Electro-chemiluminescence Nº P-034 Good Agreement between Two Methods - of 1st- and 3rd-Generation - for the Detection of Autoantibodies to the Thyrotrophin Receptor (TRAbs). Rodriguez. MP(1); Zaidman. V(2); Gazek. N(3); Herzovich. V(3); Maceiras. M(2); Dujovne. N(3); Miras. M(4); Muñoz. L(1); Pelanda. M(2); Lazzati. JM(2) and Belgorosky. A(3) (1)Laboratorio de Endocrinología. Hospital de Niños de Córdoba. Córdoba. Argentina (2)Laboratorio de Endocrinología. Hospital de Pediatría JP Garrahan. Buenos Aires. Argentina (3)Servicio de Endocrinología. Hospital de Pediatría JP Garrahan. Buenos Aires. Argentina (4)Servicio de Endocrinología. Hospital de Niños de Córdoba. Córdoba. Argentina. Introduction  The thyrotrophin receptor (TSHr) is a major autoantigen in autoimmune hyperthyroidism. Graves’ disease (GD) is the most common cause of hyperthyroidism in children. caused by autoantibodies that bind to the TSHr and stimulate the signal transduction cascade. Measurement of autoantibodies to the thyrotrophin receptor (TRAbs) is therefore used to determine the etiology of hyperthyroidism and in the follow-up of neonates born to mothers with GD. Over the last few decades methods for TRAb detection have become sensitive. 1st generation liquid-phase TRAb assays with detergent solubilized porcine TSHr was introduced into routine thyroid serology and proved to be highly specific for GD. albeit with moderate sensitivity. In the 1990s. 2nd generation solid-phase TRAb assays with immobilized porcine or recombinant human TSHr became available. Recently. 3rd generation TRAb assays have been developed. in which the human thyroid stimulating monoclonal antibody M22 replaces bovine TSH as the competing reagent for TRAb binding to TSHr. Currently. traditional radio-binding assay methods are being replaced by automated methods. Aim To evaluate agreement between two methods to measure TRAbs in different diagnosis-related groups of patients with thyroid and non-thyroid autoimmune diseases. Subjects and methods Table 1: TRAb measurement methodology descriptions. Table 2 : Demographic and serological data of all study patients. (1st-generation) RSR – RIA kit (3rd-generation) Electro-chemiluminescence immunoassay (ECLIA) Manual RIA Automated chemiluminescence two-step competitive Solubilized porcine TSHr Porcine TSH labelled Human anti-TSHr autoantibody (M22) 125I Ruthenylated MRC LATS Std.B NIBSC 90/672 Inhibition of TSH binding ( %IB) IU/L ≤10 % ≤ 1.75 IU/L 11.4 9.7% (1.73 IU/L) - 1.8 % ( 25.5 IU/L) Group M/F n Age (y) mean ± SD TSH (µIU/mL) mean ± SD fT4 (ng/dL) mean ± SD ATPO +/- (+ %) ATG +/- (+%) Total 66/32 98 9.7±5.27 3.63±5.71 1.11±0.37 36/62 (36.7) 20/78 (20.4) HT 14/26 40 11.0±4.45 2.62±4.86 1.17± 0.54 28/12 (70) 13/27 (33) NAH 9/16 25 5.7±5.88 5.34±8.46 1.12±0.21 2/23 (8) ADNT 8/15 23 10.5±4.22 2.56±1.12 1.03±0.12 0/23 (0) 1/23 (4.3) CLT 1/9 10 13.1±3.50 6.07±6.15 1.04±0.11 6/4 (60) 4/6 (40) HT: Hyperthyroidism; NAH: Non-autoimmune hypothyroidism; ADNT: Autoimmune disease with normal thyroid function; CLT: Hypothyroidism due to chronic lymphocytic thyroiditis . Results  Fig.1 Correlation between RSR (%) vs. ECLIA (IU/L). The methods showed a positive correlation (Passing & Bablok regression analysis) with significant deviation from linearity (p <0.01). Table 4: Results of disagreeing samples. Group TSH (µIU/mL) fT4 (ng/dL) T3 (ng/mL) T4 (µg/dL) ATPO (UI/mL) ATG TRAb - RSR (% IB) TRAb-ECLIA (UI/L) HT 3.91 1.04 1.35 7.5 10 20 3.0 2.0 3.27 1.01 1.33 8.9 466 23 2.1 6.6 0.86 0.89 6.9 906 1130 2.2 0.87 0.97 1.56 5.6 131 5.7 1.8 0.03 1.92 2.79 13.0 10000 2.3 1.43 1.58 10.5 531 3335 9.5 3.1 0.52 0.94 1.82 6.8 4703 33 10.0 2.7 NAH 3.16 0.95 7.0 2.5 ADNT 2.91 1.18 2.6 1.4 0.9 16 10.1 0.3 2.08 1.45 8.5 11.8 r = 0.87 CI 95% (0.81 to 0.91) p<0.0001 y = 2.3206 + 2.2646 x Fig. 2 and 3 Distribution of RSR TRAb levels in different groups. The cut-off point is shown by the dotted line for RSR and ECLIA 10% and 1.75 IU/L respectibly. Fig. 4 Distribution of discordant results found comparing both methods. Cut-off RSR 8.9 to 11.1 % Cut-off ECLIA 1.6 to 1.9 IU/L Table 3: TRAb assay comparison. Group RSR + ECLIA + RSR - ECLIA - ECLIA - RSR - Disagreement (%) Kappa Coefficient (K) Total 19 68 2 9 11.2 0.70 HT 18 14 1 7 20.0 0.61 NAH 23 8.0 - ADNT 22 4.3 CLT 0.0 1.00 Conclusions In spite of the different characteristics of the methods used. acceptable agreement was found. In the diagnosis-related groups TRAb levels were similar for both measurement methods. in samples with discordant results TRAb levels were near the cut-off point. The methodological shift to fully automated TRAb detection systems allowed for the reduction of manual procedures and seems to be reliable for the diagnosis of autoimmune thyroid diseases. The “k” coefficient was good -between 0.7 and 1.0- in all groups in which it could be evaluated; however. in 11 samples (11.2%) disagreement was observed. Disagreement was greatest in HT at 20%. In samples with discordant results TRAb levels were near the cut-off point. 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