Clinical Evaluation of the First Automated Assay for the Detection of Stimulating TSH Receptor Autoantibodies

Transcription

1 Endocrine Cre 795 Clinicl Evlution of the First Automted Assy for the Detection of Stimulting TSH Receptor Autontiodies Authors S. Allelein *, M. Ehlers *, S. Goretzki, D. Hermsen, J. Feldkmp 3, M. Hse, T. Dringenerg, C. Schmid, H. Hutzel, M. Schott Affilitions Affilition ddresses re listed t the end of the rticle Key words Grves disese utoimmune thyroiditis ridge ssy received 3.9. ccepted 9.. Biliogrphy DOI Horm Met Res ; 8: Georg Thieme Verlg KG Stuttgrt New York ISSN 8-53 Correspondence Dr. M. Schott, MD, PhD Division for Specific Endocrinology Medicl Fculty University of Düsseldorf Moorenstr. 5 5 Düsseldorf Germny Tel.: + 9//8 8 Fx: + 9//8 98 Mtthis.Schott@med.uniduesseldorf.de Introduction Grves disese (GD) is n utoimmune disese cused y utontiodies, which ind to the thyrotropin receptor (TSHR) on the surfce of thyrocytes, resulting in uncontrolled overproduction of thyroid hormones [ 3]. For quntifiction of TSHR utontiodies (TRAs) nd confirmtion of the clinicl dignosis, different types of ssy technology re commonly used in lortory medicine. The most widely used ssys mesure the competition etween inding of TRA nd TSH [, 5] or TSHR directed humn monoclonl utontiody [], respectively, t the TSHR. The ltter ssy lso exists in n utomted system []. In contrst to these in vitro TSH competition ssys, iossys mesure incresed production of cyclic AMP in cellulr systems [7 ]. These iossys lso exhiit high specificity ut re delicte nd lorious. The recently commercil- * Both uthors contriuted eqully to this work Astrct Until recently, stimulting TSH receptor utontiodies (stras) could only e mesured y iossys. A new ssy system, which directly detects stra in ser y pplying ridge technology, hs een estlished nd is now ville s utomted chemiluminescence (ridge) immunossy. We evluted the utomted ridge ssy in clinicl routine nd compred it with conventionl utomted TRA ssy (competition ssy). Altogether, Grves disese (GD), 57 utoimmune thyroiditis, 7 non-utoimmune nodulr nd 9 thyroid cncer ptients, s well s helthy were retrospectively evluted. ROC plot nlysis sed on ser of newly dignosed GD ptients reveled n re under curve of.99 (95 % CI:.99.) indicting high ssy sensitivity nd specificity. The highest sensitivity ( %) nd specificity (99 %) were seen t cut-off level of.55 IU/l. The clculted positive predictive vlue ws 9 %, wheres the negtive ws %. Applying ROC plot-derived cut-off of.3 IU/l, derived from ser of GD ptients lredy receiving ntithyroid drug therpy for months, the sensitivity ws 99 % wheres the specificity ws 98 %. Detiled comprison of oth ssy systems used reveled slightly different distriution of stra nd TRA. Mesurement of stra during follow-up reveled stedy decline over one yer of follow-up. In summry, our results demonstrte tht the new utomted ridge ssy system for detecting stra hs high sensitivity nd specificity for dignosing GD nd to discriminte from other thyroid diseses, respectively. Our study, however, does not provide full evidence tht the ridge ssy is specific for stra only. ized Thyretin iossy, which detects the stimultory ctivity of TRA y chimeric TSHR nd cyclic AMP response element (CRE)-reporter gene nd luciferse signling [8], hs een pproved y the FDA for use in the clinicl lortory. The stndrdiztion of the Thyretin iossy [9] s well s stndrdized rpid iossy with detection of thyroid stimultion using cyclic AMP-gted clcium chnnel nd equorin [] were pulished. Most recently, new ssy system hs een pulished, which directly detects the concentrtion of stra in ser of ptients y pplying ridge technology []. Within this ssy utontiodies re detected y inding with one rm to cpture receptor on the solid phse nd ridging with the other rm to detection receptor giving signl. The ssy uses chimeric TSHRs detecting thyroid stimulting immunogloulins sed on n understnding of the structure of the extrcellulr domin of the TSHR nd its interctions Allelein S et l. stra Decteted y New Automted Assy ; 8: 795 8

2 79 Endocrine Cre with nti-tshr ntiodies [, 3]. This mnul ssy hs een pulished to hve sensitivity of 99.8 % nd specificity of 99. %, respectively, with dignostic ccurcy of.998 []. Bsed on this principle, new utomted ssy for the detection of stimulting TRA (stra) hs een developed. The im of the present study ws to evlute the performnce of the new utomted ridge ssy for detection of stra in clinicl setting nd to compre it with conventionl utomted TRA ssy (competition ssy). Altogether, 7 ser of ptients with different thyroid diseses nd helthy hve een nlyzed. Ptients nd Methods Ptients Altogether, 7 individuls were included in the study. Of those, suffered from GD (8 % femles; men ge yers; rnge 8 87 yers), 57 suffered from utoimmune thyroiditis (8 % femles; men ge 7 yers; rnge 7 75 yers), 7 hd nonutoimmune nodulr thyroid disese (79 % femles; men ge 58 yers; 85 yers), 7 hd differentited thyroid cncer (37 ppillry nd folliculr), hd nplstic, nd poorly differentited thyroid cncer (5 % femles; men ge 5 yers; 8 78 yers). In ddition, nother persons (73 % femles; men ge yers; 7 yers) served s control sujects without ny history of thyroid disese. Of the GD ptients, 3 hd newly dignosed disese, wheres 9 GD ptients lredy received ntithyroid drugs. For 5 ptients, the lood smple ws tken within 3 months fter initil dignosis, for ptients within months, nd for 3 ptients within months. For ptients the lood smple ws collected for more thn months fter initil dignosis (verge 77 months). The criteri for GD were sed on initilly documented hyperthyroidism with or without ophthlmopthy nd incresed uptke in the technetium scintigrphy or hypoechogenicity nd incresed lood flow in ultrsound, respectively. The criteri for hyperthyroidism were clinicl symptoms, incresed serum concentrtions of free T, incresed free T3, nd decresed sl TSH. The criteri for n utoimmune thyroiditis were the presence of positive ntithyroperoxidse-utontiodies (nti-tpo- A) nd/or ntithyrogloulin utontiodies (nti-tg-a) without signs of GD nd sucute thyroiditis, respectively. Thyroid functionl tests nd ntiody ssys The serum concentrtions of TSH (reference rnge:.3. IU/l, lower detection limit:. lu/l), free T (norml rnge: pg/ml), nd free T3 (. 5. ng/l) were mesured y commercilly ville electrochemiluminescence ssys from Roche Dignostics. Anti-TG-A ( < IU/l) nd nti-tpo-a ( < 35 IU/l) were mesured y commercilly ville immunossys from Siemens (Immulite ). Detection of stimulting TSH receptor ntiodies The ridge ssy (Immulite, Siemens) used ws n utomted, -cycle, chemiluminescent immunossy. As descried y the mnufcturer, the ssy employs pir of recominnt htshr constructs in ridging immunossy formt. The cpture receptor is immoilized on the solid phse (polystyrene ed). The signl receptor is n lkline phosphtse leled recominnt htshr in uffer solution. In the first cycle, the smple is incuted with the solid phse for 3 min, llowing the thyroid stimulting immunogloulins in the smple to ind through one rm to the cpture receptor. Next, centrifugl wshes remove residul smple. In the second cycle, the signl receptor is dded to the rection tue nd incuted for 3 min. The complexed thyroid stimulting immunogloulins ind the signl receptor through the second rm, forming ridge. Unound signl receptor is then removed y centrifugl wshes. Finlly, chemiluminescent sustrte is dded to the rection tue nd signl is generted in direct reltion to the mount of thyroid stimulting immunogloulins in the smple. Incution cycles re 3 min. The mesuring rnge for stra is:.. IU/l. The ridge ssy ws compred with the Elecsys nti-tshr electrochemiluminescence immunossy (ECLIA) mesured on Cos e nlyzer (Roche Dignostics GmH, Mnnheim, Germny). The ssy ws performed following the mnufcturer s instructions using cut-off of.75 IU/l. Definition of cut-off nd sttisticl nlysis To otin the optiml decision threshold level for positivity, receiver-operting chrcteristic (ROC) nlysis ws performed []. Sensitivity/Specificity pirs were clculted y vrying the decision threshold levels over the entire rnge of stra vlues. Sensitivity (the true positive results) ws clculted from ptients with GD. Specificity (the true negtive results) ws clculted from helthy individuls nd 8 ptients with different thyroid diseses (excluding GD ptients) helthy. The positive predictive vlue (PPV) ws clculted s follows: PPV = numer of ntiody true positive GD ptients s frction of the totl numer of ntiody positive sujects (true nd flse positive sujects). The negtive predictive vlue (NPV) ws clculted s follows: NPV = numer of true ntiody negtive GD ptients s frction of the totl numer of ntiody negtive sujects (true nd flse negtive sujects). Comprison ws done y ANOVA-test nd Dunnett s multiple comprison test (for dt showing Gussin distriution) or Kruskl-Wllis test nd Dunn s multiple comprison test (for not normlly distriuted dt) clculted with Prism computer softwre (GrphPd Softwre Inc., Sn Diego, CA, USA). Correltion nlysis ws performed with Spermn s test. To investigte the distriution of ptients showing stra or TRA level elow or ove -times positivity, contingency tles were nlyzed y chi-squre test. A p-vlue less thn.5 ws considered sttisticlly significnt. Results Comprison of proposed nd clculted cut-off In order to independently clculte the sensitivity nd specificity of the ridge ssy, we first performed ROC plot nlysis. This nlysis hs een compred with the mnufcturer s recommendtions. Altogether helthy individuls nd 8 ptients with different thyroid diseses (excluding GD ptients) were included for clculting specificity nd 3 ptients with newly dignosed GD were used for clculting sensitivity. The re under the curve (AUC) for the utomted ridge ssy ws.999 (95 % CI:.998.; Fig. ). Optiml sensitivity (; 95 CI: 88.3 to. %) nd specificity (99; 95 CI: 9.8 to %) were seen t cut-off level of.55 IU/l. The corresponding PPV ws 9 %, wheres the NPV ws estimted to e %. These dt re in ccordnce with the mnufcturers pro- Allelein S et l. stra Decteted y New Automted Assy ; 8: 795 8

3 Endocrine Cre 797 Sensitivity [%] Frequency Fig % - Specificity [%] crosspoint of highesst sensitivity nd specificity resulting in cut-off for stra of.55 IU/l stra [IU/l] ROC plot nlysis of ptients with newly dignosed GD Are under curve (AUC):.999 (95% CI:.998.) ROC plot nd Gerhrdt plot of stra including newly dignosed Grves disese ptients. ROC plot nlysis including the dt of ptients with newly dignosed Grves disese for sensitivity nd other sujects including helthy nd ptients with utoimmune thyroiditis, non-utoimmune nodulr, nd thyroid cncer for specificity. Gerhrdt plot showing the frequency distriution of sujects dependent on the stra serum level. Grey rs represent norml s well s ptients with utoimmune thyroiditis, non-utoimmune, nd thyroid cncer. Blck rs represent newly dignosed Grves disese ptients. Bsed on these dt the clculted sensitivity (solid line) nd specificity (dshed line) is shown. The cross-point of oth lines represents the clculted cut-off of the ssy. stra vlues > IU/l were estimted s. posed threshold for stra positivity t.55 IU/l. The sum of these dt were lso clculted nd illustrted within Gerhrdt plot ( Fig. ). In ddition, we lso clculted second ROC plot nlysis y including GD ptients who hd lredy received ntithyroid drug therpy for months. This hs een done to give n impression on how the ridge ssy works in clinicl routine when ptients re included tht hve lredy een treted with ntithyroid drugs. As shown in Fig. the AUC ws By using the clculted cut-off for stra positivity of.3 IU/l the sensitivity of the ssy ws 99 % (95 CI: 9.79 to %), wheres the clculted specificity ws 98 % (95 CI: 95.7 to 99.5 %). The PPV for stra positivity ws 95 % nd the NPV ws %. By using the cut-off of.55 IU/l, which hs een clculted for purely newly dignosed GD ptients, the sensitivity of the ridge ssy (for lredy treted GD ptients) would then e 9 % (95 CI: to 99.7 %), wheres the clculted specificity would e 99 % (95 CI: 9.8 to %). The PPV for stra positivity would e 97 % nd the NPV 99 %. Clinicl evlution of the ridge ssy nd comprison with n utomted competition ssy In order to evlute the utomted ridge ssy in clinicl routine, we mesured stra nd TRA not only in ptients with GD ut lso in other thyroid diseses including utoimmune thyroiditis (), non-utoimmune, nd thyroid cncer Sensitivity [%]; Specificity [%] Sensitivity [%] Frequency Fig % - Specificity [%] crosspoint of highesst sensitivity nd specificity resulting in cut-off for stra of.3 IU/l stra [IU/l] ROC plot nlysis of ptients with GD ptients (ID mx. months go) Are under curve (AUC):.9987 (95% CI:.997.) ROC plot nd Gerhrdt plot of stra including treted Grves disese ptients with months ntithyroid drug therpy. ROC plot nlysis including the dt of ptients with Grves disese eing dignosed months efore for sensitivity nd other sujects including helthy nd ptients with utoimmune thyroiditis, non-utoimmune nodulr nd thyroid cncer for specificity. Gerhrdt plot showing the frequency distriution of sujects dependent on the stra serum level. Grey rs represent norml s well s ptients with utoimmune thyroiditis, non-utoimmune, nd thyroid cncer. Blck rs represent Grves disese ptients (initil dignosis months efore). Bsed on these dt the clculted sensitivity (solid line) nd specificity (dshed line) is shown. The cross-point of oth lines represents the clculted cut-off of the ssy. stra vlues > IU/l were estimted s. Helthy without ny history of thyroid disese served s. As shown in Fig. 3,, oth ssys significntly detected GD ptients (p <.). The utomted ridge ssy detected slightly higher percentge within the group of ll GD ptients compred to the competition ssy (8/; 8 % vs. 78/; 79 %, not significnt). Anlyzing the group of non-gd ptients some differences were lso seen: Here, stra ove the cut-off for positivity ws seen in of 57 ptients (3.5 %). Another 3 ptients nd cncer ptients hd detectle stra t low rnge, however, without reching positivity. In contrst, positive TRA were detected in out of 8 (. %) non-gd ptients (,, thyroid cncer). Of note, in nother 7 ptients (. %) TRA were detectle, however without reching vlues ove the cut-off for positivity ( Fig. 3,). Becuse of different thresholds for stra nd TRA positivity, which is cused y clirtions ginst different WHO stndrds, thresholds (cut-offs) for positivity were estimted s nd differences were expressed s multiples from this cut-off ( Fig. 3c,d). A quite similr pttern ws seen for oth ssys. A more detiled nlysis, however, reveled significnt pttern differences y compring oth ssys ( Fig. 3c,d). By using cut-off of -times positivity, the reltion of positive to negtive ptients ws significntly higher for stra in comprison to TRA (7/3 vs. 59/ %; p =.3, not shown) Sensitivity [%]; Specificity [%] Allelein S et l. stra Decteted y New Automted Assy ; 8: 795 8

4 798 Endocrine Cre c stra [IU/l] stra [IU/l] distriution of stra expressed s multiples from the cut-off for positivity ll GD ll GD GD months GD months thyroid cncer thyroid cncer TRA [IU/l] 3 Fig. Correltion etween stra nd TRA. A significnt correltion of stra to TRA levels for ll Grves disese ptients ws seen (Spermn r =.8738; p <.). Additionlly, to compre oth ssy systems, correltion nlysis of the ridge ssy nd the competition ssy ws done. A strong correltion etween stra nd TRA ws seen (Spermn r =.8738; p <., Fig. ). d TRA [IU/l] distriution of TRA expressed s multiples from the cut-off for positivity 3 3 ll GD ll GD GD months GD months thyroid cncer thyroid cncer Fig. 3 stra nd TRA levels s well s mgnitudes of the cut-off for positivity. Distriution of mesured stra nd TRA levels ( nd ) nd distriution of stra nd TRA expressed in multiples of the cut-off for positivity (c nd d) in ptients with Grves disese (ll GD) independently of the disese stge, new-onset GD (new GD), GD with ntithyroid drug therpy months, utoimmune thyroidits (), non-utoimmune ptients (), thyroid cncer, nd norml. As recommended y the mnufcturers nd sed on our nlyses the following cut-offs for stra nd TRA positivity hve een used: ridge ssy for the detection of stra:.55 IU/l; competition ssy for the detection of TRA:.75 IU/l. For ll GD, the distriution of stra multiples differs significntly from TRA multiples of the cut-off: stra levels elow -times positivity (7 % of ptients) vs. > times positivity (3 % of ptients) compred to TRA within these rnges (59 nd %; p =.5). TSH receptor utontiodies during follow-up As shown in Fig. 5, there ws cler decline of stra during follow-up of more thn months fter initil dignosis (Spermn r =.5, p =.). A similr picture ws seen in the sme group of ptients y testing for TRA in the competition ssy (Spermn r =.85, p <.) even though the competition ssy showed slightly more pronounced TRA decline (ns, Fig. 5). Correltion etween stra, TRA, nd ft stra s well s TRA vlues of ll GD ptients correlted significntly to ft vlues (stra: Spermn r =.7, p <.5; TRA: Spermn r =.3, p <.5;) even though the correltion for stra ws less pronounced (ns; Fig.,). Discussion The im of the present study ws to evlute the new utomted ridge ssy for the detection of stimulting TSH receptor utontiodies (stra) in ptients with Grves disese (GD) nd to compre these dt with those of other thyroid diseses nd helthy. Additionlly, we compred this ssy with conventionl utomted TRA ssy (competition ssy). Bsed on ROC plot nlysis we propose cut-off for positivity of.55 IU/l resulting in sensitivity of % nd specificity of 99 %. Our results demonstrte tht this new ssy system for the detection of stra hs high sensitivity for detecting GD nd specificity for discriminting GD from other thyroid diseses. We lso investigted stra nd TRA levels during fol- Allelein S et l. stra Decteted y New Automted Assy ; 8: 795 8

5 Endocrine Cre stra [IU/l] TRA [IU/l] ID ID ID 3 m. ID 3 m. 3 m. 3 m. m. m. > m. > m. Fig. 5 stra nd TRA in dependency to the time point of initil dignosis of Grves disese nd during follow-up. stra nd TRA levels in dependency to the time since initil dignosis (ID) of Grves disese re shown. The longer the intervl etween ID nd mesurement, the lower is the level of stra nd TRA, respectively (stra: Spermn r =.5; p =.; TRA: Spermn r =.85; p <.). low-up of the disese. As expected, we found decline of these ntiodies over time period of more thn months. Up to now, only mnul iossys for the detection of stra re ville [7 ]. The drwck of ll mnul ssy systems sed on cyclic AMP mesurement in cellulr systems nd independently of their individul sensitivity nd specificity is, however, their lor-intensive nd time consuming ssy procedure tking severl hours. The gret dvntge of the fully utomted stra detection system descried here is the short performnce time of out one hour without the necessity of collecting certin numer of ptient smples s dvisle for mnul ssys. Another dvntge is tht testing with the utomted stra ssy cn e integrted into the workflow on routine lortory nlysers without splitting of ptient smples. We otined threshold for stra positivity of.55 IU/l for newly dignosed GD ptients nd defined grey zone etween.3 IU/l nd <.55 IU/l for GD ptients who hd TRA [IU/l] stra [IU/l] 3 8 free thyroxine [ng/l] 8 free thyroxine [ng/l] Fig. Correltion etween stra/tra nd free thyroxine. Correltion of stra nd TRA levels to free thyroxine (ft) of ll Grves disese ptients. Both, stra nd TRA positively correlte to the ft level (stra: Spermn r =.7; p <.5; TRA: Spermn r =.3; p <.5). lredy received ntithyroid drug therpy for less thn months. For the cut-off nlysis, only 3 ptients with newly dignosed GD were ville. Still, these dt re sttisticlly significnt (AUC =.999) nd re in line with the mnufcturer (.55 IU/l) nd very similr to the study y Tozzoli et l. (.5 IU/l) [5]. Interestingly, the sme grey zone rnge ws lso found y Frnk et l., however, on the sis of the mnul stra ssy using the sme ridge technology []. In our study we noted slightly higher proportion of ptients with orderline positive TRA. In the pst, we nd others lredy reported on the existence of TRA in ptients (without defining their potentil stimulting, locking or neutrl ctivity) [,, 7]. Most recently, Khly et l. reported on stra in Allelein S et l. stra Decteted y New Automted Assy ; 8: 795 8

6 8 Endocrine Cre ptients with ssocited oritopthy [8]. Within tht study, stra were mesured y the mnul Thyretin iossy descried efore. The slightly incresed prevlence of stra compred to TRA is most likely due to the higher nlyticl performnce of the iossys for the detection of stra compred to conventionl TBII ssys. Our present study confirms this oservtion. Bsed on these dt, it needs to e discussed whether TRA positivity in such ptients my indicte yet undefined mixed immune response with fetures of oth, nd GD. Another importnt issue is the non-identicl distriution of stra nd TRA. Since oth ssys tested re clirted ginst different WHO stndrds, thresholds (cut-offs) for positivity were estimted s nd differences of oth ssys were expressed s multiples from the cut-off ( Fig. 3c,d). Within smll rnge round the cut-off for positivity, some,, nd thyroid cncer ptients were TRA positive y using the competition ssy. In contrst, on the sis of the ridge ssy, only ptients nd none of the or thyroid cncer ptients were stra positive. This phenomenon my indicte higher specificity of the ridge ssy for detecting stra compred to the competition ssy for detecting TRA. As recently descried [], the ridge ssy relily detects stra in GD nd discrimintes GD ptients from other thyroid diseses. Within the study y Frnk et l., significnt correltion etween stra nd ft titers hs een shown. Our dt, do not provide full evidence tht stra mesured y the ridge ssy re in fct thyroid-stimulting ntiodies only. Although stra titers significntly correlted with ft levels, the sme ws lso true for TRA titers ( Fig. ). Here, stronger correltion ws seen. The weker correltion etween stra nd ft levels is certinly due to the limited numer of newly dignosed GD ptients nd the high numer of GD ptients lredy treted y ntithyroid drug therpy. Nevertheless, s shown in Fig. 3c,d the distriutions of stra nd TRA levels show some differences, for exmple, ove the threshold of -times positivity. Most recently, Tozzoli et l. evluted the ility of the utomted ridge ssy to identify GD ptients, in comprison with other immunossy methods (including the utomted TRA ssy s we did) [5]. Interestingly, n cceptle greement etween the ridge ssy nd the other immunossy methods were seen. Therefore, in line with our results, full evidence tht this ssy is specific for stra only could not e provided. Nevertheless, the detection of stra cn e ssumed ecuse of the ssys principle []. Further studies need to e performed in order to clrify whether stra mesured in this ssy hve simulting cpcity. In summry, our results demonstrte the new utomted ridge ssy to detect stra with high sensitivity (in dignosing GD) nd specificity (in discriminting it from other thyroid diseses). Bsed on our dt, we propose cut-off for stra positivity of.55 IU/l. Whether this ssy will lso help for n improved outcome prediction in GD ptients [9, ] s well s in Grves ophthlmopthy [ 3] need to e investigted in future studies. Conflict of Interest The uthors declre no conflict of interest. Affilitions Division for Specific Endocrinology, Medicl Fculty, University of Düsseldorf, Düsseldorf, Germny Institute of Clinicl Chemistry nd Lortory Dignostics, Medicl Fculty, University of Düsseldorf, Duesseldorf, Germny 3 Deprtment for Endocrinology nd Dietes, Municipl Hospitl Bielefeld, Bielefeld, Germny Deprtment for Nucler Medicine, Medicl Fculty, University of Düsseldorf, Düsseldorf, Germny References Brtlen L. Dignosis nd mngement of Grves disese: glol overview. Nt Rev Endocrinol 3; 9: 7 73 Morshed SA, Dvies TF. Grves Disese Mechnisms: The Role of Stimulting, Blocking, nd Clevge Region TSH Receptor Antiodies. Horm Met Res 5; 7: Furmnik J, Snders J, Nunez MR, Rees SB. Mechnisms of Action of TSHR Autontiodies. Horm Met Res 5; 7: Shewring G, Smith BR. An improved rdioreceptor ssy for TSH receptor ntiodies. Clin Endocrinol (Oxf) 98; 7: Costgliol S, Morgenthler NG, Hoermnn R, Bdenhoop K, Struck J, Freitg D, Poertl S, Weglöhner W, Hollidt JM, Qudeck B, Dumont JE, Schumm-Dreger P-M, Bergmnn A, Mnn K, Vssrt G, Usdel K-H. Second genertion ssy for thyrotropin receptor ntiodies hs superior dignostic sensitivity for Grves disese. J Clin Endocrinol Met 999; 8: 9 97 Schott M, Hermsen D, Broecker-Preuss M, Csti M, Ms JC, Eckstein A, Gssner D, Goll R, Greer C, vn Helden J, Inomt K, Jrusch J, Krtzsch J, Miyzki N, Moreno MA, Murkmi T, Roth HJ, Stock W, Noh JY, Scherum WA, Mnn K. Clinicl vlue of the first utomted TSH receptor utontiody ssy for the dignosis of Grves disese (GD): n interntionl multicentre tril. Clin Endocrinol (Oxf) 9; 7: Giulini C, Cerrone D, Hrii N, Thornton M, Kohn LD, Dgi NM, Bucci I, Crpentieri M, Di Nenno B, Di Blsio A, Vitti P, Monco F, Npolitno G. A TSHR-LH/CGR chimer tht mesures functionl thyroid-stimulting utontiodies (TSA) cn predict remission or recurrence in Grves ptients undergoing ntithyroid drug (ATD) tretment. J Clin Endocrinol Met ; 97: E8 E87 8 Lytton SD, Ponto KA, Knitz M, Mtheis N, Kohn LD, Khly GJ. A novel thyroid stimulting immunogloulin iossy is functionl indictor of ctivity nd severity of Grves oritopthy. J Clin Endocrinol Met ; 95: Din T, Knitz M, Lehmnn M, Li Y, Olivo PD, Khly GJ. Stndrdiztion of iossy for thyrotropin receptor stimulting utontiodies. Thyroid 5; 5: 9 75 Arki N, Iid M, Amino N, Morit S, Ide A, Nishihr E, Ito M, Sito J, Nishikw T, Ktsurgi K, Miyuchi A. Rpid iossy for detection of thyroid-stimulting ntiodies using cyclic denosine monophosphte-gted clcium chnnel nd equorin. Eur Thyroid J 5; : 9 Frnk CU, Breth S, Dietrich JW, Wnjur D, Loos U. Bridge Technology with TSH Receptor Chimer for Sensitive Direct Detection of TSH Receptor Antiodies Cusing Grves Disese: Anlyticl nd Clinicl Evlution. Horm Met Res 5; 7: Thr K, Ishikw N, Ymmoto K, Hiri A, Ito K, Tmur Y, Yoshid S, Sito Y, Kohn LD. Epitopes for thyroid stimulting nd locking utontiodies on the extrcellulr domin of the humn thyrotropin receptor. Thyroid 997; 7: Rpoport B, Chzenlk GD, Jume JC, McLchln SM. The thyrotropin (TSH) receptor: interction with TSH nd utontiodies. Endocr Rev 998; 9: 73 7 Zweig MH, Cmpell G. Receiver-operting chrcteristic (ROC) plots: fundmentl evlution tool in clinicl medicine. Clin Chem 993; 39: Tozzoli R, D Aurizio F, Villlt D, Giovnell L. Evlution of the first fully utomted immunossy method for the mesurement of stimulting TSH receptor utontiodies in Grves disese. Clin Chem L Med, Jun. pii: /j/cclm.hed-of-print/cclm--97/cclm-- 97.xml doi: doi:.55/cclm--97 [Epu hed of print] Zophel K, von LP, Roggenuck D, Wunderlich G, Kotzerke J, Lckner KJ. Are porcine nd humn TSH receptor ntiody mesurements comprle? Clin L 8; 5: 8 Allelein S et l. stra Decteted y New Automted Assy ; 8: 795 8

7 Endocrine Cre 8 7 Giovnell L, Cerini L, Grncini S. Evlution of the nd genertion rdio-receptionl ssy for nti-tsh receptor ntiodies (TRA) in utoimmune thyroid diseses. Comprison with st genertion nd nti-thyroperoxidse ntiodies (ATPO). Q J Nucl Med ; 5: Khly GJ, Din T, Glng J, Knitz M, Pitz S, Konig J. Thyroid Stimulting Antiodies Are Highly Prevlent in Hshimoto s Thyroiditis nd Associted Oritopthy. J Clin Endocrinol Met ; : Schott M, Morgenthler NG, Fritzen R, Fritzen R, Feldkmp mj, Scherum WA, Seissler J. Levels of utontiodies ginst humn TSH receptor predict relpse of hyperthyroidism in Grves disese. Horm Met Res ; 3: 9 9 Tun NN, Beckett G, Zmmitt NN, Strchn MW, Seckl JR, Gi FW. Thyrotropin Receptor Antiody Levels t Dignosis nd After Thionmide Course Predict Grves' Disese Relpse. Thyroid ; : 9 Pintnid E, Li A, Sssi L, Gllo D, Sprefico E, Tnd ML, Brtlen L. Outcome Prediction of Tretment of Grves Hyperthyroidism with Antithyroid Drugs. Horm Met Res 5; 7: Bhn RS. Current Insights into the Pthogenesis of Grves Ophthlmopthy. Horm Met Res 5; 7: Slvi M, Cmpi I. Medicl Tretment of Grves Oritopthy. Horm Met Res 5; 7: Allelein S et l. stra Decteted y New Automted Assy ; 8: 795 8