Abstract
Graves’ disease (GD) is the most common cause of pediatric thyrotoxicosis; however, differentiating it from other conditions is challenging. A high free triiodothyronine-to-free thyroxine ratio (FT3/FT4) ratio has been established as an indicator of GD in adults. In this retrospective observational study, we aimed to investigate the utility of the FT3/FT4 ratio as a screening marker for pediatric GD by analyzing the medical records of 105 patients aged 1–18 years who presented with thyrotoxicosis at three hospitals in Japan. The participants were divided into a GD group (TSH receptor antibody > 2.0 IU/L and requiring antithyroid drug for at least six months; n = 70) and a non-GD group (n = 35). The median (range) of the FT3/FT4 ratio was significantly higher in the GD group than in the non-GD group (3.41 [1.90–5.22] vs. 2.92 [1.50–4.40]; p < 0.05). Receiver operating characteristic curve analysis of the FT3/FT4 ratio revealed an area under the curve of 0.693 (95% confidence interval [CI], 0.577–0.808). At the optimal cutoff value of 2.88, the FT3/FT4 ratio demonstrated a sensitivity of 0.86 (95% CI: 0.80–0.91) and a specificity of 0.51 (95% CI: 0.39–0.62) for GD screening. These findings suggest the usefulness of the FT3/FT4 ratio as a screening marker for pediatric GD.
Keywords: thyrotoxicosis, Graves’ disease, destructive thyroiditis, children, painless thyroiditis
Highlights
● This study is the first to examine the utility of the FT3/FT4 ratio as a screening marker for pediatric Graves’ disease.
● The FT3/FT4 ratio was significantly higher in the GD group than in the non-GD group.
● The FT3/FT4 ratio with the optimal cutoff value of 2.88 can be a valuable screening tool for pediatric GD.
Introduction
Thyrotoxicosis is characterized by excessively high levels of thyroid hormones in the blood. Graves’ disease (GD) is the most common cause of thyrotoxicosis in children and adults (1); however, pediatric GD accounts for < 5% of all GD cases (2, 3). Other causes of thyrotoxicosis include destructive thyroiditis (DT) and painless thyroiditis (PT). DT involves inflammatory destruction of follicles and leakage of thyroid hormones into the bloodstream (4,5,6). PT, a subtype of DT, falls within the spectrum of autoimmune thyroid disorders and is characterized by the presence of anti-thyroglobulin antibodies (TgAb) or antithyroid peroxidase antibodies (TPOAb) (7, 8). Treatment options for GD include antithyroid drug therapy, thyroidectomy, and radioactive iodine therapy. However, these options are not feasible for the treatment of DT and PT (9,10,11). Therefore, differentiating between GD and non-GD thyrotoxicosis is crucial, though it is clinically challenging. Thyroid scintigraphy may be required (12) in addition to the measurement of thyroid hormones and TSH receptor antibody (TRAb)/thyroid stimulating antibody (TSAb), which are essential for definite diagnosis of GD. However, performing scintigraphy can delay the initiation of treatment.
The Japanese Thyroid Association guidelines for the diagnosis of GD state that “the ratio of free triiodothyronine (FT3) to free thyroxine (FT4) (FT3/FT4 ratio) helps rule out PT” (12); however, it does not mention any specific cutoff value. Previously, the ratio of total triiodothyronine (TT3) to total thyroxine (TT4) (TT3/TT4 ratio) was considered helpful in differentiating GD from DT (13, 14). Currently, FT3 and FT4 levels are commonly measured because they are less influenced by thyroid hormone-binding proteins (15). Previous studies involving adult patients have reported that the FT3/FT4 ratio was useful in differentiating GD from non-GD thyrotoxicosis (16,17,18,19,20); the likelihood of GD increased as the FT3/FT4 ratio increased. An FT3/FT4 ratio > 2.4–2.6 indicated a nearly 90% sensitivity for GD (16,17,18), whereas an FT3/FT4 ratio > 3.4–4.4 indicated a specificity of > 90% (19, 20). However, the utility of the FT3/FT4 ratio has not yet been studied in children. Therefore, in the present study, we aimed to investigate the utility of the FT3/FT4 ratio in differentiating GD from non-GD thyrotoxicosis in the pediatric population.
Materials and Methods
Subjects
In this study, GD was defined by the presence of TRAb positivity requiring prolonged antithyroid drug therapy. The inclusion criteria for this study were patients who (i) visited one of the participating hospitals (Keio University Hospital, Tokyo Metropolitan Children Medical Center, or Niigata University Medical and Dental Hospital) between April 2010 and March 2021; (ii) had TSH ≤ 0.1 µIU/mL and FT4 ≥ 2.0 ng/dL on simultaneous blood testing; (iii) received at least one assessment for FT3 and TRAb; (iv) were aged 1–18 yr; (v) were not receiving antithyroid medication or levothyroxine at the time of their initial visit; and (vi) were followed up for at least six months after the initial TSH and FT4 assessments. The exclusion criteria for this study were patients (i) whose FT3 or FT4 value exceeded the maximum detection limit and (ii) who were TRAb-negative and had received methimazole (MMI) therapy for more than six months. Subjects who met all inclusion criteria and none of the exclusion criteria were enrolled.
Study design
This retrospective observational study was based on a review of patient characteristics, including sex, date of birth, length, height, and weight at diagnosis, complications, medical history, family history of thyroid disease, and date of the last visit, which were extracted from electronic medical records. Laboratory data included TSH, FT3, FT4, TRAb, TSAb, TgAb, TPOAb, and thyroglobulin (Tg). Imaging studies included thyroid echography (location, size, and superior thyroid artery blood flow) and thyroid scintigraphy (if performed). The treatment history of the patients was examined to determine whether they had received antithyroid drug therapy or oral levothyroxine therapy.
Thyroid function values before treatment were used for the analysis. Because two different assays for FT3 and FT4 were used in this study, the data were converted using linear regression based on a previous report (21). The thyroid function test data were converted using Elecsys kit (electrochemiluminescence immunoassay [ECLIA]) (Roche Diagnostics GmbH, Mannheim, Germany) in accordance with the following conversion formula: LUMIPULSE kit (chemiluminescent enzyme immunoassay [CLEIA]method) (Fuji Rebio Inc., Tokyo, Japan) FT3: (conversion value) = 1.03564 × (value measured by LUMIPULSE kit) + 0.3496; FT4: (conversion value) = 0.9993 × (value measured by LUMIPULSE kit) − 0.1525.
TRAb, TgAb, and TPOAb levels were measured by ECLIA using Elecsys kit (Roche Diagnostics, Tokyo, Japan). The cutoff values for TRAb, TgAb, and TPOAb positivity were 2, 28, and 16 IU/mL, respectively.
Methods
The participants were divided into a GD group and a non-GD group. The GD group consisted of patients with TRAb > 2.0 IU/L who had received antithyroid drug therapy for at least six months after thyrotoxicosis was confirmed. The primary endpoint was the difference in the FT3/FT4 ratio between the groups. The secondary endpoints were differences in FT3 and FT4 values between the groups and the cutoff value for the FT3/FT4 ratio separating the two groups. In addition, subjects in the non-GD group who tested positive for anti-TPO or anti-thyroglobulin antibodies were categorized into the PT group and compared with the GD group in a subgroup analysis.
Statistical analysis
Statistical analysis data are expressed as the mean ± standard deviation (SD) or median and range. Normally distributed data were compared using the Student’s t-test, and nonparametric data were compared using the Mann–Whitney U test. The proportions between the two groups were compared using the chi-square test. Quantitative variables were assessed for any correlation using Pearson’s correlation analysis. Statistical significance was set at p <0.05. Receiver operating characteristic (ROC) curve analysis was used to establish the cutoff value for the FT3/FT4 ratio separating the two groups. The optimal cutoff value was determined using Youden’s index, which maximizes the sum of sensitivity and specificity. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (R Foundation for Statistical Computing, Vienna, Austria). The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Keio University School of Medicine (approval number: 20210050).
Results
Patients’ baseline data
Of the 141 patients with thyrotoxicosis, 34 were excluded because their FT3 or FT4 level exceeded the maximum detection limit, and two were excluded because they were TRAb-negative and had received MMI therapy; therefore, a total of 105 patients were available for the final analysis (17, 36, and 52 at Keio University, Niigata University, and Tokyo Metropolitan Children’s Medical Center, respectively). The GD and non-GD groups consisted of 70 and 35 patients, respectively. The patients in the non-GD group were classified into a PT group (DT with the presence of TPOAb or TgAb, n = 19) and an “Other” group (DT without autoantibodies, n = 11; unknown, n = 5). In the non-GD group, five patients had TRAb > 2 IU/L (range: 2.6–8.9 IU/L) and received either MMI therapy within six months or no treatment.
Table 1 shows that the two groups did not differ with respect to sex, age, weight, and body mass index (BMI Z-score) at diagnosis. The median number of days of follow-up after diagnosis differed significantly at 2,192 (range: 202–5,689) and 1,492 (range: 218–3,914) in the GD and non-GD groups (p = 0.01), respectively. All subjects were observed for more than six months after the initial evaluation and received their diagnosis during or after the observation period. Thyroid echography is a practical alternative diagnostic modality to scintigraphy (22,23,24). However, the results of echography could not be analyzed because objective metrics, such as superior thyroid artery blood flow, were not assessed uniformly in the cohort.
Table 1. Basic characteristics of the GD and non-GD groups.
Biochemical test results: FT3, FT4, FT3/FT4 ratio, and Tg
The FT3, FT4, and FT3 / FT4 ratio were significantly higher in the GD group than in the non-GD group. The median (range) FT3 value in the GD group (15.2 [3.8–30.4] pg/mL) was significantly higher than that in the non-GD group (7.0 [3.0–21.6] pg/mL; p < 0.05) (Fig. 1). The median (range) FT4 value in the GD group (4.5 [2.0–9.2] ng/dL) was also significantly higher than that in the non-GD group (2.4 [1.8–5.1] ng/dL; p < 0.05) (Fig. 2). Similarly, the median (range) FT3/FT4 ratio in the GD group (3.41 [1.90–5.22]) was significantly higher than that in the non-GD group (2.92 [1.50–4.40]; p < 0.05) (Fig. 3). Outliers also existed; one patient in the GD group with a low FT3/FT4 ratio (1.9) was categorized as having mild GD owing to a high TRAb value (3.9 IU/L) and required antithyroid medication (thiamazole at a starting dosage of 5 mg [0.11 mg/kg]) for one year; however, the outlier patient showed no complications. In contrast, two patients in the non-GD group had a high FT3/FT4 ratio (4.24 and 4.40, respectively) despite being negative for TRAb and had received thiamazole therapy for only a few months, followed by levothyroxine administration.
Fig. 1.
Dot chart of free triiodothyronine values (pg/mL). GD, Graves’ disease.
Fig. 2.
Distribution of free thyroxine values (ng/dL). GD, Graves’ disease.
Fig. 3.
Distribution of the free triiodothyronine-to-free thyroxine ratio (FT3/FT4 ratio). GD, Graves’ disease.
Tg was measured in 47 and 24 patients in the GD group and non-GD group, respectively; it did not differ significantly between the two groups, with the median value being 86.8 ng/mL and 46.5 ng/mL in the respective groups (p = 0.85).
ROC curve analysis
ROC curve analysis of the FT3/FT4 ratio yielded an area under the curve of 0.693 (95% confidence interval [CI], 0.577–0.808) (Fig. 4). The optimal cutoff value for the FT3/FT4 ratio was determined as 2.88, which demonstrated a sensitivity of 0.86 (95% CI: 0.80–0.91) and a specificity of 0.51 (95% CI: 0.39–0.62). At a lower cutoff value for the FT3/FT4 ratio > 2.64, sensitivity increased to 0.90 (95% CI: 0.84–0.95) albeit with a corresponding decrease in specificity to 0.40 (95% CI: 0.29–0.49). Conversely, at a higher cutoff value for the FT3/FT4 ratio >3.78, specificity increased to 0.94 (95% CI: 0.84–0.98) although sensitivity decreased to 0.29 (95% CI: 0.23–0.31).
Fig. 4.
Receiver operating characteristic curve of the free triiodothyronine-to-free thyroxine ratio (● marks the optimal cutoff value).
Subgroup analysis: GD and PT
A subgroup analysis of patients in the non-GD group (n = 35) with PT (n = 19) with patients in the GD group found no significant differences in terms of sex, age, weight, and BMI Z-score at diagnosis; however, FT3 and FT4 values and FT3/FT4 ratio were significantly higher in the GD group than in the PT group (p < 0.05). ROC curve analysis examining the cutoff value for the FT3/FT4 ratio separating the two groups demonstrated an area under the curve (AUC) of 0.72 (95% CI: 0.58–0.86), with the FT3/FT4 ratio of 2.88 being the optimal cutoff value for the diagnosis of GD at a sensitivity of 0.86 (95% CI: 0.81–0.90) and a specificity of 0.58 (95% CI: 0.39–0.74).
Discussion
The FT3 and FT4 levels and FT3/FT4 ratio were significantly higher in the GD group than in the non-GD group. ROC curve analysis using the FT3/FT4 ratio yielded an AUC of 0.693, with the optimal cutoff value of 2.88. With a target sensitivity and specificity of 90%, the corresponding cutoff values for the FT3/FT4 ratio were 2.64 and 3.78, respectively, which aligned with previously reported values in the adult population (FT3/FT4 ratio of 2.4–2.6 for high sensitivity and 3.4–4.4 for high specificity) (16,17,18,19,20). These findings suggest the usefulness of the FT3/FT4 ratio as a screening marker for GD in the pediatric population similar to the adult population.
Although some studies addressed the utility of the FT3/FT4 ratio in differentiating GD from non-GD thyrotoxicosis in adults, other studies argued that its diagnostic utility is limited (25,26,27). Walfish (25) and Nikolai et al. (26) reported that the TT3/TT4 ratio was not reliable for distinguishing between GD and PT, owing to overlapping values. Our study is the first, to the best of our knowledge, to provide pediatric-specific data on the FT3/FT4 ratio in this context. Although considerable overlap was observed in our pediatric cohort, the FT3/FT4 ratio was significantly different between the GD and non-GD groups. Given that FT3 and FT4 assays are the most widely used thyroid function tests in clinical practice, the FT3/FT4 ratio may be a useful screening parameter for GD, particularly when rapid TRAb or TSAb results are not immediately available.
The present study has three limitations. First, 34 patients were excluded because their FT3 or FT4 level exceeded the maximum detection limit. Four, 11, and 19 patients had values above the limit for FT3, FT4, and both FT3 and FT4, respectively. All excluded patients were TRAb-positive and received antithyroid medication. These limitations are similar to those of previous studies in adults (16,17,18,19,20), which restricted the analysis to patients with measurable hormone levels. Second, the diagnosis of GD should be based on a comprehensive approach that incorporates various diagnostic tools, such as TRAb assay, ultrasound, and scintigraphy. Second- and third-generation TRAb assays are highly effective in diagnosing GD because of their sensitivity of 90–100%, depending on the cutoff value used (27, 28). Nonetheless, approximately 3–5% of patients with GD may not have detectable TRAb when evaluated using these assays (29, 30). In the present study, only four patients underwent scintigraphy, a procedure that is not widely available in pediatric hospitals in Japan. Third, to harmonize FT3 and FT4 values obtained from different assay kits, we applied a conversion formula, which might have led to variations in results depending on the measurement reagent, particularly at high concentrations. Despite these limitations, the present study is the first to describe the utility of the FT3/FT4 ratio in screening pediatric thyrotoxicosis. Future research should aim to develop a comprehensive diagnostic scoring system that incorporates the FT3/FT4 ratio, TRAb levels, and ultrasonographic findings, such as superior thyroid artery blood flow velocity, to improve diagnostic accuracy in clinical practice.
Conclusion
A high FT3/FT4 ratio is a useful screening marker for pediatric GD.
Conflict of interests
None of the authors have any potential conflicts of interest associated with this research. There was no public or commercial funding received for this study.
Acknowledgments
We thank Mr. James R. Valera for his assistance with editing of this manuscript.
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