Article by Bryan Abram Marks
Effective canine thyroid testing is composed of the following tests, which all play a role in screening dogs for thyroid disorder:
Total T4. This test measures the total amount of T4 (thyroxine) hormone circulating in the blood-both bound and unbound molecules. More than 99% of T4 hormone is “bound,” meaning that it attaches to proteins in the blood and never reaches the tissues.
Therefore, a T4 result by itself is often misleading, since it is affected by anything that changes the amount of binding proteins circulating in the blood (e.g. certain drugs). T4 is still the most popular and widely used initial screening test for thyroid disorder in dogs. As explained below in “The T4 Myth,” relying on the accuracy and sensitivity of this test alone is at the heart of the rampant misdiagnosis of canine thyroid disorder. T4 alone is not an accurate indicator of thyroid disorder in dogs, and is often affected by moderate to severe non-thyroidal illness (NTI), a disease process other than thyroid disease, and certain medications (e.g. phenobarbital, corticosteroids, and sulfonamides).
Free T4. Another essential component of canine thyroid testing is serum free T4, which represents the tiny fraction (< 0.1%) of thyroxine hormone that is unbound and therefore is biologically active. As the free T4 molecule circulates in the blood and through the pituitary gland’s sensor, the level of free T4 tells the pituitary gland whether or not it needs to make more Thyroid Stimulating Hormone (TSH). Although both the bound and free forms of T4 hormone are in circulation, the pituitary gland only recognizes the free molecule. Since protein and certain drug levels in the blood do not (or only minimally) affect free T4, it is considered a more accurate test of true thyroid activity than the total T4. Free T4 is much less likely to be influenced by NTI or drugs. Both total T4 and free T4 are lowered in cases of hypothyroidism. While endocrinologists may favor the equilibrium dialysis (ED) RIA method for measuring free T4 because earlier analog methods were less accurate, newer technologies (improved analog RIAs and non-RIA chemiluminescence and other methods) offer alternative and accurate methodology. These new assays do not require radioisotopes and so, are environmentally “green” and are also faster and less costly.
Total T3. As with total T4, total T3 represents both the bound and unbound forms of T3 circulating in the blood. Measuring serum T3 alone is not considered an accurate method of diagnosing canine thyroid disorder, as this hormone reflects tissue thyroid activity and is often influenced by concurrent NTI. This form of canine thyroid testing is, however, useful as part of a thyroid profile or health screening panel. For example, if levels of total T4, free T4, and total T3 are all quite low, the patient more likely suffers from an NTI rather than hypothyroidism.
If total T3 levels are high or very high in a dog not receiving thyroid supplementation, the patient most likely has a circulating T3 autoantibody (the most common type), which has spuriously (falsely) raised the T3 and/or free T3 level.
Free T3. As with free T4, less than 0.1% of T3 molecules circulate freely in the blood and are biologically active. The blood’s free T3 level tells the pituitary gland whether or not it needs to produce more TSH. Levels of both total and free T3 may be elevated slightly in euthyroid (normal thyroid function) dogs with increased tissue metabolic demands, and are typically spuriously high or very high in dogs with T3 autoantibodies. Both total T3 and free T3 are typically normal in cases of hypothyroidism, unless the disease has been present and undiagnosed for some time, or the dog has concurrent NTI.
Canine Thyroglobulin Autoantibodies (TgAA). Elevated thyroglobulin autoantibodies are present in the serum of dogs with autoimmune thyroiditis, which, of course, is the heritable form of hypothyroidism. As discussed earlier, as much as 90% of cases of canine hypothyroidism result from the heritable condition. TgAA is especially important in screening breeding stock for autoimmune thyroiditis, as dogs testing positive for TgAA should not be bred. The commercial TgAA test can give false negative results if the dog has received thyroid supplement within the previous 90 days, thereby allowing unscrupulous owners to test dogs while on treatment to assert their normalcy, or to obtain certification with health registries such as the OFA Thyroid Registry or Thyroid GOLD™. False negative TgAA results can also occur in about 8% of dogs verified to have high T3 autoantibody and/or T4 autoantibody. The confirmatory version of this test is preferred, as the reagents have been treated to remove any non-specific binding (NSB) proteins. Furthermore, false positive TgAA results may be obtained if the dog has been vaccinated within the previous 30-45 days for rabies, or very occasionally in cases of NTI. Vaccinating dogs with polyvalent vaccines containing rabies virus or rabies vaccine alone has been shown to induce production of antithyroglobulin autoantibodies. This represents an important finding with implications for the subsequent development of hypothyroidism.
T3 Autoantibody (T3AA)/T4 Autoantibody (T4AA). These autoantibodies affect the ability to measure T4 and T3 accurately with most canine thyroid testing methods. In the presence of high levels of circulating T3AA and/or T4AA, the autoantibody interferes with the ability of the test antibody reagent (reacting substance) to detect the hormone being measured. The result is a spuriously high reading of T3 (reported as spuriously low if measured at Michigan State University’s (MSU) Diagnostic Lab) and free T3 or T4 and free T4. However, if the free T4 is measured by the ED technique, the T4AA will be removed by the dialysis step and not be detected. Thus, the presence of T4AA may go unnoticed if free T4 is only measured by the ED canine thyroid testing method. Fortunately, most circulating antibodies are against T3 (~70%), some affect both T3 and T4 (~25%), and only a few affect T4 alone (~5%).
Most cases of autoimmune thyroiditis exhibit elevated serum TgAA levels, whereas only about 20-40% of cases have elevated circulating T3 and/or T4AA. Thus, the presence of elevated T3 and/or T4AA confirms a diagnosis of autoimmune thyroiditis, but underestimates its prevalence, as negative (non-elevated) autoantibody levels do not rule out thyroiditis.
Endogenous Canine TSH (Thyroid Stimulating Hormone). In primary hypothyroidism, as free T4 levels fall, pituitary output of TSH rises. Since about 95% of thyroid hormone regulation in humans is controlled by TSH, it is a highly accurate screening for hypothyroidism. However, only about 70% of thyroid hormone regulation in dogs is controlled by TSH, so this canine thyroid testing method shows relatively poor predictability. The remaining 30% of a dog’s thyroid regulation is controlled by growth hormone, which, like TSH, is manufactured, stored, and secreted by the pituitary gland. For this reason, the TSH test provides a false negative or false positive result in approximately 30% of canine cases. So, although elevated TSH usually indicates primary thyroid disease, there is 20-40% discordance observed between expected and actual results in normal dogs as well as in hypothyroid dogs or those with NTI.
The T4 Myth
The majority of veterinarians believe that serum T4 alone is adequate as the first screening for a thyroid problem, and that only if T4 is abnormal should further canine thyroid testing be pursued. This misconception, which is still perpetuated today by academics at veterinary schools and consultants for veterinary reference labs throughout the country, is a huge obstacle to accurately diagnosing canine thyroid disorders. Additionally, current veterinary medical textbooks have stated that if a dog has a T4 level above 2 ug/dl (26 nmol/L), there is no need to perform other canine thyroid testing because the dog has a normal thyroid. This statement is false and misleading, as the T4 result fails to identify any cases of thyroiditis in which elevated thyroid autoantibodies are present! Many dog guardians are sent away by veterinarians who insist-based solely on a normal or low-normal serum T4-that the dog does not suffer from thyroid disease. These poor people inevitably spend many months and can spend thousands of dollars trying to find out what is wrong with their dog until, hopefully, they one day come across someone who conducts a proper thyroid screening profile (see below). The only way that true progress can be made in the diagnosis of canine thyroid disorder is when veterinarians realize that serum T4 alone is not a reliable method of initial screening, as there are many circumstances in which it can provide misleading results. T4 alone can overdiagnose hypothyroidism in the presence of NTI (such as chronic yeast infections, liver and bowel disorders, and kidney disease-just a few of many examples) or with the use of certain drugs (corticosteroids, phenobarbital, and sulfonamides); it inaccurately assesses the adequacy of thyroxine therapy; and it fails to detect autoimmune thyroiditis.
Furthermore, larger veterinary reference labs are now performing T4 canine thyroid testing on an autoanalyzer along with the serum chemistry profile. These automated analyzer T4 tests frequently read too low in comparison to species-specific RIA or non-RIA T4 assays in healthy dogs. They may also read low in dogs receiving thyroxine supplement or older cats with hyperthyroidism. Thus, the clinician may increase the thyroxine dose of a dog or miss the diagnosis of a hyperthyroid cat based on inaccurate results.
The
