Detection of TNF Blockers and Anti-drug s Antibodies Levels: a Comparison of Two Commercially Available Assays

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1 MOKSLO DARBAI SCIENTIFIC PAPERS Laboratorinë medicina. 14, t. 16, Nr. 4(64), p Detection of TNF Blockers and Anti-drug s Antibodies Levels: a Comparison of Two Commercially Available Assays Inesa Arðtikytë 1, Irena Butrimienë 1, Dalius Vitkus 3, 4 Alina Prodan 4 Algirdas Venalis 1, 1 Vilnius University, Centre of Rheumatology Vilniaus universitetas, Reumatologijos centras inesa.arstikyte@santa.lt State Research Institute, Centre for Innovative Medicine Valstybinis tyrimø institutas Inovatyvios medicinos centras 3 Vilnius University, Faculty of Medicine, Department of Physiology, Biochemistry, Microbiology and Laboratory Medicine Vilniaus universiteto Medicinos fakulteto Fiziologijos, biochemijos, mikrobiologijos ir laboratorinës medicinos katedra 4 Vilnius University Hospital Santariskiu Clinics, Centre of Laboratory Medicine Vilniaus universiteto ligoninës Santariðkiø klinikø Laboratorinës medicinos centras Summary Background. For ma tion of an ti bod ies (Ab) to TNF blockers (adali mu - mab (ADA), etanercept (ETA) and infliximab (INF)) in versely cor re lates with func tional drug lev els and clin i cal ef fect. Com par i son of drug lev els and anti-drug Ab mon i tor ing is ham pered by lack of stan dard iza tion. Aim. To de ter mine the cor re la tion and agree ment be tween two different as says for mea sure ment of TNF blockers and anti-drug Ab levels. Meth ods. Se rum sam ples of 145 pa tients with au to im mune rheu - matic dis eases were eval u ated in two dif fer ent as says per formed by Sanquin (As say A), and a com mer cially avail able kit from Progenika Biopharma (As say B) per formed in the Cen tre of Lab o ra tory Med i cine at Vilnius Uni ver sity Hos pi tal Santariskiu Clin ics. Re sults. As say A found de tect able lev els of ADA in 96.3%, ETA 88.9%, INF 63.6% pa tients, whereas As say B these re sults were 96.3%, 93.7% and 7.9%, re spec tively. The good cor re la tion was ob tained when com par ing As say A vs. B in de tect ing drug lev els. Fur ther anal y ses showed good agree ment of two used as says only in INF de tec tion. Assay A de tected anti-ada Ab in 14.8%, anti-inf Ab in 3.9% pa - tients. As say B de tected anti-ada Ab in 3.7%, anti-inf Ab in 5.4% pa tients. Both as says did not de tect anti-eta Ab in tested pa tient s pop - u la tion. Conclusions. Our study showed good cor re la tion of trough lev els mea sure ments of all three TNF blockers be tween two tested as says. Agree ment be tween two as says in de tect ing se rum INF lev els was good, but not in ADA and ETA cases. In con clu sion, the agree ment be tween two as says could be cal cu lated only for de tec tion of TNF blockers lev - els, but not for anti-drug Ab as both as says use dif fer ent lab o ra tory meth ods for de tec tion of Ab. The au thors agree that ex ist ing as says to mea sure lev els of TNF blockers and anti-drug Ab need to be stan dard - ized. Keywords: rheu matic dis eases, immunogenicity, anti-tnf an ti bod ies. INTRODUCTION Tu mour ne cro sis fac tor (TNF ) blockers, such as adalimumab (ADA), etanercept (ETA) and infliximab (INF), have proven to be ef fec tive in the treatment of autoimmune rheu - matic dis eases, i. e. rheu ma toid ar thri - tis (RA), an ky los ing spondylitis (AS), psoriatic ar thri tis (PsA) and ju ve nile idiopathic arthritis (JIA). These drugs are ad min is tered at fixed dos ages and in ter vals de rived from dose find ing stud ies [1 5]. Ob ser va tional stud ies 17 LABORATORINË MEDICINA 14, t. 16, Nr. 4(64)

2 Detection of TNF Blockers and Anti-drug s Antibodies Levels: a Comparison of Two Commercially Available Assays showed that ap prox i mately one third of pa tients lose their re sponse to these drugs [6]. One fac tor as so ci ated with loss of re sponse is immunogenicity, also the pro duc tion of anti-drug an ti - bod ies (anti-drug Ab) is as so ci ated with in fu sion re ac tions and a re tarded antibody (Ab) clearance resulting in lower TNF blockers titres [7 11]. Ob - ser va tional stud ies have dem on - strated a relationship between TNF blockers con cen tra tions and the pres - ence of anti-drug Ab and clin i cal out - comes [7 11]. The role of dose in crease or short en ing of dose in ter vals re - mains un clear be cause mea sur ing drug lev els is usu ally not in cluded in previously conducted studies [1]. In observational studies, after dose esca - la tion, anti-drug Ab titers de creased without improvement of the clinical response [13]. Ther a peu tic drug and immuno ge - ni city mon i tor ing with early se rial trough and anti-drug Ab level mea - sure ments may prob a bly op ti mize treat ment with TNF blockers. Dif fer - ent as says are be ing used to mea sure drug and anti-drug Ab lev els. The most com monly used as say types are en zyme-linked immunosorbent as say (ELISA) and ra dio-immunoassay (RIA) [14 17]. How ever, stan dard iza - tion of these as says to mea sure TNF blockers lev els and anti-adalimumab (anti-ada), anti-etanercept (anti- ETA) or anti-infliximab (anti-inf) Ab is lack ing. None of the avail able as - says are able to de tect all of the sub - classes and idiotypes of anti-drug Ab [18]. Sev eral con found ing fac tors, such as drug in ter fer ence and back ground (for ex am ple, conformational changes of the ther a peu tic pro tein when bind - ing to the plate; drug anti-drug Ab com plexes for ma tion which can t be de tected by as say) can in flu ence the mea sure ment of bi o log i cal drugs and anti-drug Ab lev els [15, 18, 19]. This may result in poor specificity, sensitiv - ity and reproducibility. In this study we an a lyzed the cor re la tion be tween two com mer cially avail able as says from Sanquin (Am ster dam, Neth er - lands, as say A) and Progenica Bio - pharma (Derio, Spain, as say B) for measurement of TNF blockers lev els and anti-drug Ab. MATERIALS AND METHODS A to tal of 145 pa tients re ceiv ing treat - ment with one of TNF blockers were in cluded in this anal y sis at the Cen tre of Rheumatology of Vilnius Uni ver sity Hos pi tal Santariskiu Clin ics (VUHSK) (Ta ble 1). This was a crosssec tional ret ro spec tive ob ser va tional study ap proved by the lo cal Eth ics Com mit tee. Pa tients signed an in - formed con sent form ac cord ing to the Dec la ra tion of Helsinki before record - ing clin i cal data or tak ing blood sam - ples. We have set up a round robin ex - per i ment in which se rum sam ples were an alyzed in two different laborato ries: in the Lab o ra tory for Mono - clonal Ther a peu tics, Sanquin Di ag - nos tics (Am ster dam, Neth er lands) (As say A) and in Cen tre of Lab o ra tory Med i cine, VUHSK (As say B), us ing Promonitor ADA, Promonitor ETA and Promonitor INF test kits (Progenika, Derio, Spain) fol low ing strict ad her ence to the man u fac turer s guide lines for use [16, 17, ]. TNF blockers anti-drug Ab lev els were de - termined. Both institutions were de - liv ered se rum sam ples taken at the same time from the same pa tients with au to im mune rheu matic dis eases from the Cen tre of Rheumatology at VUHSK. Both laboratories were not fa mil iar with clin i cal data of pa tients. Blood sam ples were col lected once dur ing course of treat ment with one of TNF blockers and were stored at -8 C, un til TNF blocker and antidrug Ab were mea sured, and only used for the pur pose of this study. Pa tient s blood sam ples were col lected once dur - ing treat ment with one of the TNF blockers be fore dos ing the next sched - uled dose (ADA and ETA be fore sched uled in jec tion, INF 8 weeks af - ter last dose and just be fore next sched uled infusion). Blood samples of ADA treated pa tients were col lected me dium 6 months (interquartile range (IQR 3 18), ETA 3 months (IQR 1 54), INF me dium 4 months (IQR 1 66). In the Lab o ra tory for Monoclonal Ther a peu tics, Sanquin Di ag nos tics (Amsterdam, Netherlands) (Assay A) TNF blockers con cen tra tion was an - a lyzed us ing val i dated ELISA, and anti- drug Ab lev els were de tected with RIA [14 17]. ADA con cen tra tion.1 g/ml, ETA con cen tra tion.1 g/ml and INF con cen tra tion.3 g/ml were con sid ered as neg a - tive. Anti-ADA Ab, anti-eta Ab and anti-inf Ab lev els 1 AU/mL were con sid ered as negative. Principles of assay B [] Serum ADA and ETA assays principle Ta ble 1. Pa tient s char ac ter is tics 1 lentelë. Pacientø duomenys Characteristic Gen der: Male Fe male Promonitor ADA and Promonitor ETA are a sand wich en zyme-linked immunosorbent as say (ELISA). The mi cro- well strips were pro vided precoated with an anti-ada and anti- ETA human F(ab ) frag ment. Di luted calibrators, controls and diluted pa - tient sam ples were added to sep a rate wells, allowing TNF blocker pres ent to bind to pre-im mo bi lized anti-drug Ab. Un bound sam ple was washed away and a sec ond en zyme strepta vi - din- horse rad ish peroxidase (HRP) la beled anti-drug monoclonal Ab was added to each well. A sec ond in cu ba - tion step al lowed the HRP-la beled anti-drug monoclonal Ab to bind to the TNF blocker that be came at tached to the mi cro-wells. Af ter wash ing away the ex cess of un bound HRP-la beled anti-drug Ab, the re main ing en zyme ac tiv ity was mea sured in a spectro - photometer by add ing a chromogenic substrate and measuring the intensity of the color that de vel oped. The sig nal ob tained was pro por tional to the amount of the drug in the pa tient sam - ple. ADA con cen tra tion.4 g/ml and ETA concentration.35 g/ml were considered as negative. Serum INF assay principle Total number of pa tients, n= (46.%) 78 (53.8%) Mean age (±SD) (±13.41) Autoimmune rheu - matic dis ease: RA AS PsA JIA Concentrations for TNF blockers and anti-drug Ab, patients number: ADA ETA INF Days of treat ment, months, me dium (IQR) (intervals) ADA ETA INF 61 (4.1%) 45 (31.%) 3 (.1%) 7 (4.8%) 7 (8.6%) 63 (43.4%) 55 (37.9%) 6 (3 18) (3 84) 3 (1 54) (3 7) 4 (1 66) (3 1) Ab bre vi a tions: n num ber, RA rheu ma - toid ar thri tis, AS an ky los ing spondylitis, PsA psoriatic ar thri tis, JIA ju ve nile id - io pathic ar thri tis, anti-drug Ab anti-drug an ti bod ies, ADA adalimumab, ETA etanercept, INF infliximab, IQR interquartile range. Promonitor INF is a cap ture ELISA. The microwell strips were pro vided pre-coated with an anti-inf hu man F(ab ) frag ment bound to hu man re - combinant TNF. This for mat en sures LABORATORINË MEDICINA 14, t. 16, Nr. 4(64) 173

3 Inesa Arðtikytë, Irena Butrimienë, Dalius Vitkus, Alina Prodan, Algirdas Venalis Table. As say A (Sanquin) re sults lentelë. Metodo A (atlikta Sanquin laboratorijoje) rezultatai Patients number, n=145 Anti-ADA, n=7 Anti-ETA, n=63 Anti-INF, n=55 Positive Negative Positive Negative Positive Negative De tect able drug con cen tra tions 3 (11.1%) 3 (85.%) 56 (88.9%) 35 (63.6%) Not de tect able drug con cen tra tions 1 (3.7%) 7 (11.1%) 17 (3.9%) 3 (5.5%) Ab bre vi a tions: n num ber, ADA adalimumab, ETA etanercept, INF infliximab. Ta ble 3. As say B (VUHSC) re sults 3 lentelë. Metodo B (atlikta Vilniaus universiteto ligoninës Santariðkiø klinikø Laboratorinës medicinos cen tre) rezultatai Patients number, n=145 Anti-ADA, n=7 Anti-ETA, n=63 Anti-INF, n=55 Positive Negative Positive Negative Positive Negative De tect able drug con cen tra tions 6 (96.3%) 59 (93.7%) 39 (7.9%) Not de tect able drug con cen tra tions 1 (3.7%) 4 (6.3%) 14 (5.4%) (3.6%) Ab bre vi a tions: n num ber, ADA adalimumab, ETA etanercept, INF infliximab. that TNF struc ture is not dis rupted and is avail able to bind to INF. Di - luted calibrators, controls and diluted pa tient sam ples were added to sep a - rate wells al low ing the INF pres ent to bind to pre-im mo bi lized TNF. Un - bound sam ple was washed away and a spe cific HRP-la beled anti-inf mono - clonal Ab was added to each well. A sec ond in cu ba tion step al lowed the anti-inf Ab to bind to the INF that had be come at tached to the mi crowells. Af ter wash ing away the ex cess of un bound HRP-la beled anti-inf Ab, the re main ing en zyme ac tiv ity was mea sured by add ing a chromogenic substrate and measuring the intensity of the color that de vel oped in a spectro photometer. The sig nal ob - tained was pro por tional to the amount of the drug in the pa tient sam ple. INF con cen tra tion.35 g/ml was con - sidered as negative. Serum anti-drug Ab assays principle Promonitor anti-ada, Promonitor anti-eta and Promonitor anti-inf are bridg ing ELISA tests. The mi crowell strips were pro vided pre-coated with TNF blocker. The bridg ing ELISA takes ad van tage of the two arms of IgG sub classes 1, and 3 to crosslink the TNF blocker coated on the plane. Cal i bra tors, con trols and di - luted pa tient sam ples were added to sep a rate wells, al low ing the anti- TNF blocker Ab pres ent to bind to pre- im mo bi lized TNF blocker. Un - bound sam ple was washed away and HRP-labeled TNF blocker was added to each well. A sec ond in cu ba tion al - lowed the HRP-la beled TNF blocker to bind to the Ab that had be come at - tached to the mi cro-wells. Af ter wash - ing away un bound HRP con ju gate, the remaining enzyme activity was mea - sured by add ing a chromogenic sub - strate and mea sur ing the in ten sity of the color that de vel oped in a spectro - photometer. The sig nal ob tained was pro por tional to the amount of anti- TNF blocker Ab in the pa tient sam - ple. Anti-ADA Ab con cen tra tion 3.5 AU/mL, anti-eta Ab con cen tra - tion 14. AU/mL and anti-inf Ab con cen tra tion. AU/mL were con - sidered as negative. Statistical analysis To quan tify the cor re la tion be tween TNF blockers lev els and anti-drug Ab lev els from two as says, the Spearman s rank cor re la tion co ef fi - cient was cal cu lated, whereby a value of 1 rep re sents an ideal cor re la tion be - tween two meth ods. To quan tify the agree ment be tween two as says in de - tecting TNF blockers and anti-drug Ab lev els, the intraclass cor re la tion co - efficient (ICC) was calculated using the two-way mixed sin gle mea sures test (ab so lute agree ment) whereby a value of 1 rep re sents com plete agree - ment, >.9 high agree ment,.7.89 ques tion able agree ment;.69 shows no agree ment. Data are shown in a Bland Altman plot in which the dif fer ence be tween two mea sure ments is plot ted on the Y-axis and the av er age of two mea sure ments on the X-axis. This plot al lows com par - ing of two as say meth ods. An ideal agree ment be tween two as say meth - ods is rep re sented by a flat line in the Bland-Altman plot. Moun tain plots, Pass ing-bablok and Deming re gres - sion was used ad di tion ally to an a lyze agree ment between two different assays. A p-value of <.5 was con sid - ered statistically significant. The sta tis ti cal soft ware pack age SPSS17., Microsoft Of fice Ex cel 7 and MedCalc (free trial ver - sion) were used for all anal y ses. RESULTS As say A found de tect able lev els of ADA in 6 from 7 (96.3%), ETA in 56 from 63 (88.9%), INF in 35 from 55 (63.6%) pa tients, whereas As say B these re sults were 96.3%, 93.7% and 7.9%, re spec tively. As say A de tected anti-ada Ab in 4 from 7 (14.8%), anti-inf Ab in 17 from 55 (3.9%) pa tients. As say B de tected anti-ada Ab in 3.7%, anti-inf Ab in 5.4% pa - tients. Anti-ETA Ab were not de tected by both as says. Re sults of As say A and As say B anal y ses are pre sented in Ta - bles and 3. As say A found 3 from 7 (11.1%) pa tients with pos i tive ADA and anti- ADA lev els, whereas it was not de - tected by As say B. As say A found 11.1% ETA and 5.5% INF treated pa - tients with no de tect able drug or antidrug Ab lev els, whereas As say B these pa tients were 6.3% and 3.6%, re spec - tively. When an a lyz ing blood sera re - sults of INF treated pa tients, we found that As say A did not de tect trough INF lev els in 5.4% pa tients, whereas As - say B in 3.9% pa tients with positive anti-inf Ab. We did not an a lyze the cor re la tion and agreement between positive antidrug Ab val ues ob tained with the two dif fer ent as says be cause we found it inappropriate two assays used dif - fer ent meth ods for de tec tion of antidrug Ab and the cut-off lim its dif fered significantly (for example the lower pos i tive limit for anti-inf Ab in Assay A is 1 AU/mL, whereas in As - say B it is AU/mL). Pos i tive anti- ETA Ab was not de tected in our patient s blood samples. The cor re la tion be tween TNF blockers lev els of the dif fer ent as says was ex pressed us ing a Spearman s rank correlation coefficient (Pearson 174 LABORATORINË MEDICINA 14, t. 16, Nr. 4(64)

4 Detection of TNF Blockers and Anti-drug s Antibodies Levels: a Comparison of Two Commercially Available Assays Fig. 1. The cor re la tion be tween ADA lev els of two as says (X axis (A) Sanquin, Y axis (B) VUHSC) 1 pav. Dviem metodais nustatytø ADA koncentracijø koreliacija (X aðyje (A) Sanquin laboratorijos rezultatai, Y aðyje (B) Vil - niaus universiteto ligoninës Santariðkiø klinikø Laboratorinës me di ci nos centro rezultatai) Fig.. The cor re la tion be tween ETA lev els of two as says (X axis (A) Sanquin, Y axis (B) VUHSC) pav. Dviem metodais nustatytø ETA koncentracijø koreliacija (X aðyje (A) Sanquin laboratorijos rezultatai, Y aðyje (B) Vil - niaus universiteto ligoninës Santariðkiø klinikø Laboratorinës me di ci nos centro rezultatai) r cor re la tion co ef fi cient could n t be used be cause TNF blockers and anti-drug antibodies levels, measured by two dif fer ent as says were not dis - trib uted by a nor mal curve). We ex - cluded data of 11 blood sam ples of pa - tients treated with ADA and 1 INF sam ple from fur ther anal y sis. 1 INF sam ple was ex cluded, be cause it was found spike se rum drug con cen tra - tion. 11 ADA sam ples were ex cluded be cause As say B de tected se rum drug con cen tra tion 1 g/ml (up per limit of the mea sure ment pro ce dure) and higher con cen tra tions, whereas Assay A mea sured higher ADA lev els due to higher limit of the mea sure - ment pro ce dure. The stron gest cor re la tion was ob - tained when com par ing INF as say A vs. B, which gave a Spearman s rank correlation co ef fi cient (r).96 (p<.1), al though all drug s con - cen tra tions as says showed a lin ear quan ti ta tive cor re la tion (r=.93, p<.1 for ETA and r=.765, p=.1 for ADA) (Figures 1 3). Spearman s rank cor re la tion co ef fi cient is an in ap pro pri ate mea sure of re li - abil ity be cause the strength of lin ear as so ci a - tion, and not agree ment, is mea sured (it is pos si ble to have a high de gree of cor re la tion when agree ment is poor). The agree - ment be tween TNF blockers lev els of two as says was ex pressed us - ing an intraclass cor re la tion co ef fi - cient (ICC). Com par ing INF as say Fig. 3. The cor re la tion be tween INF lev els of two as says (X axis (A) Sanquin, Y axis (B) VUHSC) 3 pav. Dviem metodais nustatytø INF koncentracijø koreliacija (X aðyje (A) Sanquin laboratorijos rezultatai, Y aðyje (B) Vil - niaus universiteto ligoninës Santariðkiø klinikø Laboratorinës medicinos centro rezultatai) B vs. A gave the best agree ment with an ICC=.95 (95% CI ; p<.1); questionable agreement in Mean of A and B SD,9 Mean -, SD -5, Fig. 4. Bland-Altman and Moun tains plots of ADA lev els to com pare two as say meth ods In the first graphic: the dif fer ence be tween two mea sure ments ( g/ml) is plot ted on the Y-axis and the av er age of the two mea sure - ments ( g/ml) on the X-axis. 4 pav. Bland-Altman o ir Mountain grafikai, lyginantys ADA koncentracijas, nustatytas dviem metodais Pirmajame grafike: Y aðyje pateikiama dviejø metodø rezultatø skirtumas ( g/ml), X aðyje dviejø metodø rezultatø vidurkis ( g/ml). LABORATORINË MEDICINA 14, t. 16, Nr. 4(64) 175 Percentile

5 Inesa Arðtikytë, Irena Butrimienë, Dalius Vitkus, Alina Prodan, Algirdas Venalis SD -1, Mean of A and B SD,9 Mean -, Fig. 5. Bland-Altman and Moun tain plots of INF lev els to com pare two as say meth ods In the first graphic: the dif fer ence be tween two mea sure ments ( g/ml) is plot ted on the Y-axis and the av er age of the two mea sure - ments ( g/ml) on the X-axis. 5 pav. Bland-Altman o ir Mountain grafikai, lyginantys INF koncentracijas, nustatytas dviem metodais Pirmajame grafike: Y aðyje pateikiama dviejø metodø rezultatø skirtumas ( g/ml), X aðyje dviejø metodø rezultatø vidurkis ( g/ml). Percentile SD 1, SD -1-9, Mean of A and B Mean -4, Fig. 6. Bland-Altman and Moun tain plots of ETA lev els to com pare two as say meth ods In the first graphic: the dif fer ence be tween two mea sure ments ( g/ml) is plot ted on the Y-axis and the av er age of the two mea sure - ments ( g/ml) on the X-axis. 6 pav. Bland-Altman o ir Mountain grafikai, lyginantys ETA koncentracijas, nustatytas dviem metodais Pirmajame grafike: Y aðyje pateikiama dviejø metodø rezultatø skirtumas ( g/ml), X aðyje dviejø metodø rezultatø vidurkis ( g/ml). Percentile 3 1 ADA (ICC=.73; 95% CI ; p<.1) and no agree ment in ETA assays (ICC=.47; 95% CI ; p<.1). An al ter na tive and sup port ing way of ex plor ing the re li abil ity of the mea - sure ments be tween the two as says is a Bland-Altman plot. This ap proach is based on anal y sis of the dif fer ences be - tween mea sure ments, sug gest ing that esti mates of agree ment be tween two as says may be better than re li abil ity co ef fi cients. When the av er age of the dif fer ences is close to in a Bland-Altman plot, this in di cates that the both as says pro duce sim i lar re - sults. Two as says pro vide good agree - ment in de tect ing trough INF lev els, al though ADA and ETA de tec tion by two as says dif fer (Fig ures 4 6). Moun - tain plot provides information about the dis tribution of the differences between two as says. If two as says are un bi ased with re spect to each other, the moun tain will be cen tered over zero; long tails in the plot re flect large dif fer ences be tween the meth ods. Moun tain plots of ADA, and ETA con - cen tra tions (B vs. A) show that two as - says used to mea sure con cen tra tions of these two TNF blockers have sig - nif i cant dif fer ences; in INF case the dif fer ence in re sults of two dif fer ent assays is insignificant (Figures 4 6). Passing Bablok regression can be used as an al ter na tive method to com - pare two dif fer ent as says of mea sure - ment of TNF blockers con cen tra - tions. It should only be used on vari - ables that have a lin ear re la tion ship and are highly cor re lated. Since it is a non- para met ric pro ce dure, Pass ing- Ta ble 4. Pass ing-bablok re gres sion val ues, used to com pare two dif fer ent as says of trough TNF blockers lev els 4 lentelë. Pass ing-bablok o regresijos reikðmës, gautos lyginant TNF blokatoriø koncentracijas, nustatytas dviem skir tin - gais laboratoriniais metodais TNF blockers In ter cept A value (95% CI) Slope B value (95% CI) RSD (from RSD to RSD) Cusum test, p ADA.487 ( ) ( ).965 (±1.886).95 ETA -.35 ( ) 4.34 ( ).3618 (±.791).78 INF.8183 ( ) 1.1 ( ).4663 (±.914) <.1 Ab bre vi a tions: ADA adalimumab, ETA etanercept, INF infliximab, 95% CI 95 per cent con fi dence in ter val, RSD re sid ual stan - dard de vi a tion, p p value of Cusum test. 176 LABORATORINË MEDICINA 14, t. 16, Nr. 4(64)

6 Detection of TNF Blockers and Anti-drug s Antibodies Levels: a Comparison of Two Commercially Available Assays B ADA ETA INF A B A A Fig. 7. The Scat ter di a gram with the re gres sion line (solid line), the con fi dence in ter val for the re gres sion line (dashed lines) and iden tity line (x=y, dot ted line) (Pass ing-bablok re gres sion for ADA, ETA and INF trough lev els) 7 pav. Scater tipo diagrama su regresijos linija (iðtisinë linija), regresijos linijos pasikliautiniai intervalai (brûkðninë linija), identiðkumo (atitikimo) linija (x=y, taðkinë linija) (Pass ing-bablok regresijos metodas ADA, ETA ir INF koncentracijø atitikimo vertinimui) B Bablok re gres sion is not in flu enced by the pres ence of one or rel a tive few out - liers. The in ter cept A is a mea sure of the sys tem atic dif fer ences be tween the two meth ods. The 95% con fi dence in - ter val for the in ter cept A can be used to test the hy poth e sis that A=. This hy poth e sis is ac cepted if the con fi - dence in ter val for A con tains the value. If the hy poth e sis is re jected, then it is con cluded that A is sig nif i - cantly dif fer ent from and both meth - ods dif fer at least by a con stant amount. We did not found any sys tem - atic dif fer ences be tween the two as say meth ods in INF, ADA and ETA test ing (Table 4). The slope B is a mea sure of the pro - portional dif fer ences be tween the two meth ods. The 95% con fi dence in ter val for the slope B can be used to test the hy poth e sis that B=1. This hy poth e sis is ac cepted if the con fi dence in ter val for B con tains the value 1. If the hy - poth e sis is re jected, then it is con - cluded that B is sig nif i cantly dif fer ent from 1 and there is at least a pro por - tional dif fer ence be tween the two meth ods. Our re sults showed that as - says for INF, ADA and ETA test ing have pro por tional dif fer ences (Ta - ble 4). The re sid ual stan dard de vi a tion (RSD) is a mea sure of the ran dom dif - fer ences be tween the two meth ods. 95% of ran dom dif fer ences are ex - pected to lie in the in ter val from RSD to RSD. If this in ter - val is large, the two meth ods may not be com pa ra ble. In our case the larg est RSD in ter val was found for ADA; in - ter vals for ETA and INF was sig nif i - cantly smaller (Ta ble 4). The Cusum test for lin ear ity is used to eval u ate how well a lin ear model fits the data. In our case, sig nif i - cant de vi a tion from lin ear ity was found in INF, but not in ETA and ADA pa tient s sam ples (Fig. 7, Ta ble 4). Deming re gres sion al lows to com - pare two as says us ing the re gres sion model; it takes into ac count mea sure - ment er rors for both meth ods, whereas the or di nary lin ear re gres - sion method as sumes that only the Y measurements are associated with ran dom measurement errors. Coefficient of variation (standard deviation di vided by the mean) in both as says for test ing INF con cen tra tion ex ceeds 1%, there fore Deming re gres sion can t be used. In our anal y sis, as says for ADA and ETA had no sys temic dif - fer ences, but they had pro por tional dif fer ences (Table 5). DISCUSION Blood sam ples of 145 pa tients from the Cen tre of Rheumatology at VUHSC were an a lyzed for TNF blockers and anti- drug Ab lev els in two dif fer ent lab o ra to ries in de pend ently: in Sanquin Di ag nos tics (Am ster dam, Neth er lands (As say A); TNF blockers concentration was analyzed us ing val i dated ELISA, anti-drug Ab levels were de tected with RIA); in the Centre of Laboratory Medicine at VUHSC (As say B, us ing sand wich ELISA for ADA and ETA, cap ture ELISA for INF and bridg ing ELISA for anti-drug Ab lev els de tec tion). A good cor re la tion and agree ment of TNF blockers lev els mea sured with two as says was found. Nev er the less, As say B found de tect able ETA and INF lev els in more pa tients sam ples, whereas As say A de tected more sam - ples with anti-ada Ab and anti-inf Ab. Only As say A de tected 11.1% sam - ples with pos i tive anti-ada and ADA lev els. In all these cases the trough lev els of anti-drug Ab and con cen tra - tions of TNF blockers were low. These re sults pro vide grounds for sus - pect ing fact, that conformational changes in the ther a peu tic pro tein pos si bly have taken place, as well as low avidity immunoglobulin M (or immu no glob u lin G) rheu ma toid fac tor bind ing to the Fc part of anti-drug Ab. Therefore the detection and quantifica tion of anti-drug Ab is not al ways pos si ble when de tect able TNF blockers lev els are pres ent and those sam ples should be con sid ered in con - clu sive for anti-drug Ab [18]. As say A found 11.1% ETA treated pa tients and 5.55% of INF treated pa - tients with no de tect able drug and anti- drug Ab lev els. As say B de tected 6.3% of ETA treated pa tients and also 3.6% INF treated pa tients with no de - tect able drug and anti-drug Ab lev els. All pa tients had good clin i cal re sponse and treat ment was not changed. In this case drug interference (formation of drug com plexes to gether with antidrug Ab) is one of the rea sons for these re sults [18]. An other ex pla na tion could be the in ad e quate dos age of TNF blockers re sult ing in ab sence of trough drug lev els. Ta ble 5. Deming re gres sion val ues, used to com pare two dif fer ent as says of trough TNF blockers lev els 5 lentelë. Deming o regresijos reikðmës, gautos lyginant TNF blokatoriø kon cen - tra ci jas, nustatytas dviem skirtingais laboratoriniais metodais TNF blockers In ter cept value (95% CI) Slope value (95% CI) ADA.941 ( ) ( ) ETA.33 ( ) ( ) Ab bre vi a tions: ADA adalimumab, ETA etanercept, 95% CI 95 per cent con fi dence in - ter val. LABORATORINË MEDICINA 14, t. 16, Nr. 4(64) 177

7 Inesa Arðtikytë, Irena Butrimienë, Dalius Vitkus, Alina Prodan, Algirdas Venalis Both as says were use ful in de tect - ing anti-ada Ab and anti-inf Ab, with a slight ad van tage for the As - say A, in de tect ing low anti-drug Ab and ADA con cen tra tions with no de - tect able lev els of TNF blockers, as RIA is less sen si tive to drug in ter fer - ence than the ELISA, it is able to de - tect low anti-drug Ab con cen tra tions in the pres ence of the drug [15]. Thus, an ti body de vel op ment may be de - tected a bit ear lier. How ever, in vivo these low lev els of anti-drug Ab may only in flu ence drug con cen tra tions to a lim ited ex tent [18]. Agree ment and cor re la tion of anti-drug Ab mea sure - ments by two as says was not cal cu - lated, be cause of the dif fer ent meth ods (ELISA vs. RIA). In this study we did not cor re late the re sults of the tests with the clin i cal effec tive ness of treat ment (cor re la tion was per formed in the sub-study with the same pa tients, re sults in print). The main goal of this com par a tive test was to eval u ate the abil ity of the tests to detect typical, clinical meaningful lev els of TNF blockers and anti-drug Ab. A num ber of clin i cal stud ies anal y - ses clin i cal rel e vance of TNF blockers and anti-drug AB lev els [13, 1 3]. Fur ther more, anti-drug Ab have been as so ci ated with a higher in - cidence of in fu sion re ac tions, ad verse events and an in creased risk for loss of re sponse [6, 13, 4 8]. As low se rum drug lev els are as so ci ated with lack or loss of clin i cal re sponse, ther a peu tic drug mon i tor ing would be an im por - tant tool in clin i cal de ci sion mak ing in pa tients with rheu matic dis eases. For this purpose a preliminary algorithm in tro duc ing as sess ment in the man - age ment of RA pa tients is pro posed [9]. Au thors sugested that RA pa - tients should be eval u ated for drug trough lev els and ther a peu tic re - sponse (Eu ro pean Leaque Against Rheumatism criteria) every 3 months. When drug lev els are un de tect able, as sess ment of anti-drug Ab should be per formed and treat ment de ci sions made [9]. Rou tine de ter mi na tion of TNF blocker and anti-drug Ab lev els could aid in elucidating determinants for re - sponse and nonresponse and in clin i - cal decision-making because the pres - ence or ab sence of anti-drug Ab has im pli ca tions for the re sponse to a sec - ond TNF blocker [9 31]. In pa tients with out Ab to their first TNF blocker and de tect able se rum drug lev els, a sec ond TNF blocker is less likely to be ef fec tive. These pa tients might ben - e fit from bi o log i cals with an other mode of ac tion [3, 31]. In con trast, pa - tients who did de velop Ab to their first TNF blocker may re spond to a sec ond drug, how ever, they are more prone to de velop anti-drug Ab to their sec ond TNF blocker [3, 31]. Trough lev els can in di cate whether the pa tient is a pri mary (no re sponse de spite ad e - quate drug con cen tra tions) or sec ond - ary (no re sponse de spite high drug dose and de tect able anti-drug Ab lev - els) nonresponder. Im por tant for def i - ni tions of lack or loss of re sponse is the aware ness that anti-drug Ab are al - ready pres ent dur ing the first weeks of treat ment in a pro por tion of the pa - tients [13]. In spite of the re sults of this com - par a tive eval u a tion, our study was lim ited by the fol low ing: the small sam ple size, blood sam ples were col - lected at the dif fer ent time points af ter be gin ning of the treat ment, the im pact of the re sults which will be re stricted lo cally be cause of the kit s avail abil ity and the fact that we have not used healthy con trol se rum and known drug spiked sam ples. Un til now there are no gold stan dards or guide lines avail able to mon i tor these drugs. For these rea sons, fur ther stud ies will be needed to es tab lish their use in clin i - cal practice and to optimize treatment. CONCLUSIONS Our study showed good cor re la tion of all three TNF blockers trough lev els measurements between the assays de - veloped by Sanquin Diagnostics (Am - sterdam, Netherlands, Assay A) and the com mer cially avail able kit from Progenika (Derio, Spain, As say B). There is good agree ment be tween two as says in de tect ing se rum INF lev els, Santrauka but it s not the case for the test ing of ADA and ETA trough con cen tra tions. In con clu sion, the agree ment be tween two as says could be cal cu lated only for detection of TNF blockers lev els, but not for anti-drug Ab as the both as says use different laboratory methods for de tec tion of Ab and re port dif fer ent cut-off lim its. The au thors agree that ex ist ing as - says to mea sure lev els of TNF blockers and anti-drug Ab need to be standardized. Financial support This study was sup ported by a re - search grant from the Lith u a nian Ar - thritis Foundation, Promonitor kits were pro vided by Phizer Luxemburg SARL Lithuania. Conflicts of interest Inesa Arstikyte, Irena Butrimiene, Al - gir das Venalis have re ceived speaker hon o raria or con sul tan cies from phar - ma ceu ti cal com pa nies pro duc ing TNF blockers or dis trib ut ing them in Lith u a nia (Abbvie, Phizer, MSD). This study was sup ported by an re search grant from the Lith u a nian Ar thri tis Foun da tion. Dalius Vitkus and Alina Pro dan have no con flicts of in ter est to de clare. Acknowledgements Au thors thank the phy si cians and pa - tients who par tic i pated in this study. Gauta: Priimta spaudai: TNF BLOKATORIØ IR PRIEÐ JUOS SUSIDARIUSIØ ANTIKÛNØ KONCENTRACIJØ NUSTATYMAS: DVIEJØ LABORATORINIØ METODØ PALYGINIMAS Inesa Arðtikytë, Irena Butrimienë, Dalius Vitkus, Alina Prodan, Algirdas Venalis Literatûros duomenimis, nustatomos an - ti kû niø prieð TNF blo ka to rius (ada li - mumabà (ADA), etanerceptà (ETA) ir in - flik si ma bà (INF)) kon cen tra ci jos at virkð - èiai ko re liuo ja su funk ci niu vais to ly giu se ru me bei kli ni ki niu efek ty vu mu. Pa - saulyje TNF blo ka to riø ir prieð juos su - sidariusiø antikûnø tyrimo metodai nëra stan dar ti zuo ti. Tiks las. Áver tin ti dvie jø tai ky tø la - bo ra to ri niø me to dø re zul ta tø ko re lia ci jà ir suderinamumà, nustatant TNF blo - ka to riø ir prieð juos su si da riu siø an ti kû - nø koncentracijas. Me to dai. Reumatinëmis ligomis ser - gan èiø 145 pa cien tø krau jo se ru mai bu vo ana li zuo ti dviem skir tin gais me to dais: Sanquin la bo ra to ri jo je (me to das A) bei Vil niaus uni ver si te to li go ni nës San ta rið - kiø kli ni kø La bo ra to ri nës me di ci nos cen - tre, naudojant tyrimø rinkinius ið Proge - nika Biopharma (metodas B). Re zul ta tai. Me to du A nu sta ty tos vais tø kon cen tra ci jos: ADA 96,3 %, ETA 88,9 %, INF 63,6 % pa cien tø, me to du B ati tin ka mai 96,3 %, 93,7 % ir 7,9 % pa cien tø. Ly gi nant abu vais tø kon cen tra ci jø nu sta ty mo me to dus, gau LABORATORINË MEDICINA 14, t. 16, Nr. 4(64)

8 Detection of TNF Blockers and Anti-drug s Antibodies Levels: a Comparison of Two Commercially Available Assays ta ge ra ko re lia ci ja, ta èiau ge ras me to dø su de ri na mu mas nu sta ty tas tik INF gru - pë je. Me to du A ap tik ti an ti-ada an ti kû - nai 14,8 %, an ti-inf an ti kû nai 3,9 % pa cien tø, me to du B ati tin ka mai 3,7 % ir 5,4 % pa cien tø. An ti-eta an ti kû nai ne bu vo nu sta ty ti abiem tai ky tais me to - dais. Ið va dos. At lik tas ty ri mas pa ro dë dvie - jø laboratoriniø metodø, taikytø TNF blo - katoriø koncentracijoms serumuose nusta - ty ti, ko re lia ci jà, ta èiau ge ras su de ri na mu - mas ras tas tik INF gru pë je. Dvie jø ty ri mø metodø suderinamumas gali bûti tiriamas tik nu sta tant TNF blo ka to riø, bet ne an ti - kûnø koncentracijas, nes pastariesiems ap - tik ti nau do ja mi skir tin gi la bo ra to ri niai metodai. 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