Reference range of thyroid function (FT3, FT4 and TSH)

Clinical Biochemistry 46 (2013) 341–345

Contents lists available at SciVerse ScienceDirect

Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem

Reference range of thyroid function (FT3, FT4 and TSH) among Indian adults Raman Kumar Marwaha a,⁎, 1, Nikhil Tandon c, 1, Mohd Ashraf Ganie e, Neena Mehan c, d, Aparna Sastry a, M.K. Garg b, Kuntal Bhadra a, Satveer Singh a a

Department of Endocrinology and Thyroid Research Centre, Institute of Nuclear Medicine and Allied Sciences, Timarpur, India Army Referral and Research Hospital, New Delhi, India Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, New Delhi 110054, India d B. R. Sur Homeopathy Medical College, New Delhi, India e Department of Endocrinology Sher-i-Kashmir Institute of Medical Sciences Srinagar, J & K, India b c

a r t i c l e

i n f o

Article history: Received 30 January 2012 Received in Revised form 7 September 2012 Accepted 23 September 2012 Available online 3 October 2012 Keywords: FT3 FT4 TSH Thyroid function Reference norms Indian adults

a b s t r a c t Objectives: To generate thyroid hormone reference norms using electro-chemiluminescence technique. Design and methods: Cross sectional study on apparently normal 4349 Delhi adults (18–86 years). Predetermined exclusion criteria (goiter, hypoechogenicity or nodularity on ultrasound, elevated antithyroid peroxidase antibody, hypo or hyperthyroidism and family history of thyroid dysfunction) excluded 2433 subjects leaving 1916 (916 males and 1000 females) as the reference population. Results: Mean age and BMI of the reference population were 41.2 ± 18.1 years and 24.5 ± 4.4 kg/m2 respectively. Median urinary iodine excretion was 233.6 μg/L (79–458;3rd–97th centile). The population was categorized into various age groups (18–30, 31–40, 41–50, 51–60, 61–70 and ≥ 70 years). Overall FT3 and FT4 values in the reference population irrespective of age, ranged from 2.4–8.8 (mean 4.6 ± 0.9) pmol/L and 10.1–24.8 (mean 15.40 ± 2.0) pmol/L, respectively. Mean TSH value in the reference population was 2.2 ± 0.9 mIU/L which was significantly lower than that of total population (3.8 ± 6.1; p b 0.001). Conclusion: FT3 values were observed to be significantly higher in men than women (p = 0.001). The centiles (3rd, 5th, 10th, 25th, 50th, 75th, 90th, 95th and 97th) of FT3, FT4 and TSH were derived for reference purposes in Indian adults. This community based study in Indian adults has established mean reference intervals for FT3, FT4 and TSH for different age groups for both sexes separately using strict exclusion criteria. These can be used as reference norms for Indian adults. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction One third of the world's population lives in areas with risk of iodine deficiency (ID) and its complications [1,2]. Iodine deficiency is known to result in thyroid functional abnormalities, ranging from sub-clinical to overt hypothyroidism, across all age groups [3–5]. Thyroid function tests, routinely used in clinical practice to diagnose thyroid disorders, are known to be influenced by age, ethnicity, geographical and climatic conditions, and other biological variables including nutrition and lifestyle [6–11]. In view of this, age and gender specific reference intervals generated from an iodine sufficient population, are an important prerequisite for interpreting thyroid hormone measurements [12,13]. In India, the programme of Universal Salt Iodization (USI) was instituted in 1984 and has achieved considerable success with large parts of the country becoming iodine sufficient [14,15]. Several studies have

⁎ Corresponding author at: Department of Endocrinology and Thyroid Research Centre, Institute of Nuclear Medicine and Allied Sciences, Lucknow Road, Timarpur, New Delhi-110054, India. Fax: +91 11 23919509. E-mail address: [email protected] (R.K. Marwaha). 1 R.K.M. and N.T. are to be considered as joint first authors.

demonstrated the city of Delhi to be iodine sufficient for over the last decade [14,16,17]. Since we have now achieved iodine sufficiency, we felt that it was appropriate time to generate thyroid hormone reference norms for our population. While we have previously published thyroid hormone reference range in healthy Indian school-age children [18,19] and pregnant women [20] there are no normative data available for adults. In view of this, we decided to generate thyroid hormone reference norms using electro-chemiluminescence technique from cohort of healthy adult subjects with no known risk factors for thyroid dysfunction. Materials and methods Study subjects This cross sectional study was conducted in adults, aged 18–86 years, between December 2007 and January, 2010. All residents from five residential colonies (one each from 5 different geographical zones of Delhi) from different parts of Delhi were invited through their Resident Welfare Associations to participate in a general health check-up, which included a clinical evaluation by a physician, and

0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.09.021

342

R.K. Marwaha et al. / Clinical Biochemistry 46 (2013) 341–345

blood sampling for routine blood biochemistry and thyroid hormone profile. Only those who agreed to participate were included as part of the study. The present study includes 4349 consecutive subjects who gave their written informed consent. Subjects with history of thyroid disease (treated or untreated, n = 60) were excluded from further evaluation. Individuals with family history of thyroid disease, use of medications known to interfere thyroid function, systemic illness, goiter on palpation, abnormal thyroid ultrasonographic picture (nodules/ hypoechogenicity), or elevated thyroid antibody levels and hypo/or hyperthyroidism were excluded from the cohort used to generate the reference range. On the basis of these pre-determined exclusion criteria, 2433 subjects in both sexes were excluded and only 1916 (44.1%) subjects (916 males, 1000 females) were included as the reference population in the study. On the basis of predetermined exclusion criteria, two thousand four hundred thirty three subjects (721 men and 1712 women) who either had goiter, thyroid dysfunction, anti TPO antibody or positive family history were excluded from the study. The remaining 1916 subjects (916 men and 1000 women) were finally undertaken for making norms for FT3, FT4 and TSH. This stepwise exclusion of subjects to generate reference population is depicted in the flow chart (Fig. 1).

The study was approved by the Institutional Ethics committee of the Army Referral and Research Hospital, New Delhi. Methods All adults were evaluated by taking a detailed clinical history and general physical examination, including anthropometry. Family history of known thyroid dysfunction in first degree relatives of study subjects was noted. Thyroid palpation was carried out independently by two endocrinologists with experience in thyroid epidemiology. Goiter was graded according to WHO/UNICEF/ICCIDD recommendations [21]. Body weight was measured to the nearest 0.1 kg using a beam balance-weighing scale. The adults were weighed wearing the light clothing but without shoes or any other items found on them. Height was measured to the nearest 0.1 cm using the height scale. Ultrasonography (USG) of thyroid gland was performed with subjects in supine position with neck hyper-extended by a single sonologist who was blinded to the results of thyroid palpation, using a portable ultrasound machine (Sonosite Titan, Germany) with a 7.5 MHz transducer. The USG was used to define presence and size of any nodules

Ethical clearance

Subjects selected from areas of Delhi n=4409

Questionnaire provided to each subject 60 individuals were excluded since they had present or past history of thyroid disease

4349 (total population)

Clinical examination – goitre by WHO classification Ultrasound of the thyroid – echogenicity or nodularity Blood sample - FT3, FT4, TSH and TPO Ab Urine sample for Urinary iodine excretion

Positive family history of thyroid/medication n=194

Positive TPO Ab n=589

Goiter N= 414

Hyper/ Hypothyroidism N=981

Clin /USG nodularity n=302

Some subjects had more than one exclusion factors

2433 excluded

N= 1916 formed part of study (Reference Population), Men =916 and Women= 1000 Fig. 1. Diagram showing methodology and the process for identifying reference population.

Hypoechogenicity USG n=1330


and echogenecity. The gain settings of the ultrasound scanner were adjusted so that the lumina of the carotid artery and internal jugular vein were free of echoes. Hypoechogenicity was diagnosed if echogenecity of the thyroid was uniformly less than that of the connective tissue and similar to or less than that of the neck muscles. Assays All adults were subjected to blood sampling and 5 mL of blood was collected from each subject between 8–10 AM, and serum stored at −20 °C in a central laboratory. FT3, FT4, TSH and thyroid peroxidase autoantibody (TPO-Ab) were analyzed using electrochemiluminescence assay (Cobas-Roche Elecys 1010 analyzer). The analytical sensitivity of TPO Ab was b 5 IU/mL and range of measurement, 5–600 IU/mL. The TPO-Ab was defined positive above a cutoff of 34 IU/mL. The intra and inter-assay coefficients of variation (CV) were b4.2% and b7.2% respectively. Subclinical or overt hypo or hyperthyroidism was defined by the reference ranges given in the package inserts of kits. Ranges for FT4, FT3 and TSH were 12.0–22.0 pmol/L, 2.8–7.1 pmol/L and 0.28–4.2 mIU/L respectively, with intra assay and inter assay CV being less than 7% for all three parameters. Iodine excretion was evaluated in spot urine samples collected in every second participant using kits based on the Sandell– Kolthoff reaction, which were purchased from Bioclone Australia Pty Limited, NSW, Australia. The inter assay and intra assay CV were (b6%) within the kit prescribed limits. Urinary iodine excretion (UIE) of b 100 μg/L was considered as iodine deficiency and >300 μg/L as iodine excess. Statistical analysis SPSS 11.5 version (Chicago, IL, USA) was used to analyze the data. Quantitative variables have been described as mean ± SD and median (range) or percentiles as appropriate. Subjects were grouped at ten yearly age intervals. However, as the number of subjects at either end of the age distribution was small, wider age categories were generated namely 18–30 years and > 70 years. FT3, FT4 and TSH of the reference population were compared across the age categories using one way ANOVA followed by Bonferroni correction for pair-wise comparison. FT3, FT4 and TSH of the reference men and women were compared using Student's t-test for independent samples. A p value of b0.05 was taken as significant. Results A total of 4349 (1637 men and 2712 women) who consented to be a part of the study were further evaluated in detail for clinical, biochemical, sonographic examination and UIE for iodine intake. The comparative description of clinical, biochemical and sonographic evaluation done between the total population studied vs. the reference population showed significant differences. Mean ages

343

Table 2 Percentile distribution of FT3 values in different age groups in the reference population of adult men and women. Age group (years)

n

18–30 31–40 41–50 51–60 61–70 ≥71 Total

FT3 percentiles in men 3

5

10

25

50

75

90

95

97

258 182 88 58 207 123 916

3.80 3.49 3.38 3.28 3.11 3.01 3.17

4.00 3.70 3.53 3.84 3.27 3.07 3.39

4.30 4.0 0 4.09 4.08 3.58 3.37 3.70

4.70 4.40 4.33 4.41 4.10 3.78 4.20

5.00 4.80 4.80 4.72 4.60 4.30 4.70

5.60 5.30 5.36 5.07 4.96 4.80 5.14

6.80 6.10 5.80 5.52 5.34 5.30 5.70

7.20 6.90 6.48 6.14 5.70 6.26 6.40

7.30 7.25 6.86 6.22 6.04 6.61 6.90

Age group (years)

n

FT3 percentiles in women

18–30 31–40 41–50 51–60 61–70 ≥71 Total

425 201 143 124 82 25 1000

2.96 3.15 3.14 3.10 2.97 2.78 2.81

4.40 4.50 4.60 4.46 4.20 3.90 4.32

4.90 4.99 5.23 4.99 4.80 4.22 4.90

5.30 5.39 6.02 5.56 5.36 4.58 5.36

5.50 5.79 6.58 5.99 5.99 4.74 5.70

5.76 5.96 6.76 6.48 6.25 4.75 6.07

3.10 3.32 3.25 3.14 3.06 2.79 3.01

3.25 3.60 3.51 3.39 3.23 2.95 3.28

3.71 4.10 4.00 3.82 3.67 3.50 3.80

of the reference and total population were 41.2 ± 18.1 and 44.5 ± 18.6 years respectively. The median UIE in the total and reference population was 221.0 μg/L (41–664) and 224.0 μg/L (42–664) respectively (p = 0.78). Iodine deficiency was observed in only 10.55% of subjects in total population and 10.30% in reference population. The TSH values and TPO-Ab titers were significantly higher in the total population when compared with the reference group (3.8 ± 6.1 vs. 2.2 ± 0.9 mIU/L; p = 0.0001 and 42.0 ± 108.8 vs. 9.3 ± 4.9 IU/mL; p = 0.001). The reference population was further categorized into various age groups as shown in Table 1. One way ANOVA was done to compare the FT3, FT4 and TSH between various age categories. TSH did not show any significant difference between any age categories in both men and women. FT4 showed a significant difference on comparing 18–30 years age category with 61–70 years (p = 0.001) and > 71 years (p = 0.03) in women and 18–30 years age category with > 71 years in men (p = 0.01). FT3 however showed significant difference other categories and >71 with other categories in both men and women (p b 0.05). FT3 and FT4 values in the reference population ranged from 2.4–8.8 (mean 4.6 ± 0.9) pmol/L and 10.1–24.8 (mean 15.40 ± 2.0) pmol/L, respectively. Overall FT3 was significantly higher in men than women (4.85 ± 0.9 pmol/L vs. 4.42 ± 0.81 pmol/L; p = 0.001) while there was no significant difference in FT4 and TSH values (p = 0.55 and p = 0.23 respectively) using Student's t-test. The FT3 values were significantly higher in men than in women in each age category (p b 0.05) except in the 5th decade (p = 0.19). FT4 and TSH were not significantly different in the sexes in the respective age groups except in the sixth decade where the values were higher in women than in men (Table 1).

Table 1 Comparison of FT3, FT4 and TSH values in men and women among reference population. Age group (years)

18–30 31–40 41–50 51–60 61–70 ≥71

Women

Men

FT3 (pmol/L)

FT4 (pmol/L)

TSH mIU/L

FT3 (pmol/L)

FT4 (pmol/L)

TSH (mIU/L)

4.32 ± 0.85 4.54 ± 0.71 4.71 ± 1.11 4.47 ± 0.83 4.31 ± 0.83 3.85 ± 0.53x

15.11 ± 1.80 15.40 ± 2.17 15.32 ± 1.95 15.56 ± 2.08 15.85 ± 2.28y 15.66 ± 2.47y

2.17 ± 0.86 2.10 ± 0.93 2.34 ± 0.95 2.33 ± 0.94 2.21 ± 0.95 2.12 ± 0.95

5.26 ± 0.94 a 4.94 ± 0.88a 4.81 ± 0.83 4.78 ± 0.60a 4.53 ± 0.74a 4.37 ± 0.87a,x

14.97 ± 1.99 15.61 ± 1.89 15.74 ± 2.15 14.92 ± 1.93 b 15.52 ± 1.89 15.72 ± 3.29y

2.25 ± 0.86 2.35 ± 0.92 2.17 ± 1.0 1.98 ± 0.94c 2.14 ± 0.90 2.00 ± 0.92

a, b or c marked in case of p value of b0.05 in each age category. a = Student's t-test between FT3 of men and women, b = t-test between FT4 of men and women and c = t-test between TSH of men and women. Using one way ANOVA, p b 0.05 between age-groups: x for FT3 and y for FT4.

344


Table 3 Percentile distribution of FT4 values across different age groups in the reference population of adult men and women. Age group (years)

n

18–30 31–40 41–50 51–60 61–70 ≥71 Total

FT4 percentiles in men

258 182 88 58 207 123 916

3

5

10

25

50

75

90

95

97

12.00 12.46 12.13 12.17 12.61 12.40 11.51

12.20 12.79 12.70 12.48 12.87 12.52 12.19

12.50 13.00 13.09 12.69 13.29 13.21 12.70

13.50 14.21 14.37 13.29 14.10 14.00 13.71

15.00 15.60 15.56 14.64 15.30 15.35 15.10

16.28 16.99 17.07 15.92 16.60 16.90 16.50

17.67 18.25 18.22 17.14 18.18 19.04 18.0

18.53 19.17 19.17 18.46 19.20 20.58 19.0

19.08 19.7 20.68 20.81 19.96 21.34 19.72

12.60 12.20 12.52 12.61 13.04 12.39 12.10

12.94 12.80 12.80 13.20 13.35 12.75 12.60

13.80 13.70 13.84 14.36 14.09 13.73 13.64

15.00 15.10 15.20 15.20 15.44 15.54 14.94

16.21 16.85 16.40 16.59 17.33 16.48 16.42

17.40 18.30 18.23 18.50 18.45 19.49 17.90

18.30 18.90 18.97 19.95 19.75 22.46 18.90

18.89 19.87 19.66 20.25 22.19 23.20 19.81

Age group (years)

FT4 percentiles in women

18–30 31–40 41–50 51–60 61–70 ≥71 Total

425 201 143 124 82 25 1000

12.40 12.10 12.38 12.26 12.86 12.33 11.67

The distribution of FT3, FT4 and TSH (3rd to 97th centiles) for the reference population was determined for the entire group and each age category for both men and women (Tables 2–4). Hypoechogenicity on USG was seen in 1330 subjects, who were excluded from further analysis. Median TSH was significantly high in this group as compared to those 3019 subjects with normal echogenecity (3.54 vs. 2.48 mIU/mL, p =0.04). FT3, FT4 and TPO levels were not significantly different between these two groups. Discussion Consequent to the USI program, India is making a transition from being an iodine-deficient to an iodine-replete nation. The present study was conducted in Delhi, which is iodine replete for at least the last decade [14–17]. In view of changes in thyroid function anticipated in response to improved iodine nutrition, we evaluated 4349 adult subjects from Delhi for various parameters related to thyroid functions. An important feature of the present study has been stringent exclusion criteria to generate norms for different age groups as recommended by International Federation of Clinical Chemistry (IFCC) which adopted exclusion criteria namely, goiter, family history of thyroid disease, endocrine disorders, obesity, etc. [22,23] and also autoimmunity as suggested by others [24,25]. The other limitation of previous

Table 4 Percentile distribution of TSH values in different age groups in the reference population of adult men and women. Age group (years) N

TSH percentiles in men 3

5

10

25

50

75

90

95

97

18–30 31–40 41–50 51–60 61–70 ≥71 Total

0.71 0.65 0.36 0.40 0.64 0.32 0.70

1.00 0.80 0.64 0.59 0.84 0.70 0.81

1.10 1.00 1.00 0.80 1.06 1.00 1.00

1.60 1.63 1.34 1.38 1.40 1.33 1.10

2.20 2.40 2.08 1.70 2.00 1.90 1.60

2.80 3.02 2.99 2.67 2.80 2.49 2.40

3.40 3.57 3.40 3.53 3.45 3.33 3.51

3.90 3.90 3.95 3.80 3.83 3.93 5.71

4.00 4.00 4.12 3.88 4.00 4.17 8.11

2.12 2.18 2.35 2.31 2.12 1.96 1.74

2.89 2.90 3.14 3.13 3.08 2.78 2.68

3.36 3.49 3.59 3.61 3.62 3.71 4.10

3.70 3.88 3.83 3.91 3.85 4.00 6.70

3.80 3.96 4.00 4.01 3.98 4.00 9.78

258 182 88 58 207 123 916

Age group (years) TSH percentiles in women 18–30 31–40 41–50 51–60 61–70 ≥71 Total

425 201 143 124 82 25 1000

0.70 0.80 0.64 0.35 0.62 0.02 0.68

0.90 0.83 0.77 0.76 0.70 0.26 0.86

1.06 1.00 0.98 1.09 1.02 0.89 1.00

1.50 1.48 1.67 1.54 1.48 1.53 1.19

studies has been the use of hospital-based data for making reference norms, where the major disadvantage is that information is retrospective and subjects could have had illnesses effecting thyroid function. The United States National Health and Nutrition Examination Survey III (NHANES III) used most of these exclusion criteria for thyroid function evaluation in adolescents and adults [26,27]. The present study, besides using strict exclusion criteria, has used reliable and sensitive electro chemiluminescence hormone assay technique for the purpose of making normal reference ranges of FT3, FT4 and TSH. However, the use of strict exclusion criteria has been questioned by some authors, especially for TSH reference intervals, as it may result in narrowing of reference intervals and unnecessarily increase the quantum of subclinical thyroid disease. Völzke et al. [28] similarly evaluated an event free population of 1053 subjects and observed significantly lower TSH values in this as compared to the general population. However, no significant differences were observed with respect to serum FT3 and FT4 reference intervals in the two populations. Our observation is in consistent with the findings of NHANES III study and Volzke et al. [26–28]. We, therefore, believe that only reference population should be used to make norms. Furthermore use of different analytical methods prevents comparisons between studies [29]. Adult reference values for thyroid functions have been well established. However, the upper limit of normal TSH values is currently debatable [29]. Different studies in the past have suggested different values for FT3, FT4 and TSH with age and sex, though they lack uniformity in study population and assay techniques [30,31]. Tunbridge et al. in the Whickham survey [24,25] found that serum TSH levels did not vary with age in men but increased markedly in TPO-Ab positive women aged above 45 years. In the present study, the only difference between sexes was noted in FT3 values, which were higher in men. Most other investigators did not find any sex-related differences in thyroid functions for the entire study group and for the smaller age-specific groups [32,33]. Our results cannot be compared to any previous data because of a) comprehensive exclusion criteria, b) different age groups and analytic methods used for hormone assays, and c) different source of samples. In addition, authors in most studies have also not provided details that would allow comparison by age. The strengths of the present study are a) large community based study evaluating more than 4000 adult subjects, b) use of strict exclusion criteria, and c) usage of most recent, reliable and sensitive electrochemiluminescence assay technique. Therefore, this study should form the basis for interpretation of thyroid function tests in adults from our country for both clinical and epidemiological


purposes. However, the normal reference ranges may be biased by using the kit reference ranges to exclude subclinical and overt hypo- or hyperthyroidism as was done in the present study. This can be considered a limitation of the study. In conclusion, this is the first community based study to determine reference intervals for FT3, FT4 and TSH, in different age categories, for males and females using strict exclusion criteria. In view of the significant difference in mean TSH values between the total and reference population, we support the concept that reference ranges for FT3, FT4 and TSH should be established using a defined population at minimal risk for thyroid disease. Conflict of interest The authors have nothing to declare. Grant: Defence Research and Development Organization (DRDO), India. References [1] Hetzel BS, Dunn JT. The iodine deficiency disorders: their nature and prevention. Annu Rev Nutr 1989;9:21-38. [2] Delange F. Iodine deficiency. In: Braverman LE, Utiger RD, editors. The thyroid. 8th ed. Philadelphia: Lippincott; 2000. p. 295-316. [3] Konno N, Makita H, Yuri K, Iizuka N, Kawasaki K. Association between dietary iodine intake and prevalence of subclinical hypothyroidism in the coastal regions of Japan. J Clin Endocrinol Metab 1994;78:393-7. [4] Fradkin J, Wolff J. Iodide-induced thyrotoxicosis. Medicine 1983;62:1–21. [5] Stanbury JB, Ermans AE, Bourdoux P, Todd C, Oken E, Tonglet R, et al. Iodine-induced hyperthyroidism: occurrence and epidemiology. Thyroid 1998;8:83–100. [6] Demers LM, Spencer CA. Laboratory medicine practice guidelines: laboratory support for the diagnosis and monitoring of thyroid disease. Thyroid 2003;13:1–104. [7] Szabolcs I, Ploenes C, Beyer M, Bernard W, Herrmann J. Reference intervals for serum thyrotropin: dependence on the population investigated. Exp Clin Endocrinol 1991;98:23-31. [8] Jensen E, Hyltoft Petersen P, Blaabjerg O, Hansen PS, Brix TH, Kyvik KO, et al. Establishment of a serum thyroid stimulating hormone (TSH) reference interval in healthy adults. The importance of environmental factors, including thyroid antibodies. Clin Chem Lab Med 2004;42:824-32. [9] Boucai L, Hollowell JG, Surks MI. An approach for development of age-, gender-, and ethnicity-specific thyrotropin reference limits. Thyroid 2011;21:5–11. [10] Penny R, Spencer CA, Frazier D, Nicoloff JT. Thyroid-stimulating hormone and thyroglobulin levels decrease with chronological age in children and adolescents. J Clin Endocrinol Metab 1983;56:177-80. [11] Sari R, Balci MK, Altunbas H, Karayalcin U. The effect of body weight and weight loss on thyroid volume and function in obese women. Clin Endocrinol (Oxf) 2003;59:258-62. [12] Solberg HE. International Federation of Clinical Chemistry, Expert Panel on Theory of Reference Values, and International Committee for Standardization in Hematology, Standing Committee on Reference Values. Approved recommendation on the theory of reference values. Part 5. Statistical treatment of collected reference values. Determination of reference limits. Clin Chim Acta 1987;170:S13-32. [13] Dybkær R, Solberg HE. International Federation of Clinical Chemistry, Expert Panel on Theory of Reference Values, and International Committee for Standardization in Haematology, Standing Committee on Reference Values. Approved recommendation on the theory of reference values. Part 6. Presentation of observed values related to reference values. Clin Chim Acta 1987;170:S33-42.

345

[14] Marwaha RK, Tandon N, Desai A, Kanwar R, Mani K. Iodine nutrition in upper socioeconomic school children of Delhi. Indian Pediatr 2010;47:335-8. [15] Brahmbhatt SR, Fernley R, Brahmbhatt RM, Eastman CJB. Study of biochemical prevalence of indicators for the assessment of iodine deficiency disorders in adults at field conditions at Gujarat (India). Asia Pac J Clin Nutr 2001;10:51-7. [16] Kapil U, Sharma TD, Singh P. Iodine status and goiter prevalence after 40 years of salt iodisation in the Kangra District, India. Indian J Pediatr 2007;74:135-7. [17] Sood A, Pandav CS, Anand K, Sankar R, Karmarkar MG. Relevance and importance of universal salt iodization in India. Natl Med J India 1997;10:290-3. [18] Marwaha RK, Tandon N, Desai AK, Kanwar R, Aggarwal R, Sastry A, et al. Reference range of thyroid hormones in healthy school-age children: country-wide data from India. Clin Biochem 2010;43:51-6. [19] Marwaha RK, Tandon N, Desai A, Kanwar R, Grewal K, Aggarwal R, et al. Reference range of thyroid hormones in normal Indian school-age children. Clin Endocrinol (Oxf) 2008;68:369-74. [20] Marwaha RK, Chopra S, Gopalakrishnan S, Sharma B, Kanwar RS, Sastry A, et al. Establishment of reference range for thyroid hormones in normal pregnant Indian women. BJOG 2008;115:602-6. [21] International Council for Control of Iodine Deficiency Disorders, UNICEF, World Health Organization. Assessment of iodine deficiency disorders and monitoring their elimination: a guide for programme managers. 2nd ed.Geneva: World Health Organization; 2001. [22] Solberg HE, Stamm D. International Federation of Clinical Chemistry, Expert Panel on Theory of Reference Values. IFCC recommendation. The theory of reference values. Part 4. Control of analytical variation in the production, transfer and application of reference values. Clin Chim Acta 1991;202:S5–S12. [23] Baloch Z, Carayon P, Conte-Devolx B, Demers LM, Feldt-Rasmussen U, Henry JF, et al. Guidelines Committee, National Academy of Clinical Biochemistry. Laboratory medicine practice guidelines. Laboratory support for the diagnosis and monitoring of thyroid disease. Thyroid 2003;13:3–126. [24] Tunbridge WM, Evered DC, Hall R, Appleton D, Brewis M, Clark F, et al. The spectrum of thyroid disease in acommunity: the Whickham survey. Clin Endocrinol (Oxf) 1977;7:481-93. [25] Vanderpump MP, Tunbridge WM, French JM, Appleton D, Bates D, Clark F, et al. The incidence of thyroid disorders in the community: a twenty-year follow-up of the Whickham Survey. Clin Endocrinol (Oxf) 1995;43:55-68. [26] Hollowell JG, Staehlingm NW, Flanders WD, et al. Serum TSH, T(4), and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 2002;87:489-99. [27] Spencer CA, Hollowell JG, Kazarosyan M, Braverman LE. National Health and Nutrition Examination Survey III thyroid-stimulating hormone (TSH)-thyroperoxidase antibody relationships demonstrate that TSH upper reference limits may be skewed by occult thyroid dysfunction. J Clin Endocrinol Metab 2007;92:4236-40. [28] Völzke H, Alte D, Kohlmann T, Lüdemann J, Nauck M, John U, et al. Reference intervals of serum thyroid function tests in a previously iodine-deficient area. Thyroid 2005;15:279-85. [29] Zophel K, Wunderlich G, Kotzerke J. Should we really determine a reference population for the definition of thyroid-stimulating hormone reference interval? Clin Chem 2006;52:329-30. [30] Kratzsch J, Fiedler GM, Leichtle A, Brugel M, Buchbinder S, Otto L, et al. New reference intervals for thyrotropin and thyroid hormones based on National Academy of Clinical Biochemistry criteria and regular ultrasonography of the thyroid. Clin Chem 2005;51:1480-6. [31] Wiedemann G, Jonetz-Mentzel L, Panse R. Establishment of reference ranges for thyrotropin, triiodothyronine, thyroxine and free thyroxine in neonates, infants, children and adolescents. Eur J Clin Chem Clin Biochem 1993;31:277-8. [32] Canaris GJ, Manowitz MR, Mayor G, Ridgway EC. The Colorado thyroid disease prevalence study. Arch Intern Med 2000;160:526-34 ((Oxf) 70:788–93). [33] Estaquio C, Valeix P, Leenhardt L, Modigliani E, Boutron-Ruault MC, Chérié-Challine L, et al. Serum thyrotropin and free thyroxine reference ranges as defined in a disease-free sample of French middle-aged adults. Clin Chem Lab Med 2009;47(12):1497-505.