Our findings highlight the diagnostic value of the FT4/TSH ratio in GD, demonstrating its high specificity (99.4%) and PPV of 92.3%, with an AUC of 0.740. These results position the FT4/TSH ratio as a valuable, non-invasive alternative diagnostic marker for GD—particularly in resource-limited settings where access to TRAb testing is constrained. Although the FT4/TSH ratio showed lower sensitivity, its diagnostic utility can be significantly enhanced when combined with clinical symptoms, imaging studies, and other supportive diagnostic indicators.
While both FT4 and the FT4/TSH ratio exhibited comparable specificity, the notably higher AUC observed for the FT4/TSH ratio underscores its superior diagnostic potential. Furthermore, the strong positive correlation between FT4/TSH ratio and TRAb levels found in our study supports its role as a reliable surrogate biomarker in the diagnosis of GD.
Interestingly, our analysis revealed that FT3, FT3/FT4, and FT3/TSH ratios did not reach statistical significance (P > 0.05), indicating that these markers may not be ideal for diagnosing GD. This may be attributed to the smaller sample size and the limited routine use of FT3 testing. Additionally, the higher cost of FT3 assays, as compared to TSH and FT4, restricts their application to selective, reflective testing in specific clinical scenarios. This finding further emphasizes the practicality and accessibility of FT4 and TSH measurements, which are widely available and routinely requested in thyroid function assessments across Malaysia.
In the current healthcare landscape, timely and accurate diagnosis of GD is essential for improving patient outcomes. In Malaysian public healthcare settings, TRAb testing is centralized and processed through a single reference center to optimize costs. However, this approach results in significant delays—often exceeding three months—before results are available. These logistical challenges reinforce the urgent need for reliable, rapid, and cost-effective diagnostic alternatives that can be readily adopted in decentralized healthcare facilities.
Previous studies have explored the diagnostic potential of ratios such as FT3/FT4 (
9-
13) and FT3/TSH (
8) in differentiating GD from other thyroid disorders. A recent study by Khan et al. (conducted from November 2023 to May 2024) reported that the FT3/FT4 ratio achieved a sensitivity of 87%, specificity of 85%, and an AUC of 0.92 in distinguishing GD from subacute thyroiditis (
15). Similarly, another study found an AUC of 0.936 for the FT3/FT4 ratio, with sensitivity and specificity values of 89.7% and 91.8%, respectively (
16). Despite these promising results, the studies were limited by small sample sizes and single-center designs, potentially restricting the generalizability of their findings.
Moreover, there is variability in the diagnostic confirmation methods employed across studies. For example, a study by Ibrahim and Hamam (
16) used confirmatory imaging techniques such as Technetium-99m thyroid scans, while others relied on TRAb testing. This lack of standardization underscores the need for consistent diagnostic criteria in future research to better validate the diagnostic performance of hormone ratios in GD.
In contrast, Shigemasa et al. demonstrated that the total T3/T4 ratio effectively distinguishes GD from painless thyroiditis (PT) (
17). However, the FT3/FT4 ratio showed significant overlap between the two groups (
17), making it a less reliable diagnostic marker. This discrepancy arises because the T3/T4 ratio reflects total hormone levels (both bound and unbound), which are influenced by variations in thyroxine-binding globulin (TBG) and prealbumin (TBPA). In contrast, the FT3/FT4 ratio focuses exclusively on unbound hormones, which are more directly affected by changes in binding capacity.
Our findings align with those of Zhang et al. in China, who identified the FT4/TSH ratio as a valuable diagnostic marker for GD. Their study reported an AUC of 0.846 (
14), with high specificity (90.16%), sensitivity (71.52%), a PPV of 90.77%, and an overall diagnostic accuracy of 79.44% at a cut-off value of > 5731.286 pmol/mIU. Similarly, an earlier five-year study (2015 - 2019) demonstrated that the FT4/TSH ratio was statistically significant (P < 0.0001) in differentiating GD from autoimmune thyroiditis (
8). That study reported an AUC of 0.83 (95% CI: 0.81 - 0.86, P < 0.0001), with an optimal cut-off of 5.99 pmol/mIU, achieving 76% sensitivity and 75% specificity.
A key distinction between our study and these prior investigations lies in the higher specificity and PPV observed in our findings. High specificity is essential for minimizing false-positive results, which helps prevent misdiagnosis and unnecessary treatment with anti-thyroid medications. This not only alleviates psychological stress for patients and their families but also enhances the accuracy of clinical decision-making. Furthermore, the high PPV indicates that most positive test results in our study are true positives, offering clinicians greater diagnostic confidence while reducing the need for additional confirmatory testing. As a result, healthcare resources are conserved, and timely, appropriate management of GD is facilitated.
While previous studies often relied on RAIU or 99mTcO4 thyroid scintigraphy to confirm GD, these methods are resource-intensive and not universally accessible. Additionally, those studies were conducted exclusively in populations of Chinese ethnicity, highlighting the importance of population-specific research. Genetic and environmental factors can influence optimal diagnostic thresholds, reinforcing the relevance of studies such as ours that focus on Southeast Asian populations. Our study also benefits from a larger sample size, which improves statistical power, reduces bias, and enhances the reliability of the findings.
In our cohort, GD cases were characterized by significantly elevated FT4 levels and TSH values near the assay's lower detection limit, resulting in markedly elevated FT4/TSH ratios. This pattern reinforces the utility of the FT4/TSH ratio as a clear marker of GD, especially in distinguishing it from NGD hyperthyroidism—an essential clinical consideration. Given the prolonged delays in TRAb test availability in Malaysia, the FT4/TSH ratio serves as a valuable and timely diagnostic alternative.
Minimizing dependence on TRAb testing allows clinicians to reduce costs, expedite diagnosis, and improve patient care by avoiding the risks associated with delayed intervention. While TRAb assays are highly sensitive, their lower specificity at commonly used cut-off values may lead to false-positive results (
7). Although increasing the TRAb cut-off improves specificity, the FT4/TSH ratio provides a simpler, more efficient, and cost-effective alternative. It maintains diagnostic reliability while reducing the need for additional confirmatory testing.
Our study employed a third-generation chemiluminescence immunoassay for TSH and FT4 measurements, minimizing interference from protein-binding abnormalities such as hypoalbuminemia or low TBG. This approach reduced the risk of falsely low FT4 results. The assays demonstrated high precision, with consistently low coefficients of variation (CVs), thereby ensuring reliable and reproducible data.
The high-sensitivity TSH assay, with a detection limit of 0.005 mIU/L, accurately identified the suppressed TSH levels characteristic of thyrotoxicosis, effectively capturing the strong inhibitory feedback exerted by elevated FT4 on TSH in GD. Diagnostic accuracy was further enhanced by correlating clinical findings with TRAb positivity, minimizing the risk of falsely low TSH values. To maintain measurement precision, patients with known protein-binding abnormalities were excluded from the study.
The FT4/TSH ratio cut-off was derived from robust FT4 and TSH measurements. Notably, the median TSH value in the study cohort was 0.01 mIU/L, well above the assay’s detection limit, ensuring precise ratio calculations and reinforcing diagnostic reliability.
A key strength of our study is the use of a well-defined patient cohort and standardized FT4 and TSH measurements, which significantly enhance the reliability and reproducibility of the findings. The application of stringent inclusion and exclusion criteria helped to minimize potential confounding factors, ensuring that the observed association between the FT4/TSH ratio and GD was clinically meaningful.
However, several limitations must be acknowledged. The retrospective design may introduce bias, as data were collected from existing records rather than through a prospective approach. Additionally, the small sample size for FT3 measurements limited the statistical power to draw definitive conclusions regarding the diagnostic utility of FT3/FT4 and FT3/TSH ratios. The study population, drawn exclusively from the East Coast of Malaysia, may not fully reflect the ethnic and genetic diversity of the broader Malaysian population, highlighting the need for larger, multi-centre studies to enhance the generalizability of the results.
While the FT4/TSH ratio proved highly effective for diagnosing GD, its reliability may diminish in cases of subclinical hyperthyroidism or when TSH suppression is mild. In such instances, TRAb levels may be normal or only slightly elevated. Moreover, the FT4/TSH ratio alone cannot distinguish GD from other causes of NGD hyperthyroidism, such as toxic multinodular goitre or thyroiditis, which share overlapping clinical features but necessitate different management approaches.
Free thyroxine remains a highly sensitive marker of TH feedback, especially when TSH levels are markedly suppressed (
14). However, FT4 readings can be affected by conditions such as hypoalbuminemia or altered TBG levels. One limitation of our study is the absence of total T4 measurements, which would have provided a means to validate FT4 accuracy. Correlating FT4 with total T4 is recommended, as discrepancies between the two can indicate underlying protein-binding issues.
Future studies should incorporate total T4 assessments to better evaluate the accuracy of FT4 assays. Additionally, prospective, multi-centre research involving diverse ethnicities, age groups, and varying disease severities is essential to validate the FT4/TSH ratio as a diagnostic tool for GD. Expanding the diagnostic framework to include non-invasive modalities such as thyroid ultrasonography or scintigraphy, in conjunction with the FT4/TSH ratio, may further refine diagnostic algorithms. Future investigations should also prioritize differentiating GD from conditions with overlapping presentations, such as subacute thyroiditis, which have distinct etiologies and therapeutic implications (
14).
5.1. Conclusions
This study highlights the FT4/TSH ratio as a promising diagnostic tool for the early identification of GD, particularly in healthcare settings where access to TRAb testing is limited or delayed. By streamlining the diagnostic process, the FT4/TSH ratio offers the potential to facilitate faster, more efficient clinical decision-making, ultimately contributing to improved management and patient outcomes in GD.