خبرگزاری آیصد (جدیدترین و جذاب ترین اخبار ایران و جهان)

Comparative study on determination of plasma thyroid hormones using the three immunoassay methods in sheep

Behrad Eshratkhah

Department of Veterinary Medicine, Shabestar Branch, Islamic Azad University, Shabestar, East Aazarbaijan, Iran.

Received 16January 2012; Accepted 4 February 2012; Available Online 5 February 2012

Background:There are various methods to determine of plasma thyroid hormones in human and animals, which among them; the radioimmunoassay (RIA) method is commonly used in livestock. But, the RIA has some disadvantages compare to the other methods. In recent years, new and more safety immunoassay methods such as chemiluminescence (CLIA) and electrochemiluminescence (ECLIA) are introduced to deter-mine of the thyroid hormones in medical diagnostic laboratories.

Objective:The aim of this study was to compare the concentrations of plasma thyroid hormones e.g. thyroxine (T₄)and triiodothyronine (T₃) in sheep when determined using the RIA, CLIA and ECLIA methods.

Methods:This study was performed on bloodsamples from 40 clinically healthy and non-pregnant Iranian fat-tailed adult sheep. Blood samples were collected from the jugular vein and the separated plasma was used to determine of T₄ and T₃ concentrations by RIA, CLIA and ECLIA methods.

Results:There were significant differences between all the used immunoassay methods concerning the both T₄ and T₃ concentration. The higher concentration of T₄ was observed when determined using the RIA; but, the concentration of T₃ was higher when determined using the ECLIA method. The linear regression analysis of T₄ and T₃ values revealed that the results of CLIA with the ECLIA (T₄: P = 0.010, r = 0.626) (T₃: P = 0.0001, r = 0.922), and also of the RIA with CLIA (T₄: P = 0.017, r = 0.584) (T₃: P = 0.007, r = 0.701) and ECLIA (T₄: P = 0.001, r = 0.688) (T₃: P = 0.006, r = 0.688) were significantly correlated.

Conclusion:CLIA and ECLIA can be used as alternative methods for determination of T₄ and T₃ in sheep, but, the results of ECLIA was more consistent to the RIA. Therefore, ECLIA can be introduced as a good and safe alternative method for assaying sheep T₄ and T₃ concentrations in veterinary diagnostic laboratories and research centers.

Key words Thyroxine, triiodothyronine, radioimmunoassay, chemiluminescence immunoassay, electrochemiluminescence immunoassay, plasma, sheep

Introduction

The thyroid gland secret some of the most important endocrine hormones e.g. thyroxine and triiodothyronine, affect the metabolism of various macromolecules such as carbohydrate, protein and lipid in most body tissues. The thyroid hormones have increasing effect on basal metabolism, glucose availability to cells, protein synthesis and lipid metabolism (Wheeler and Lazarus, 1994; Todini et al., 2007). Therefore, appropriate activity of thyroid gland is crucial to sustain a good productive performance in livestock. The thyroxine hormone (T₄) is the main product secreted by the thyroid gland in many species (Wheeler and Lazarus, 1994; Pfannenstiel and Saller, 1981; Eshratkhah et al., 2011a, b), and the main hormone of thyroid gland in adult sheep is T₄ (approximately 90.4 %) which the major part of blood T₄ is present in protein-bound form (Eshratkhah et al., 2011a). Triiodothyronine (T₃) is mainly formed extrathyroidally, in the liver, and is the hormone principally responsible for the development of the effects of the thyroid hormones on the various target tissues (Todini et al., 2007). T₃ comprises 8.8 % of total thyroid hormones of the thyroid gland in adult sheep and similar to T₄, more than 99.5 % of T₃ circulate in plasma bound to plasma proteins (Eshratkhah et al., 2011a). Measurement of plasma T₄ and T₃ concentrations are generally regarded as important in-vitro diagnostic tests for assessing thyroid function. There are some disorders of thyroid gland in ruminant, which between them; the hypothyroidism is the most common type of its disorders which the possible causes are low iodine intake or an interference with dietary iodine absorption and utilization (Guyot et al., 2007; Gupta et al., 2010). Many methods have been used to determine of plasma thyroid hormone concentration in livestock which among them, radioimmunoassay (RIA) has been used routinely in veterinary medicine (Singh et al., 1997; Eshratkhah et al., 2011a, c). Use of the RIA has some disadvantages compare to the newer developed non-isotopic immunoassays determination methods such as chemiluminescence (CLIA) and electrochemiluminescence (ECLIA). RIA is more harmful to the operators in a long time as the use of radioisotopes, therefore, above mentioned new methods are used routinely in medical diagnostic laboratories in recent years (Sanchez-Carbayo et al., 1999; Eshratkhah et al., 2010 a). The CLIA and ECLIA methods have been used for determination of plasma thyroid hormones in ruminant (sheep, cow and calves) and poultry (Eshratkhah et al., 2010 a, b; 2011d-f). The aim of this study was to compare the concentrations of plasma T₄ and T₃ using the RIA, CLIA and ECLIA methods in sheep.

Materials and Methods

Experimental animals

This study was performed on blood samples from 40 Iranian fat-tailed adult sheep, aged 1-3 years at the animal house at the Islamic Azad University, Shabestar branch, East Azarbaijan, Iran. All the animals were treated with febendazol (Razak Co., Iran; 10 mg/kg) before the study, and also were clinically healthy, non-pregnant and free from internal and external parasites.

Blood sampling

Blood samples were collected from the jugular vein, using heparinized syringes (1000 Iu/ml) from the animals at 9-11 A.M. The study was carried out in spring and the mean ambient temperature was 23°C.The plasma was separated by centrifugation at 750 × g for 15 min and then frozen at -20°C until used.

Sample analysis and assay performance

Theconcentration of plasmaT₄ and T₃ were measured in duplicate by Cobas ECLIA kits (Roche Boeringer-Mannheim, USA) with the Elecsys 2010 analyzer, RIA kits (Biosource Europe SA & Belgium) with the Kontron analyzer (Kontron Co., Sweden), and DiaSorin CLIA kits (Strada per Cresentino-13040 Saluggia (Vercelli), Italy) according to the kit manufacturer recommendations. As the hypothyroidism is the most common type of thyroid disorder in ruminants (Gupta et al., 2010), intra-run precisions of all the used immunoassays methods were determined by evaluating 10 pooled plasma samples with low T₄ and T₃ concentration, 3 times within the same run of assay by all methods. Then, the percentages coefficient of variation (CV %) were calculated. Determination of inter-run precisions was performed using the 3 pooled plasma samples with low concentration of T₄ and T₃ concentration, which analyzed every day for 10 days. For the determination of assay linearity, two pooled plasma samples with low and moderate concentration of T₄ and T₃ concentration were used. Each pooled samples were diluted 1/2, 1/5 and 1/10 with saline. Then, each dilution was determined in duplicate by RIA, CLIA and ECLIA methods. The evaluations were made by the percentage of difference between the expected and observed values. For recovery studies, at first a pooled sample with low T₄ and T₃ concentration was selected and the levels of studied hormones were determined 5 times 4 day by RIA, CLIA and ECLIA methods. Different amounts of this sample were added to the two plasma samples at different concentrations of T₄ and T₃ concentration. The recovery percentages were calculated by the differences between the expected and observed values.

Statistical analysis

The data was analyzed by ANOVA method for determination of significant difference between RIA, CLIA and ECLIA values at the P< 0.05 level using SPSS v. 16 software. The linear regression analysis was performed to determine the percentage coefficient of variation (CV %), coefficient of determination (r ²), correlation coefficient (r), 95 % confidence interval (CI) and the slope of the curve. All the values are shown as mean ± standard deviation (SD).

Results

The results of present study regarded that there was a significant difference among the used immunoassays methods for determ-ination of T₄ and T₃ concentration in sheep. The mean ± SD and 95 % CI of the plasma concentrations of thyroid hormones in sheep are presented in Table 1. The results exhibited significant differences between all the used methods for T₄ and T₃ determination in Sheep, and the higher values of T₄ and T₃ observed when using the RIA and ECLIA methods, respectively.

The existence of significant differences for T₄ and T₃ values using the three immunoassay methods are shown in Table 2. The intra- and inter-run percentage coefficients of variations results of T₄ and T₃ determination using the CLIA, ECLIA and RIA methods are presented in Table 3.

The recovery and linearity analysis of obtained values of T₄ and T₃ using the RIA, CLIA and ECLIA methods are presented in Table 4 and 5, and also, the linear regression and analysis of curve the studied thyroid hormones by the above mentioned immunoassays methods are shown in Figures 1- 4. We found only a little overlap concerning the 95 % CI values of T₄ when determined using the ECLIA and RIA methods.

According to our results, the distribution of T₄ and T₃ were linear when CLIA and ECLIA values were plotted against the RIA value. Similar state was observed when the CLIA values were plotted against the ECLIA values. As well as, we showed significant positive correlation concerning the values of studied thyroid hormones when measured using the three immunoassay methods. The results from Kolmogrov – Smirnov test indicated that CLIA, ECLIA and RIA had a normal and relatively similar distribution. Validation of the RIA method for determination of the plasma T₄ and T₃ has been performed by Nazifi et al.. (2008) in sheep.

Discussion

The determination of thyroid hormones plays an important role in the evaluation of ruminant performance. In this present study, the higher values of TT₄ and T₃ observed when determined using RIA and ECLIA methods and their lower level provided using CLIA and RIA methods, respectively. Our results were consistent with the previous reports in sheep (Eshratkhah et al., 2011 a, d). However, the higher values of these hormones in calves were obtained using the RIA compared to the CLIA method (Eshratkhah et al., 2010 a). Results from this study showed that the intra- and inter- run % CV of T₄ and T₃ were < 10 % in the CLIA, ECLIA and RIA methods which implied that there were not more than 10 % difference concerning the values of each hormone of a sample which was analyzed in the same and between run using the above mentioned immunoassay methods. As the obtained CVs from imprecision studies were about < 10 %, demonstrated a good precise of the assays. As well as, we found more than 90 % recovery for the T₄ and T₃ which indicated on good precision of CLIA, ECLIA and RIA methods for determination of plasma T₄ and T₃ concentrations in sheep. According to the coefficient of determination (r ²) of T₄ and T₃ which obtained from the regression equation, about 39.2 % and 98.4 % of the variation in the T₄ and T₃ values using the CLIA method explained by the ECLIA method, respectively. As well as, approximately 34.1 % and 47.3 % of the variation in the T₄ value and also, 49.1 % and 47.3 % of the variation in the T₃ values using the CLIA and ECLIA methods explained by the RIA method, respectively. These finding regarded that the regression equation of T₃ appear to be strongly useful for making prediction about its concentration only using the CLIA and ECLIA methods. But, the regression equations of T₄ and T₃ values revealed moderate or relatively strongly useful for making prediction about their concentrations using the aforementioned methods versus the RIA method. The principle of the DiaSorin CLIA kits for the quantitative determination of T₄ and T₃ is based on competitive CLIA where a specific polyclonal antibody of T₄ and T₃is coated on the solid phase (solid phase antigen linked technique). These hormones are linked to isoluminol- conjugate which during the incubation the aforementioned hormones dissociated from its binding protein and compete with labeled hormone for binding site on the antibody. But, the Cobas ECLIA kits employ a competitive test principle assaying anti-antibody specifically directed T₄ and T₃. During the test, endogenous - derivative T₄ and T₃ released by the of 8-anilino-1-naphthalene sulfonic acid, compete with the added biotinylated T₄- and T₃- derivative for binding sites on the antibodies labeled with the ruthenium complex. The test principle of Biosource RIA kits is based on competition a fixed amount of I¹²⁵ labeled T₄ and T₃ with the T₄ and T₃ hormonesto be measured present in the sample or in the calibrator for a fixed amount of anti- T₄ and T₃ antibody sites, respectively, which are bound to the goat anti mouse antibodies immobilized to the wall of a polystyrene tube. It seems that, the type of antibody, binding affinity of T₄ and T₃hormones for sites on the labeled antibodies and the concentrations of calibrators were effective factors on the results. The observed differences in the T₄ and T₃ values when determined using the CLIA, ECLIA and RIA methods may be related to separate or complex effects of the above mentioned effective factors. As, unlike to the RIA and CLIA methods, the sheep anti- T₄ and T₃ antibodies (labeled with ruthenium complex) are used for determination of T₄ and T₃ in ECLIA method. As well as, likely tend of plasma T₄ and T₃ for binding to anti- T₄ and T₃ antibodies labeled with ruthenium complex may be higher compared to the isoluminol- antibody conjugate in CLIA method. Our results showed that, T₄ and T₃ values in sheep were consistent with ones reported by the kits manufacturers in man. It is particularly important when T₄ and T₃ assays used in sheep to yield accurate and precise T₄ and T₃ values at their low concentrations. According to the manufacturer recommendations, pregnancy, haemolysis, lipemia, bilirubinemia, some drugs (such as salicylate, testosterone) and biotin (especially in ECLIA method) are potentially interfering substances. However, in this study, as all the animals were clinically healthy and non-pregnant, the assay performance was not affected by the above mentioned factors. The exact explanation of these findings is not possible at this time as various endogenous (such as age and sex) and exogenous factors and related physiological changes may effects on the results. Indeed, it needs to do more studies on quality control, limitations and interfering factors which effects on the obtained results from the used immunoassay methods in sheep.

The comparison of the RIA, CLIA and ECLIA performances suggested that, CLIA and ECLIA can be used as alternative methods for quantitation of T₄ and T₃ in sheep. The higher level of T₄ and T₃ provided by RIA and ECLIA methods and their lower level provided by CLIA and RIA methods, respectively. Although, all the aforementioned methods exhibited approximately similar good precision for determination of T₄ and T₃ values in sheep. But, it seems that, as the sheep anti- T₄ and T₃ antibodies are used for T₄ and T₃ quantitation in ECLIA method, the provided values of T₄ and T₃ using the ECLIA method are more acceptable than other methods in sheep.

Therefore, veterinary clinicians and researchers must be aware of the differences between these immunoassay methods when assaying T₄ and T₃