PetSavers – Understanding the stability of thyroxine in feline blood samples

3 December 2021

Feline hyperthyroidism is a common endocrinopathy and one of the most prevalent diseases of geriatric domestic cats in the UK. The measurement of total serum thyroxine (tT4) is performed regularly, not only as an aid in the diagnosis of feline hyperthyroidism, but also as part of regular feline screening programs to identify occult disease. Pre-analytical errors, made between the point of sampling and laboratory analysis, constitute a large proportion of errors in any biological sample testing. For example, they are estimated to account for up to 70% of errors identified with human samples. In clinical practice, short delays to sample analysis are inevitable, either because of time constraints, or because of the lack of in-house equipment required to perform the analysis. These delays can last from a few hours to a number of days. This storage time and the conditions, particularly temperature fluctuations, to which these samples are subjected are major sources of pre-analytical error in veterinary practice and can consequently cause false results and misdiagnosis.

When testing for hyperthyroidism it is commonly assumed that feline serum samples can be left at room temperature for several days without this affecting the results of tT4 analysis. However, this assumption is based on limited numbers of studies performed in human and non-feline domestic mammal species. For example, a study conducted in dogs has shown that tT4 concentration decreases with storage at 37°C after five days, but not when kept at ambient room temperature, prompting the conclusion that serum samples can be shipped without cooling if assayed within five days. These studies generally suggest that tT4 is stable during short-term storage.

However, similar studies have not been performed to assess the stability of tT4 in feline blood samples. Evidence suggests that the stability of certain hormones in serum samples is not maintained across mammalian species. For example, the serum insulin concentration in horses was found to be only minimally altered after storage at room temperature for 72 hours, while in cows it decreased by as much as 62% after four days, and in humans by 25.7% in just 24 hours. These striking differences are suggested to be a result of variability in hormone structure and complexity, as well as interactions with other serum constituents.

Therefore, the objectives of this project were to evaluate the changes in serum tT4 (measured using the Microgenics enzyme immunoassay method) on samples stored for a number of days at two common temperature conditions seen in clinical practice: room temperature and 4°C. An assessment of changes in tT4 associated with storage over a total period of one week was also made, with assays performed at intervals within this period that may reflect reasonable delays to analysis due to, for example, delayed transportation to an external laboratory or transportation over a weekend. Our results indicated that serum tT4 increased significantly over time up to 168 hours (1 week) at both room temperature and 4°C, and that this daily increase is more pronounced at room temperature. At 4°C, tT4 increased by approximately 0.34 nmol/L per day, while at room temperature the average daily increase in tT4 was approximately 0.79 nmol/L. We hypothesise that this increase reflects changes in the pH of the sample over time, which in turn impacts the chemical reaction used to measure the tT4, rather than reflecting a true increase in tT4 over time. While this increase was statistically significant and noted consistently between groups, the overall magnitude of the increase was not large enough to be likely to change a clinician’s decision with regard to pursuing further diagnostic tests, such as serum-free T4 (fT4), for borderline or unexpected cases.

Determining these effects of short-term sample storage on the stability of feline tT4 in serum has provided clinically relevant data to veterinary surgeons in practice. Although short-term storage at room temperature is unlikely to change a clinician’s approach to a case, we have determined that rapid analysis of feline serum and storage at 4°C is preferable to obtain more accurate results. This is particularly important for cats that may have tT4 at the borderline between normal and high values. We expect our results to contribute to improving feline health and welfare by aiding clinicians in the interpretation of tT4 results following delays in analysis, thereby assisting in early identification and treatment of mild hyperthyroid cases or preventing an incorrect diagnosis and further unnecessary testing.

Applying for a Student Project Grant with BSAVA PetSavers

As a student, I found it very simple to apply for a grant with BSAVA PetSavers to fund my project. In the process of getting project approval, I had to submit project proposals and ethics and welfare committee forms. The BSAVA PetSavers application follows a very similar format, but with the main focus on the potential benefits to pet health and welfare, which I think is already a guiding factor when many students first begin to plan their projects. On submission, BSAVA PetSavers were quick to respond, and direct and concise with their instructions on how they would provide support. Regular reports on progress were required, but were not overwhelming and followed a simple, repeated format. I am extremely grateful to BSAVA PetSavers for funding my student project and for making their process and support as simple and accessible as possible.