduced cellular metabolic function caused by deficiency of thyroid hormone. In more than 95% of cases this is associated with destruction of the thyroid gland itself (primary hypothyroidism) as a consequence of immune-mediated destruction (lymphocytic thyroiditis) or idiopathic atrophy. Clinical signs of hypothyroidism are very variable (see Table 1) and may occur together or a single sign may predominate. As a consequence hypothyroidism is a differential diagnosis in very many conditions. Problems of over diagnosis can arise because reduced thyroid hormone output is a very common finding in a wide variety of systemic non-thyroidal illnesses (NTI). In addition, some cases of obesity, exercise intolerance and dermatopathy may respond to some extent to thyroid hormone supplementation, even in the absence of hypothyroidism.

Diagnosis therefore currently requires:

• Presence of compatible clinical signs
• Exclusion of other systemic disease
• Demonstration of low (low normal) total and/or free T4
• TSH measurement
• Evaluation of the clinical response to the introduction and withdrawal of T4 supplementation

Long term studies into the significance of thyroglobulin autoantibodies in normal dogs are not complete. Their presence in dogs with compatible signs and equivocal laboratory results however significantly increases the index of suspicion for hypothyroidism associated with lymphatic thyroiditis.

Table 1: Clinical Manifestations of Hypothyroidism in the adult dog

Profile Changes in Hypothyroidism

Routine laboratory test abnormalities are very common in hypothyroidism and include mild normocytic, normochromic, non-regenerative anaemia (40% of cases), hypercholesterolaemia (60-80%), which can be mild to marked, hypertrigylceridaemia (80%), mild increases in alanine transferase (ALT), alkaline phosphatase (ALP) and creatinine kinase (CK) (35%).

While none of these changes are specific, their presence is supportive of hypothyroidism in a dog with appropriate signs. Profiles are also useful in excluding other differential diagnosis, and for identifying other disease conditions that may significantly affect thyroid function (Cushing’s, diabetes mellitus, and other systemic disease).

Where Cushing’s could be a differential diagnosis clinically, it is important to exclude this endocrinopathy before evaluating thyroid function. Cushing’s can significantly reduce basal T4, free T4, TSH and TRH responsiveness resulting in a potentially erroneous diagnosis of hypothyroidism.

Total Thyroxine (T4)

Thyroid gland hormone production comprises T4 predominantly with small amounts of triiodothyroinine (T3). More than 99% of T4 is bound to serum proteins (thyroid binding globulin (TBG) and albumin) leaving 0.1% unbound and available to tissues for intracellular conversion to T3, the metabolically active form. Measurement of total T4 includes both the free and bound T4.

Hypothyroidism, Cushing's, systemic illness and drug therapy can all be associated with alterations in the relative proportions of bound and free T4.

In early hypothyroidism, basal T4 is often within normal limits but declines as the condition progresses. There is however, complete overlap between the T4 concentrations seen in hypothyroidism, NTI and those associated with drug therapy (see Table 2).

Table 2: Factors and conditions associated with reduced total T4

Therefore, middle range or higher basal T4 concentrations help to exclude hypothyroidism whereas low normal or reduced concentrations are not specific for hypothyroidism and require further diagnostic evaluation (see Table 3).

Sample required: 1 ml separated serum or heparin plasma.

Accuracy: As assessed against results of TSH stimulation and therapeutic response, T4 has been reported to correlate with hypothyroidism or euthyroidism in approximately 85% of cases.

Table 3: Interpretation of Basal T4 Concentration (Immulite Canine Thyroxine)

Total Triiodothyronine (T3)

Although T3 is the metabolically active hormone at a cellular level most of the conversion of T4 → T3 occurs intracellularly. Consequently serum T3 is not a good guide to its level within cells. Low, normal and occasionally increased T3 concentrations have been reported in canine hypothyroidism, the latter reflecting the relative shift to T3 synthesis from T4 in the failing thyroid.

Anti-T3 antibodies are more common than anti-T4 antibodies and are often associated with lymphocytic thyroiditis. When present, spuriously high T3 concentrations are evident in some RIA and chemiluminescent assay systems. Their presence is not pathognomic for hypothyroidism however.

Free T4 (FT4)

FT4 theoretically has greater diagnostic value than T4 in that it better reflects available hormone at a cellular level and is less likely to be affected by drugs or NTI. As a consequence, in man it is used extensively to the virtual exclusion of T4.

Endogenous Canine TSH assay (cTSH)

In man, detection of increased TSH concentrations in combination with low FT4 reliably diagnoses primary hypothyroidism.

In dogs only 63-80% of hypothyroid cases have increased cTSH while 8-13% of dogs with NTI have increased cTSH concentrations. As a result cTSH results should be only interpreted in the light of simultaneous T4 measurement after clinical evaluation for the presence of NTI.

Interpretation: Combination of normal cTSH and T4 is very likely to reflect the presence of normal thyroid function. Low T4 with markedly increased cTSH (>2.5ng/ml) is consistent with primary hypothyroidism. Low/low normal T4 with normal/mildly increased cTSH may reflect hypothyroidism, presence of NTI or effects of prior sulphonamide, glucocorticoid or chronic anticonvulsant therapy.

Accuracy: cTSH sensitivity 63-87%, specificity 82-93%, accuracy 80-84%
T4 and cTSH – sensitivity 63-67%, specificity 98-100%, accuracy 82-88%

Thyroid Autoantibodies

Approximately half of all naturally occurring cases of hypothyroidism result from lymphocytic thyroiditis. This condition, analogous to Hashimoto’s thyroiditis in man, is considered to be immune-mediated and is associated with serum autoantibodies directed against the thyroid products thyroglobulin, T3 and less commonly T4.

Previous methods of measuring thyroglobulin antibodies (TGAA) were cumbersome and associated with frequent false positive results in dogs with NTI.
Newer, more specific ELISA methods have been shown to be suitable for diagnostic use however.

Interpretation: Presence of TGAA with clinical and laboratory findings consistent with hypothyroidism confirm lymphocytic thyroiditis.

Euthyroid dogs can have TGAA. The predictive value of TGAA for the subsequent development of hypothyroidism in dogs however is not fully clear.

Provocative Thyroid Function Tests

Thyroid Stimulating Hormone (TSH) Response Test

TSH stimulation test has been regarded as the gold standard test for the diagnosis of canine hypothyroidism for a long time.1 In general, this test is recommended when other means of diagnosis of hypothyroidism have not been able to provide a diagnosis, and the suspicion of hypothyroidism persists. Before testing for hypothyroidism, it is important to exclude canine hyperadrenocorticism, severe non-thyroidal illness and discontinue drugs which can influence the thyroid function (i.e. glucocorticoids, anticonvulsants, NSAIDs) for at least 4-6 weeks, as these can result in reduced responsiveness to TSH. Please note that sight hounds can have naturally very low or even undetectable thyroid levels which can hamper the diagnosis of hypothyroidism in these breeds.

Notes
The TSH we supply is reconstituted recombinant human TSH (rhTSH) which is an approved medical product whose use has been validated in dogs2. Since IDEXX is not licensed as a pharmacy, the reagent is supplied on a named patient basis and must be used as part of a diagnostic test run by IDEXX Laboratories. The cost of the reagent therefore includes T4 assays on the basal and post injection samples.

One vial of TSH contains 75ug of reconstituted rhTSH.
On receipt of the vial(s) it/they should be stored at 4°C and should be used as soon as possible.
Please note the TSH reagent, is non returnable, and will be dispatched Monday to Thursday by Royal Mail Special delivery at no extra charge.

• Collect 1-2 ml blood, as a baseline sample, into a plain or gel serum separation tube
• Inject the contents of one or two vials* of the supplied rhTSH intravenously using normal aseptic technique
• 4-6 hours post injection collect 1-2ml of blood into second plain or gel serum tube
• Label tubes with name and time of sample
• Ensure the samples have clotted fully and separate the serum 30-120 minutes after collection
• Submit the two separated serum samples to the lab requesting TSH stimulation test on the laboratory submission form

* Recent scientific literature indicates that in dogs over 20 kg body weight, the use of 150ug instead of 75ug rhTSH results in a better stimulation and improved the differentiation between primary hypothyroidism and non-thyroidal disease.³

¹ Scott-Moncreiff JCR, Guptill-Yoran L Hypothyroidism p1425 In Textbook of Veterinary Internal Medicine. Ettinger SJ, Feldman EC Eds 5th Edition WB Saunders Philadelphia
² Sauve F, Paradis M Canadian Vet Journal 2000, 41:215-219
³ Boretti FS et al. Comparison of 2 Doses of Recombinant Human Thyrotropin for Thyroid Function Testing in Healthy and Suspected Hypothyroid dogs. J Vet Intern Med 2009: 23: 856-861

Monitoring T4 Replacement Therapy

Pre- and post-pill sampling for T4 can be very useful in the assessment of therapeutic adequacy in dogs on replacement therapy with T4. Considerable variation has been reported in intestinal absorption, rate of metabolism of T4 and in the bioavailability of T4 in differing preparations. As a consequence all dogs on replacement therapy should be sampled after two weeks on therapy, and as necessary thereafter, to optimise the dose and formulation for the individual.

Peak concentrations should be in the upper half or slightly above the top of the normal range. The exact time of peak concentrations post-pill also shows considerable individual variation. However single sampling 4-6 hours post-pill has been recommended. Pre and post-pill testing can be useful in dogs showing a poor response to therapy to check for poor absorption and rapid metabolism.

Feline Hyperthyroidism

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Hyperthyroidism is a common, multisystemic disorder resulting from excessive circulating concentrations of T4 and T3. In 98-99% of cases it is caused by benign thyroid neoplasia (adenomas {adenomatous hyperplasia}). However, 1–2% of cases are associated with functional thyroid carcinoma.
While the condition is recognised in cats as young as 6 years, more than 90% of cases are aged 10 or older. Clinical findings are very variable (see Table 6). Palpable goitre, weight loss and involvement of more than one body system are common features. Common laboratory profile changes in hyperthyroidism are listed in Table 7. Increases in ALT and ALP are very common findings, however normal concentrations of these enzymes do not rule out hyperthyroidism. A variety of other conditions (gastrointestinal disease, diabetes mellitus, cardiac disease, and progressive lymphocytic cholangitis) may show similar profile changes however, and are important differentials clinically.

Table 6: Clinical findings in feline hyperthyroidism

Table 7: Routine profile changes in feline hyperthyroidism

Diagnosis is normally straightforward and is based on the history, clinical findings and presence of increased basal T4. More challenging cases include those with apathetic hyperthyroidism in which unusual clinical signs predominate (reduced activity, anorexia, ventral neck flexion), those in which hyperthyroidism is an incidental finding unrelated to the presenting illness, and those cases with classic clinical signs but an apparently normal basal T4.

Further diagnostic evaluation of the latter group may include a repeat T4 after 2-3 weeks, checking for concurrent illness, drug therapy which may reduce T4 concentration into the normal range in a hyperthyroid individual, or occasionally the use of suppression or stimulation tests.

Where cases are managed medically, periodic monitoring of T4 is useful in assessing the adequacy of therapy. T4 concentrations should be in the lower half of the reference range in cats on therapy.

Total T4

Interpretation: Increased T4 in cats aged 6 years or greater is diagnostic of hyperthyroidism where there are consistent clinical signs. 10-15% of hyperthyroid cats have T4 concentrations in the normal range however. This reflects the fluctuating T4 levels into and above the normal range seen in early disease. Other causes include effects of concurrent non-thyroidal illness or drug therapy which may depress basal T4 into the normal range.

In cats with suspicious signs in which an initial basal T4 is normal (but within the upper half of the normal range), further diagnostic evaluation may still be indicated. Repeat basal T4 after a minimum of 14 days may yield a diagnosis. In other cases in which an early diagnosis is required, a FT4 may be useful to run in addition to the T4. When FT4 is run alone, the diagnostic specificity will be lost because up to 12% of cats with non-thyroidal illness and some euthyroid geriatric cats can have increased FT4 concentrations.

Free T4

Interpretation: Increased FT4 concentrations as determined by equilibrium dialysis (EQ) are found in a similar proportion of hyperthyroid cats to those with elevated T4, i.e. sensitivity of FT4(EQ) and T4 for hyperthyroidism is similar. However, small numbers of cats with non-thyroidal illness have increased FT4 concentrations despite having normal thyroid function. As a consequence, the finding of increased FT4(EQ) is less specific for hyperthyroidism than increased T4.

T4 is therefore preferred as a diagnostic screening test for hyperthyroidism over FT4(EQ), and FT4 should be run with corresponding T4 levels.

Provocative Thyroid Function Tests

T3 Suppression Test

Principle: Release of TSH from the pituitary is inhibited by increased concentrations of T3 which may be induced by administration of oral T3 tablets. In euthyroid cats T4 concentration falls following the reduction in TSH secretion induced by increased T3. In hyperthyroid cats however, when T4 secretion is relatively independent of TSH, little change in T4 is noted following T3 administration.

• Baseline separated serum sample is taken and stored frozen
• T3 (liothyronine 25µg) is given tid per os for 2 days
• On the morning of the third day a second dose is given
• A second sample is taken 2-4 hours post-pill.

Samples are assayed for T4 and T3.

Interpretation: T3 concentrations rise following liothyronine administration and are used to monitor owner compliance with drug administration. Normal cats show a 50% or greater reduction in T4 concentration following liothyronine, and have concentrations of 20 nmol/l or less. Hyperthyroid cats show minimal change in T4 concentration, and have post liothyronine T4 levels in excess of 25 nmol/l. Values between 20-25 nmol/L are non diagnostic.

Accuracy: T3 suppression test is useful in differentiating hyperthyroid from euthyroid cats and has similar sensitivity and specificity to the TRH stimulation test. However, it is a prolonged test and very dependent on good owner compliance. Failure to ingest the liothyronine may result in apparent failure of T4 to suppress and false positive diagnosis of hyperthyroidism.

TRH Stimulation Test

Principle: TRH stimulates the release of TSH from the pituitary and results in increased T4 concentrations in normal cats. In hyperthyroid cats release of TSH is inhibited by high T4 concentrations. As a result hyperthyroid cats stimulate very poorly in response to TRH.

• Baseline separated serum sample is obtained
• TRH (0.1 mg/kg iv) is administered
• A second sample is taken 4 hours later.

T4 only is routinely assayed in this test.

Interpretation: Normal cats and those with non-thyroidal illness have been demonstrated to show an approximately two-fold increase in T4 in response to TRH. Hyperthyroid cats show little increase (<50%) in response to TRH. Increases in T4 of >60% are seen in cats with normal thyroid function. Responses between 50-60% are regarded as equivocal.

Accuracy: TRH stimulation and T3 suppression tests evaluate the pituitary thyroid axis in different ways but have been shown to have similar diagnostic value. Both tests potentially may produce equivocal results in a small percentage of cases however.

Warning: Administration of TRH i.v. is commonly associated with central nervous system signs including salivation, vomiting, tachypnoea and defaecation. These signs are transient and resolve by the time of the second sample.