The initial screening test used to evaluate thyroid function is to measure the basal concentration of total T4 (TT4) in the serum. The concentration of total T4 comprises the fraction of thyroxine bound to plasma proteins (T4) and the free fraction (fT4). The techniques most widely used in laboratories to assay this hormone are the radioimmunoassay (RIA), considered the gold standard method, the enzyme-linked immunosorbent assay (ELISA) and the chemoluminescence assay (CEIA). The reference ranges considered normal for the concentration of T4 in the dog vary between laboratories because of differences between the reagents supplied in the commercial kits and between the techniques and manual skills in individual laboratories. Most laboratories consider dogs with a serum concentration of T4 between 13 and 50 nmol/l as being euthyroid. It is, however, worth emphasising that there are age-related physiological differences in the concentration of thyroid hormones: puppies and dogs up to 6 years old have higher concentrations of hormones than older subjects; there also size- and breed-related differences with medium-sized to large dogs having lower concentrations and Greyhounds, Whippets, Sloughis, Siberian Huskies, Alaskan Huskies and Basenji sometimes having concentrations below the reference range.
The finding of a basal level of T4 higher than 19 nmol/l is strongly suggestive of a state of euthyroidism (with the only exception of cases [1%] in which there are antibodies to T4 which can falsely cause an increase in basal values in dogs with lymphocyte thyroiditis). Hypothyroid dogs usually have basal serum levels of T4 below 19 nmol/l but euthyroid subjects can also have basal levels of T4 below this level if they have concomitant, chronic debilitating disorders (euthyroid sick syndrome) or are being treated with drugs that influence thyroid activity (glucocorticoids, phenobarbital, non-steroidal anti-inflammatory drugs, sulphonamides). In these cases it is the history, clinical examination and laboratory findings (together with the basal levels of fT4 , TSH and the TSH stimulation test) that guide the clinician towards the correct interpretation of the results.
MEASURING FREE T4
Two different methods can currently be used to measure fT4: the radioimmunological assay (RIA) and modified equilibrium dialysis (MED). In all the studies performed, the accuracy of this latter method has been found to be greater than 90%, exceeding that of the T4 assay (75-85%). Compared to the RIA for measuring fT4, the MED technique for fT4 has the advantage of not being influenced by the presence of any circulating anti-thyroid hormone antibodies. From the preliminary studies on the chemoluminescence technique, this method does not seem to be suitable for assaying fT4.
Euthyroid dogs usually have fT4 values above 1.5 ng/dl. Values below 0.8 ng/dl in dogs with clinically suspected hypothyroidism are considered strongly supportive of the diagnosis of this endocrine disorder.
The accuracy of the RIA technique for fT4 varies in the range from 65% to 85%, and its sensitivity and specificity in evaluating thyroid function is equivalent to those of the RIA method for measuring T4. Many of the difficulties encountered in interpreting the values of TT4 are the same as those for fT4, even if this latter parameter is influenced less by the presence of concomitant pathologies. Although a significant difference has been found between the mean values of fT4 in healthy dogs and hypothyroid ones, also in this case the range of values of fT4 in these two categories of animals overlap partially.
MEASURING CANINE THYROID-STIMULATING HORMONE
Three different methods are currently available for measuring the basal concentration of canine TSH (cTSH): (i) an immunoradiometric technique (Coat-A Count Canine TSH IRMA); (ii) a chemoluminescent immunometric technique (Immulite Canine TSH); and (iii) an enzymatic immunometric technique (Milenia canine TSH). All three techniques are considered reliable, although the chemoluminescent one has been shown to be most precise. Since this is a species-specific test, it is not possible to make use of laboratories for human medicine.
Various studies have demonstrated the high specificity (greater than 90%) of this test in the diagnosis of hypothyroidism in the dog; furthermore, if, in association with cTSH, another parameter of thyroid function, such as T4 or fT4 , is also measured, the specificity of the test is greater than when only the basal concentration of pituitary thyrotropin is considered. This is due predominantly to the low sensitivity of basal TSH measurements (60%); indeed, in some hypothyroid dogs (20-40%) the basal values of cTSH are still within the reference range for euthyroid dogs. In most laboratories the upper limit of TSH is set at 0.6 ng/ml.
The finding of decreased basal values of T4 or fT4 in the serum, together with an increase of TSH in a dog with clinically suspected hypothyroidism can be considered strong support for the diagnosis. In contrast, when all these parameters are within the reference range, a state of hypothyroidism can be definitely excluded. A rare finding is increased values of TSH and normal concentrations of T4 or fT4; these findings may reflect an early stage of the disease, as happens in humans. In these cases it is not appropriate to give the subject replacement treatment with levothyroxine, but it is advisable to repeat the test 3 to 6 months later.
TSH STIMULATION TEST
This test, which is intended to evaluate the hormone stores in the thyroid gland by administering exogenous TSH, is currently the most reliable diagnostic test of thyroid function. The test evaluates residual thyroid function by comparing the circulating levels of thyroxine before and after a dose of exogenous pituitary thyrotropin. The major advantage of this test over the others listed above is that in the dog it can differentiate hypothyroidism from other non-thyroid disorders that can cause a reduction in the circulating levels of thyroid hormones, particularly in the early stages of the endocrine pathology. Until a few years ago this test could be carried out using TSH of bovine origin (bTSH). bTSH is no longer available and only in recent years has a substitute become commercially available: recombinant human TSH (rhTSH), which is produced by genetic engineering.
Performance of the test
Blood samples are taken for the T4 assays immediately before and 4 to 6 hours after the intravenous administration of 75-100μg of rhTSH.
Interpretation of the results
In healthy dogs the post-stimulation concentration of T4 in the serum is higher than 32 nmol/l and exceeds the basal (pre-stimulation) value of T4 by at least 1.5 times. Post-stimulation values of T4 below 20 nmol/l and less than 1.5 times the basal value are considered diagnostic of hypothyroidism. If the post-stimulation T4 is between 20 and 32 nmol/L or is higher than 32 nmol/l but is not more than 1.5 times the basal value, the test result is considered equivocal; this can occur occasionally in subjects with severe, debilitating non-thyroid disorders. In these cases it is advisable to continue the diagnostic investigations by carrying out scintigraphy of the thyroid.
Suggested readings
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- Feldman E.C. , Nelson R.W.: Hypothyroidism. In:Canine and Feline Endocrinology and Reproduction, III edizione Saunders, pg. 87-151, 2004.
- Gaughan K.R. Bruyette D.S.: Thyroid function testing in Greyhound. J Am Vet Med Ass, 215, 1999.
- Hill R.C. et al.: Effects of racing and training on serum thyroid hormone concentration in racing Greyhound. Am J Vet Res, 62, 2001.
- Marca M.C.: Evaluation of canine serum thyrotropin (TSH) concentration: comparison of three analytical procedures.J Vet Diagn Inv , 13(2), 1992.
- Nelson R.W. et al.:Serum free thyroxine concentration in healthy dogs, dogs with hypothyroidism, and euthyroid dogs with concurrent illness. J Am Vet Med Ass ,198, (8), 1991.
- Reimers T.J. et al.: Effects of age, sex, and body size on serum concentration of thyroid and adrenocortical hormones in dogs. J Am Vet Res, 51, 1990.
- Peterson L.B. et al.: Measurement of serum total thyroxine, triiodothyronine, free thyroxin, and thyrotropin concentrations for diagnosis of hypothyroidism in dogs. J Am Vet Med Ass, 21, (11), 1997.