Establishment of Reference Intervals for Plasma TSH Across the Lifespan in an Algerian Mediterranean Population

ABSTRACT

Background

Thyroid‐stimulating hormone (TSH) serves as a sensitive marker of thyroid function; however, its interpretation is subject to variation across different populations due to physiological, environmental, and methodological factors. Currently, no validated TSH reference intervals (RIs) have been established for the Algerian population.

Objective

This study aimed to establish age‐specific, assay‐validated reference intervals for plasma TSH levels in an Algerian Mediterranean population using a standardized immunometric platform.

Methods

A cross‐sectional study was conducted on 3012 apparently healthy participants (1 day to 93 years). After excluding outliers and antibody‐positive individuals, 2882 participants were analyzed. Plasma TSH levels were measured using the Mindray CL‐900i chemiluminescent assay. Nonparametric percentile methods were applied to estimate age‐ and sex‐specific RIs with bootstrap‐derived confidence intervals.

Results

Age‐specific TSH reference intervals varied markedly across the lifespan. The widest limits were observed in neonates (2.5th–97.5th percentiles: 0.50–22.41 mIU/L), followed by progressive narrowing through childhood and adulthood (19–50 years: 0.54–4.68 mIU/L). In older individuals, the upper reference limit increased modestly, reaching 5.17 mIU/L after age 70. All reference intervals were derived using non‐parametric percentile methods following outlier exclusion.

Conclusion

This study provides the first locally validated age‐specific TSH reference intervals for Algeria. Age was the principal determinant of TSH variation, whereas sex‐related differences were not clinically significant. The results enhance the diagnostic precision for thyroid disorders and support assay‐specific standardization in North African laboratories.

This study establishes the first age‐specific reference intervals for plasma thyroid‐stimulating hormone (TSH) in an Algerian Mediterranean population using the Mindray CL‐900i platform. Analysis of 2893 healthy individuals revealed a strong age dependency of TSH values, with highest levels in neonates and mild elevation in older adults, while sex‐related differences were minimal. These findings provide assay‐validated, population‐specific reference values to improve thyroid disorder diagnosis and promote standardization across North African laboratories.

1. Introduction

Thyroid disorders are among the most prevalent endocrine conditions worldwide, affecting a significant proportion of the population [1]. These conditions often manifest with nonspecific and subtle symptoms, making early diagnosis challenging. Laboratory testing plays a central role in the diagnosis and long‐term management of thyroid dysfunction, with thyroid‐stimulating hormone (TSH) measurement widely accepted as the initial and most informative test owing to its high sensitivity, clinical relevance, and accessibility [2, 3, 4].

However, TSH concentrations are influenced by multiple physiological and environmental factors [5, 6]. In addition to these biological determinants, analytical variability poses a significant challenge to clinical interpretation. Variations in assay design, calibration, and antibody specificity across different immunoassay platforms contribute to inter‐method bias, impeding the harmonization of TSH results [7, 8]. Despite the efforts of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and its Working Group for Standardization of Thyroid Function Tests to introduce reference measurement procedures and calibration materials for better comparability [9], significant inter‐assay discrepancies persist [7]. As a result, establishing universally applicable reference intervals (RIs) remains challenging, and laboratories continue to depend on manufacturer‐supplied or literature‐based reference ranges that may not accurately reflect the characteristics of the local population.

Iodine status significantly influences population‐level TSH variations, leading to notable geographic disparities in the reference values. Consequently, there is a need to establish population‐specific, assay‐validated TSH reference intervals [10]. In the Mediterranean region, research has been conducted in Southern Europe [11, 12] and parts of the Eastern Mediterranean [13]; however, data from North Africa remain limited. Notably, no study has established validated assay‐specific TSH reference intervals for the Algerian population, despite national data indicating sufficient iodine intake.

Algerian laboratories currently employ various immunoassay platforms that utilize manufacturer‐provided reference ranges, which have not undergone independent verification for local applicability. This practice raises concerns about potential diagnostic inaccuracies due to mismatches between the population and the assay. To address this issue, the present study aimed to establish assay‐specific RIs for serum TSH using a single third‐generation immunometric platform in a large, well‐defined Algerian cohort. The study encompassed a broad age range, from neonates to 93 years, and represents the first comprehensive effort to define locally validated RIs for TSH in Algeria.

2. Materials and Methods

A cross‐sectional study was conducted between March 2023 and August 2024 at the Immunology Department of the Beni‐Messous University Hospital Center (Algiers, Algeria). The primary objective of this study was to establish age‐specific RIs for plasma TSH in an apparently healthy Algerian population, following the recommendations of the National Academy of Clinical Biochemistry (NACB) [4] and the Clinical and Laboratory Standards Institute (CLSI C28‐A3) [14]. Ethical approval was granted by the Institutional Ethics Committee (approval no. [19/CE/2025]). Written informed consent was obtained from all adult participants or the legal guardians of minors, with assent secured where appropriate.

2.1. Study Population

Participants aged 1 days to 93 years were recruited from various settings within the Beni‐Messous University Hospital Center and its surrounding areas. This region is considered to be relatively homogeneous in terms of genetic background and environmental exposure. No known regional differences in iodine intake have been reported in this area, which is regarded as iodine sufficient according to national public health data.

Neonates were enrolled from the maternity ward, and infants, children, and adolescents were recruited from pediatric outpatient clinics and vaccination centers. Adults were recruited from hospital staff, outpatient clinics, and community volunteers. Only one blood sample was collected per participant for TSH measurement. All TSH measurements were performed in a single laboratory using a single analyzer.

2.1.1. A Priori Inclusion

The participants were clinically asymptomatic and stable, with no prior diagnosis of thyroid disease. The eligibility criteria required the absence of clinical or historical evidence of thyroid dysfunction, such as goiter, nodules, previous thyroid surgery, or treatment for hypothyroidism or hyperthyroidism. Goiter was screened solely by palpation, without ultrasound, across all study populations. Furthermore, participants could not be pregnant or postpartum within the preceding six months, nor could they have an acute systemic illness, active infection, or chronic non‐thyroidal disease, including renal, hepatic, or autoimmune diseases.

Age‐specific requirements were also applied:

• –Neonates and infants (≤ 1 year) were required to have been born full‐term, with normal birth weight, no perinatal complications or systemic illness. Inclusion was limited to neonates born to mothers whose TSH levels remained within trimester‐specific reference ranges established for pregnancy, as confirmed by a comprehensive review of their medical records.
• –Children and adolescents (1–19 years) were expected to exhibit normal growth and development according to the WHO standards, with no chronic or endocrine disorders and regular school attendance. Adolescents with clinically detectable goiters were excluded. No additional age‐specific criteria were applied to this population.
• –Adults (> 19 years) were required to have no personal or family history of thyroid disease, not be breastfeeding, and not be taking medications that are known to interfere with thyroid function.

2.1.2. A Posteriori Exclusion

After recruitment, participants were excluded if they tested positive for thyroid autoantibodies, defined as TPOAb > 9 IU/mL and/or TgAb > 4 IU/mL. TPOAb, TgAb, and FT4 levels were systematically measured in all study participants.

2.2. Age Stratification

To accommodate physiological variations in TSH secretion, participants were categorized into 11 predefined age groups: neonates (1–7 days), infants (1 week–1 year), young children (1–5 years), children (5–12 years), adolescents (12–19 years), young adults (19–30 years), and subsequent decades for adults (30–40, 40–50, 50–60, and 60–70 years), with a final category for the elderly (> 70 years). This stratification adhered to the CLSI C28‐A3 guidelines, with a minimum target of 120 participants per group to ensure statistical validity [14].

2.3. Sample Collection and Processing

Venous blood was collected in lithium‐heparin tubes under standardized pre‐analytical conditions. The fasting duration was adjusted according to the age group, with approximately 4 h for neonates and 6–8 h for children and adults. Sampling was conducted between 08:00 and 10:00 am to minimize the diurnal variation. Plasma was separated within 30 min of collection by centrifugation at 4000 rpm for 10 min. Samples that were hemolyzed, lipemic, or icteric were excluded. All analyses were conducted on the day of collection to ensure the stability of the analytes.

2.4. Laboratory Analysis

Plasma concentrations of TSH, FT4, TPOAb, and TgAb were quantified using a Mindray CL‐900i chemiluminescence immunoassay analyzer, employing reagent kits validated by the manufacturer. The TSH assay is a two‐site sandwich immunoassay with chemiluminescent detection based on the alkaline phosphatase activity. The assay's performance was verified against the WHO International Standard for TSH (NIBSC code: 81/565), yielding a measured value of 8.36 μIU/mL compared to a defined value of 9.00 μIU/mL, indicating a 7.11% deviation, which was within the ±7.8% acceptance limit [9, 15]. Precision was assessed in accordance with the NCCLS EP5‐A2 guidelines, involving duplicate testing of two control levels over a 20‐day period, resulting in within‐device coefficients of variation (CVs) of 3.13% (0.75 μIU/mL) and 2.32% (62.47 μIU/mL). Throughout the study, a three‐level Bio‐Rad Immunoassay quality control system was employed to ensure the continuous monitoring of analytical performance and assay reliability.

2.5. Statisticals Analysis

Data analysis was performed using R software (version 4.4.1). Plasma TSH concentrations were summarized by age group using descriptive statistics, including median, interquartile range, minimum, and maximum values. The distribution was evaluated visually through quantile–quantile (Q–Q) plots and analytically via the Anderson–Darling test. Given that several age groups demonstrated non‐normal distributions, non‐parametric methods were utilized for both reference interval estimation and group comparisons. Reference intervals were defined as the 2.5th–97.5th percentiles, with 90% confidence intervals calculated using bootstrap resampling (10,000 iterations). Outliers were identified and excluded according to CLSI recommendations, specifically employing the Tukey and Horn approaches. Sex‐related differences in TSH levels were examined using the Wilcoxon rank‐sum test.

3. Results

Of the 3186 subjects initially recruited, 3012 were retained following the exclusion of individuals with abnormal FT4 values or positivity for TPOAb/TgAb antibodies. Subsequent analysis of TSH values across different age groups led to the identification and exclusion of 130 aberrant measurements using the Tukey method, resulting in a final analytical sample of 2882 participants. Table 1 shows the descriptive distribution of TSH values by age group, both prior to and after excluding outliers.

TABLE 1.

Descriptive statistics of TSH values by age group: before and after outlir removal in the reference population.

TSH (μ TSH (mUI/L)UI/mL)
Partition	Before outlier detection					Outlier characteristics		After outlier removal
	N	Average (SD)	Median (Q25 _Q75)	Minimum value	Maximum value	Number of outliers	Intervals of outliers a	N′	Average (SD)	Median (Q25 _Q75)	Minimum value	Maximum value
1_7 Days	129	9.23 (9.594)	5.40 (2.66–12.04)	0.112	42.37	9	[30.5–42.4]	120	7.22 (6.294)	4.90 (2.64–10.11)	0.112	25.99
1 Week_1 Year	160	3.724 (2.444)	3.319 (2.174–4.406)	0.27	16.55	11	[8.14–16.6]	149	3.23 (1.56)	3.21 (2.11–4.20)	0.27	7.56
1_5 Years	340	3.465 (2.181)	2.89 (2.066–4.126)	0.47	16.65	21	[7.27–16.65]	319	3.062 (1.441)	2.748 (2.041–3.868)	0.47	7.17
5_12 Years	154	2.969 (1.851)	2.661 (1.909–3.677)	0.17	10.73	8	[6.52–10.7]	146	2.659 (1.286)	2.545 (1.896–3.498)	0.17	6.112
12_19 Years	142	2.473 (1.49)	2.083 (1.53–2.95)	0.266	8.100	9	[5.23–8.1]	133	2.195 (1.028)	1.953 (1.51–2.829)	0.266	4.922
19_30 Years	532	2.313 (1.272)	2.107 (1.401–2.895)	0.313	8.506	16	[5.34–8.51]	516	2.18 (1.025)	2.027 (1.393–2.773)	0.313	5.118
30_40 Years	385	2.292 (1.322)	1.96 (1.384–2.197)	0.310	8.641	13	[5.43–8.64]	372	2.147 (1.075)	1.92 (1.352–2.783)	0.310	5.187
40_50 Years	358	2.239 (1.257)	1.901 (1.348–2.873)	0.33	6.921	8	[5.36–6.92]	350	2.149 (1.118)	1.858 (1.34–2.781)	0.33	5.16
50_60 Years	355	2.315 (1.243)	2.04 (1.349–3.038)	0.294	8.166	5	[5.75–8.17]	350	2.25 (1.118)	2.014 (1.344–2.992)	0.294	5.563
60_70 Years	254	2.266 (1.517)	1.938 (1.23–2.83)	0.270	8.894	14	[5.31–8.89]	240	2.011 (1.087)	1.778 (1.124–2.712)	0.27	5.16
> 70 Years	203	2.405 (1.800)	1.905 (1.135–2.883)	0.26	8.876	16	[5.51–8.88]	187	2.010 (1.200)	1.600 (1.097–2.612)	0.26	5.397

Note:  N′: Final number after exclusion of aberrant values.

^a Range of values that have been identified as outlier.

The data distribution was assessed both visually with Q–Q plots and quantitatively through the Anderson–Darling test. Both methods revealed significant deviations from normality, prompting the choice of a nonparametric percentile method. Reference intervals, encompassing the central 95% of the population within each age subgroup, were derived from the 2.5th and 97.5th percentiles. These limits, along with their 90% and 95% bootstrap confidence intervals, are detailed in Table 2.

TABLE 2.

Reference intervals for TSH by age subgroups.

N = 2882	TSH (mUI/L)
	N	2.5th percentile	IC 90%	IC 95%	97.5th percentile	IC 90%	IC 95%
1_7 Days	120	0.50	0.24–1.36	0.24–1.55	22.41	20.57–25.54	20.45–25.54
1 Week_1 Year	149	0.61	0.28–0.79	0.17–0.79	7.38	7.23–8.71	7.23–8.76
1_5 Years	319	0.88	0.62–1.01	0.59–1.05	6.57	6.06–7.21	6.06–7.25
5_12 Years	146	0.33	0.27–0.58	0.27–0.63	5.31	4.88–5.72	4.73–5.82
12_19 Years	133	0.74	0.62–0.89	0.42–0.90	4.55	4.30–4.89	4.27–4.89
19_30 Years	516	0.67	0.63–0.74	0.63–0.75	4.58	4.33–4.71	4.29–4.72
30_40 Years	372	0.65	0.47–0.74	0.44–0.74	4.68	4.47–4.90	4.46–4.94
40_50 Years	350	0.54	0.46–0.61	0.45–0.62	4.65	4.47–4.85	4.42–4.88
50_60 Years	350	0.58	0.49–0.71	0.46–0.73	4.74	4.53–4.88	4.51–5.00
60_70 Years	240	0.42	0.36–0.58	0.36–0.58	4.50	4.21–4.78	4.12–4.78
> 70 Years	187	0.51	0.30–0.59	0.29–0.64	5.17	4.52–5.35	4.42–5.35

The effect of sex on serum TSH concentrations was examined using statistical analyses in different age groups. The neonatal (1–7 days) and early infancy (1 week–1 year) cohorts were omitted from this analysis due to their small sample sizes and considerable physiological variability in TSH levels, which could obscure sex‐based comparisons.

Among individuals older than 1 year, sex‐specific comparisons of TSH distributions were performed within each predefined age group using the Wilcoxon rank‐sum test. The Hodges–Lehmann estimator of the median difference, along with the corresponding 95% confidence intervals, was calculated to assess the effect size. The reference intervals, specifically the 2.5th and 97.5th percentiles, were determined using a non‐parametric method when the sample size was greater than 120 (n > 120), and a robust method when the sample size was less than 120 (n < 120). These calculations were conducted for each group following the exclusion of outliers, which were identified and removed using the Tukey method. Table 3 summarizes these sex‐specific comparisons and the corresponding reference limits (2.5th–97.5th percentiles) with 90% confidence intervals, stratified by age group and sex.

TABLE 3.

Reference intervals for serum TSH (mUI/L) stratified by age group and sex.

Partition	Females (n = 1626)					Males (n = 987)
	N	2.5th percentile	IC 90%	97.5th percentile	IC 90%	N	2.5th percentile	IC 90%	97.5th percentile	IC 90%
1_5 Years	145	0.707	0.438–0.803	6.177	5.616–6.647	174 a	1.19	0.946–1.508	6.99	6.857–7.885
5_12 Years	74	0.299	0.181–0.582	5.199	4.579–5.900	72	0.250	0.177–0.335	5.588	4.951–5.976
12_19 Years	82	0.672	0.494–0.725	4.493	4.096–4.723	51	0.782	0.562–0.834	4.842	4.763–5.260
19_30 Years	379 a	0.670	0.599–0.709	4.599	4.472–5.067	137	0.695	0.542–0.871	4.607	4.201–4.872
30_40 Years	270	0.531	0.354–0.646	4.64	4.239–4.803	102	0.710	0.663–1.013	4.862	4.775–5.287
40_50 Years	231	0.540	0.479–0.631	4.809	4.628–5.159	119	0.469	0.218–0.508	4.648	4.136–5.066
50_60 Years	224	0.660	0.550–0.766	4.742	4.607–4.982	126	0.442	0.259–0.579	4.961	4.700–5.425
60_70 Years	123	0.469	0.360–0.546	4.588	4.401–5.064	117	0.358	0.139–0.447	4.795	4.431–5.353
> 70 Years	98	0.367	0.116–0.474	5.351	5.305–5.577	89	0.517	0.427–0.735	4.699	4.268–5.288

^a Groups in which outlier values were detected and excluded prior to calculating the reference intervals.

Although statistically significant differences (p < 0.05) were observed in some age groups (19–30 and 40–50 years), none of the median differences exceeded 0.30 μIU/mL. This magnitude falls below the commonly recognized thresholds for clinical relevance, indicating that the observed sex‐based differences are unlikely to be of practical significance. The results are presented in Figure 1, which shows the overall serum TSH distribution by sex, as well as age‐group‐specific overlaid density plots comparing female and male TSH distributions within each age category.

FIGURE 1.

Sex‐ and age‐stratified distribution of serum TSH. (A) Overlaid density curves showing the overall distribution of TSH concentrations in females and males. (B) Age‐group–specific density curves comparing TSH distributions between females and males within each age category (TSH, mIU/L).

4. Discussion

Establishing reliable population‐specific RIs for TSH is essential for the accurate diagnosis and management of thyroid disorders in the general population [16]. Although TSH is the most sensitive indicator of thyroid function, its interpretation is affected by physiological factors such as age, sex, and iodine status, as well as interassay variability across different laboratory platforms. In many iodine‐sufficient countries, including Algeria, clinical laboratories frequently rely on reference values derived from foreign populations or manufacturer‐supplied ranges that may not accurately reflect local physiological values. This reliance can potentially lead to misclassifications, particularly in borderline cases.

The primary aim of this study was to establish robust, age‐specific reference intervals for serum TSH concentrations in a well‐defined euthyroid population. By employing nonparametric percentile estimation and bootstrap‐derived confidence intervals, we identified significant age‐related variability in the TSH distribution. Although sex‐based differences were statistically significant in certain age groups, their magnitude did not reach clinically meaningful thresholds in any age group.

A primary finding of this study is that serum TSH concentrations exhibit significant variability across different age groups, underscoring the necessity for age‐specific reference intervals. The population was initially stratified into 11 distinct age groups, ranging from neonates (1–7 days) to adults aged > 70 years. This detailed stratification facilitated an in‐depth examination of TSH distribution across critical developmental and physiological stages, including infancy, childhood, adolescence, adulthood, and aging. Using this methodology, we identified notable differences in the lower and upper percentiles of TSH values. For instance, the 97.5th percentile varied from 22.41 μIU/mL in neonates to 4.68 μIU/mL in adults aged 30–40 years, with subtle variations even between adjacent decades of adulthood. These findings emphasize the continuous non‐linear progression of thyroid function with advancing age.

Statistical analysis revealed that several adjacent age categories did not show significant differences, indicating a potential issue of over‐segmentation. To rectify this, we implemented a revised partitioning scheme consisting of five groups, merging certain age categories where TSH levels were both statistically and clinically consistent. The newly defined categories (1–7 days, 1 week–5 years, 5–12 years, 12–60 years, and > 60 years) maintain critical developmental milestones while improving the robustness of the reference intervals by increasing subgroup sample sizes and narrowing confidence intervals. Importantly, the adult age range of 12–60 years displayed consistent TSH percentiles, supporting its consolidation. This refined system effectively balances the need for granularity and statistical power. Both partitioning strategies provide valuable insights: the original 11‐group stratification allows for detailed age‐specific analysis when necessary, whereas the revised 5‐group system is potentially more suitable for clinical practice and routine reporting.

The identification of 130 statistical outliers among 3012 participants (≈4%) raises both methodological and biological considerations. Outliers were detected using Tukey's method, which is purely statistical and does not account for physiological variability. Some values, although lying beyond the conventional percentiles (2.5th–97.5th), may represent true physiological extremes attributable to genetic polymorphisms [17, 18, 19], interindividual variation in hypothalamic–pituitary–thyroid (HPT) axis sensitivity [20, 21], or other unmeasured factors. Excluding such values is consistent with international recommendations for defining reference intervals, which are conventionally restricted to the central 95% of the reference population. Nevertheless, removing these data points may obscure biologically meaningful variability, particularly in small subgroups, where extreme values exert a disproportionate influence on distribution estimates. Future studies could analyze these values separately, not as statistical artifacts, but as potential indicators of subpopulations with distinct endocrine regulation or metabolic traits.

The assessment of plasma TSH levels by sex revealed significant differences in certain age groups. However, these differences never exceeded 0.30 μIU/mL, remaining below the threshold that is generally considered clinically relevant. However, the international literature on this topic is inconsistent. Large‐scale studies have reported small but statistically significant sex differences, typically showing slightly higher TSH levels in females [22, 23]. These have been attributed to hormonal influences, such as the modulatory effects of estrogen. Other studies suggest that these disparities may be more pronounced in postmenopausal women or influenced by iodine status, smoking, or the higher prevalence of thyroid autoantibodies in women [5].

Conversely, several cohorts have found no significant sex differences after adjusting for confounding factors. Some researchers argue that discrepancies between studies may reflect selection bias, methodological heterogeneity, or environmental imbalances. These divergent findings underscore the need to interpret TSH data in light of the demographic, nutritional, and methodological context of each study.

Placing our findings in a global context enhances their interpretability but requires caution in generalizing the results. International differences in TSH reference intervals arise from genetic, environmental, and demographic factors, particularly iodine status and age distribution, as well as methodological variability [24].

Given the known inter‐assay variability in TSH measurements, we began by comparing our RIs specifically with those provided by Mindray, the analytical platform used in this study. In the pediatric population, our age‐stratified upper limits were broadly aligned with those of Yao et al. [25] using Mindray, although our neonatal upper limit (22.41 μIU/mL for Days 1–7) more accurately captured the early postnatal surge than the manufacturer's pooled 0–1 month interval (11.35 μIU/mL), which likely underestimates the physiological peaks. For children aged 5–12 years, our upper limit (5.24 μIU/mL) was lower than Mindray's published value (7.42 μIU/mL), possibly reflecting demographic or iodine‐related differences. In adults, the Mindray RI of 0.56–5.91 μIU/mL is not stratified by age and may encompass individuals with early or subclinical thyroid dysfunctions. In contrast, our adult RIs, based on strict selection criteria, were narrower (upper limit of 4.54 μIU/mL for 18–60 years). These findings highlight both the clinical relevance of local, age‐specific RIs and the limitations of using manufacturer‐provided intervals, particularly in pediatric and older adult populations, without population‐specific validation.

In neonates, a characteristic postnatal surge in TSH occurs within the first days of life. In our Algerian cohort, the 97.5th percentile reached 22.4 μIU/mL at 1–7 days after birth, substantially exceeding the values reported in Kenyan newborns (7.9 μIU/mL at 2–4 days, 6.3 μIU/mL at 5–7 days) obtained using the Siemens Atellica IM platform [26]. Comparable early life peaks above 15 μIU/mL have been described in Chinese cohorts [25], while the CALIPER study using the Ortho VITROS 5600 reported an upper limit of 31.5 mIU/L during the first week of life [27]. These discrepancies highlight TSH's strong dependence on sampling time, analytical platform, and population iodine status.

Following this neonatal surge, the TSH concentration progressively declines with age. The 97.5th percentile values were 6.09 μIU/mL in infants and toddlers (1 week–5 years) and 5.24 μIU/mL in older children (5–12 years). These intervals are consistent with CALIPER pediatric reference limits across analytical systems [28], including the Abbott ARCHITECT (4.77 mIU/L for < 6 months; 4.17 mIU/L for < 14 years) and Beckman DxI (5.85 mIU/L for < 12 years). This trend is also supported by the work of Iwaku et al. and Mitsumatsu et al., who, using ECLIA technology, reported that TSH levels in children aged 4–12 years were significantly higher than those in adults. Specifically, Iwaku et al. observed an upper limit of 5.16 μU/mL in the 7–8‐year‐old group [29], while Mitsumatsu et al. identified a peak of 5.01 μU/mL for the same age range [30]. This agreement with our upper limit of 5.24 μIU/mL for the 5–12‐year‐old group strengthens the validity of age‐based partitioning in the pediatric population.

Our stratification, grounded in statistically significant distributional shifts, follows the CALIPER framework, which emphasizes refined age partitioning to prevent diagnostic misclassification during childhood growth and thyroid maturation.

For individuals aged 12–19 years, the upper reference limit (97.5ᵗʰ percentile) of serum TSH was established at 4.59 mIU/L. This upper limit aligns with values reported in previous studies, including those by Chaler et al. [31] (4.65–4.88 mIU/L), Kapelari et al. [32] (4.33–4.61 mIU/L), Gannagé‐Yared et al. [33] (4.36–4.62 mIU/L), and Argente del Castillo et al. [34] (4.26 mIU/L). However, it remains slightly higher than the narrower reference interval reported by the CALIPER study (3.35–3.41 mIU/L) [28], as well as the results of Mitsumatsu et al. [30], who observed upper limits ranging from 3.49 to 4.02 μU/mL in individuals aged 13–19 years. Mitsumatsu et al. also pointed out that in their cohort, the upper limit of TSH in adolescents aged 11–19 years was lower than that in the adults. These variations between studies may reflect differences in ethnic backgrounds, iodine intakes, or subject selection methodologies. Despite these nuances, the partitioning of the 12–19 age group appears to be highly consistent across various investigations, marking the transition phase toward young adult levels.

An upper reference limit of 4.58 μIU/mL for serum TSH was identified in adults aged 19–60 years. This value is comparable to the findings of international studies using broader population criteria. A Lebanese study reported upper limits of up to 5.10 μIU/mL in the 51–60 age group [35], while East Asian cohorts, such as those in Korea and Japan [36, 37], showed even higher values, likely due to their iodine‐rich diets. In contrast, stricter studies, such as that of Chaker et al. [38], using the NACB criteria, reported lower limits (3.77–3.94 mIU/mL), as did German [39] and Sudanese [40] data (approximately 3.10–3.29 mIU/mL). While many European countries use reference intervals of 0.4–4.0 or 0.4–4.5 mIU/L, the NHANES III study in the U.S. found a range of 0.4–4.1 mIU/mL in antibody‐negative adults [41], with ethnic variability.

In individuals aged > 70 years, the TSH reference interval observed in this dataset was 0.51–5.17 μIU/mL. This finding is consistent with the growing evidence that TSH concentrations gradually increase with age and that applying fixed adult reference intervals may lead to the overdiagnosis of subclinical hypothyroidism (SCH) in older adults [12]. Several population‐based studies have supported this age‐related shift. The Australian ASPREE cohort proposed a narrower interval of 0.34–3.75 mIU/L for healthy individuals aged ≥ 70 years [42], with no association between TSH values within this range and adverse clinical outcomes over an 11‐year follow‐up. Similarly, Yeap et al. reported a reference range of 0.67–4.98 mIU/L in healthy men aged 70–89 years [43]. In Lebanon, the upper limit of TSH continued to rise with age, reaching 6.15 mIU/L in individuals over 80 years old [31], further confirming this physiological trend.

Recent investigations have highlighted the potential value of composite reference models that integrate both TSH and FT4 levels. Studies by Ross et al. [44] and Li et al. [45] demonstrated that such bidimensional approaches reduce the number of individuals classified with SCH compared to conventional univariate thresholds, providing a more conservative and physiologically accurate framework for assessing thyroid function in older adults.

Variability in findings on the effects of age and sex on TSH levels arises from both biological and methodological factors. Genetically, TSH is more heritable in women than in men and is modulated by sex hormones, autoimmunity, and ethnicity. Environmental influences, particularly iodine status, BMI, smoking, and exposure to pollutants, also contribute to inter‐individual and population‐level variations. Methodological differences, including inconsistent definitions of “healthy” reference populations, varying statistical models, and differences in assay standardization and sampling conditions, further account for discrepancies across studies. Thus, age, particularly in adults and older individuals, and sex effects may appear divergent across studies without indicating a true biological contradiction.

This study had several limitations. First, while age and sex were carefully considered, other relevant confounding factors, such as body mass index, smoking status, menopausal status, and iodine intake, were not systematically evaluated. These may influence TSH variability and should be considered in future research. In pediatric subjects, insufficient stratification of age may conceal nuanced developmental changes in the thyroid physiology. Second, TSH measurements were performed using a single immunoassay platform. Although this ensured consistency within the study, it limited the applicability of the reference intervals across different laboratory systems. Given the known inter‐assay variability, caution is required when applying these values elsewhere. Nonetheless, future efforts in assay harmonization and standardization may support wider transferability. Third, the cross‐sectional design provides only a snapshot of TSH levels at a single time point, without accounting for longitudinal changes in individuals over time. Finally, the study population was drawn from a specific region in Algeria, which may limit the generalizability of the results to populations with different genetic, environmental, or nutritional backgrounds. However, the methodology used in this study offers a strong framework for similar studies in other settings.

5. Conclusion

This study provides the first age‐specific reference intervals for TSH in the Algerian population based on a rigorously selected cohort and a standardized Mindray assay methodology. TSH levels were highest in neonates and older adults and remained stable throughout adolescence and adulthood. Minor sex differences were observed but lacked clinical relevance, confirming age as the main determinant of TSH variations.

By establishing assay‐ and population‐specific reference values, this study fills a critical gap for North Africa, where Mindray platforms are widely used, but local data are limited. These results support a more accurate diagnosis of thyroid disorders in Algeria and reduce the reliance on non‐local reference ranges. Further longitudinal studies are encouraged to refine neonatal and elderly values and assess the genetic, nutritional, and environmental influences on TSH levels.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. The data are not publicly available due to ethical and privacy restrictions related to human participants.