SPECIAL EdItIon 1 – 2010

The 7th International Congress on AutoimmunityMay 5 – 9, 2010 Ljubljana, Slovenia

2 JournAL

EliA Journal is the Journal of

Phadia GmbHMunzinger Straße 7, D-79111 Freiburg

Editor: Nina OlschowkaContributors: Authors of posters and oral presentations shown at the 7th International Congress on AutoimmunityLayout: Melanie Tritschler, Tom BernhardNumbers printed: 7,000

Editorial

At the beginning of May 2010, blue-bag-wearing people roamed through the charming city of Ljubljana, the capital of Slovenia, on the way to or from the 7th International Congress on Autoimmunity. Although volcano Eyjafjallajökull made the trip by airplane to a risky game, the congress organization announced a record number of almost 2000 participants. This congress is the biggest meeting of autoimmunity specialists worldwide, and takes place every second year.

Phadia is one of the main sponsors of this congress. At our booth we had the opportunity to welcome many customers and opinion leaders in Autoimmunity, we offered the possibility to learn more about our Phadia 250 instrument, which was installed at the booth, we discussed our products and our newest developments and, last but not least, offered wonderful coffee.

One of the best attended sessions of the congress was the EASI conference, a session held on the Saturday afternoon. The Chairmen Yehuda Shoenfeld, Michael Haass and Jan Damoiseaux led through the hot issue of ANA testing: which methods should be recommended, what are their pitfalls and which algorithms in ANA testing could be applicable. One of the speakers, Henry Homburger from the Mayo Clinics could not attend the meeting because of the volcano ashes but was connected electronically and while he sat in front of a camera in Rochester, USA, some hundred participants in Ljubljana followed his presentation. The presentations of this EASI conference are accessible on www.easi-network.com.

A high number of posters and oral presentations at the congress were about Phadia products such as our new EliA CTD Screen or the highly specific EliA GliadinDP. Almost all posters which involve Phadia products are summarized in this special edition of the EliA Journal. In most cases, the posters come from independent labs and do not always reflect our opinion. However, our products seemed to convince with their excellent performance, and the results confirmed our strategy of producing specificity-focused, high-quality products.

Enjoy reading,

EDITORIAL __________________________________________

CONTENT3 Role of a new FEIA assay in systemic connective tissue

disease diagnosis

4 Performance of a new screening test for connective tissue disease specific antibodies compared to HEp2 screening

5 A comparison between two enzyme-linked immuno-sorbent assays and an indirect immunofluorescence test for Antinuclear Antibodies screening

6 Comparison of performance of HEp-2 and an automated CTD Screening test in a routine setting

7 Performance of a New One-Step Diagnostic Test for Accurate Screening of Connective Tissue Diseases

8 Evaluation of CTD Screen on the ImmunoCAP 100 System: Confirmed Diagnosis Samples and Consecutive Unknown Routine Samples

10 Evaluation of a new enzyme fluoroimmunoassay for ANA detection in systemic sclerosis

11 Detection of Ribosomal P (Rib-P) autoantibodies in SLE

12 Diagnostic value of antibodies against ribosomal protein P in SLE

12 Quantitative evaluation of anti-topoisomerase I antibodies in systemic sclerosis

14 A comparison of anti-CCP and rheumatoid factor in the diagnosis of rheumatoid arthritis

15 Preliminary evaluation of anti-cyclic citrullinated peptide antibodies of IgA and IgM isotypes using a fully automated fluoroenzymeimmunoassay (EliA) in Rheumatoid Arthritis

16 High sensitivity of anti-deamidated gliadin peptide IgG antibodies for diagnosis of celiac disease in children

17 Evaluation of a new fluorescent enzyme immunoassay in celiac disease diagnosis based on the use of deamidated gliadin peptides

18 Good sensitivity of tests for antibodies to deamidated gliadin peptides in children – correlation with jejunal morphology

20 Diagnostic performance of IgG anti-DGP assays is comparable to IgA anti-tTG in celiac disease

21 Comparison of two cardiolipin antibodies tests and two β2 glycoprotein 1 antibodies tests

22 Comparative evaluation of the fully automated new PR3-ANCA sensitive test (EliA PR3S), for the diagnosis of the ANCA-associated systemic vasculitis (AASV)

3JournAL

_________________________________________ CTD SCREEn

Role of a new FEIA assay in systemic connective tissue disease diagnosisC. Alpini1, S. Valaperta2, S. Avalle1, V. Ramoni3, C. Bonino3, C. Montecucco3, R. Moratti1

1 Clinical Chemistry Laboratories, IRCCS Policlinico S. Matteo Foundation, Pavia, Italy, 2 Clinical Investigation Laboratory, IRCCS Istituto Clinico Humanitas, Rozzano (MI), Italy, 3 Division of Rheumatology IRCCS Policlinico S. Matteo Foundation, Pavia, Italy

Objective: To evaluate the diagnostic efficiency of a new screening test for CTD based upon a wide antibodies profile (EliA CTD Screen - Phadia).

Patients and Methods: 157 samples (52 with known CTD, 105 with different autoimmune disease (non-CTD)). All the samples were tested for ANA IFI on Hep-2 cells (ImmunoConcepts) and ENA (EliA Symphony and EliA CTD Screen, Phadia) and anti-dsDNA (EliA dsDNA, Phadia) on Immuno-CAP 250 analyzer.

Additionally, the specific antibody characterization has been performed on ENA positive samples with commercial and research methods (Phadia).

Results: 69 samples were negative for ANA and ENA with both methods. Of the 88 ANA positive samples, all the ENA EliA Symphony positive samples were also positive for EliA CTD Screen, but 13 additional samples were positive only for EliA CTD Screen due to the wider antibody profile. In fact, 6 samples were subsequently found positive for dsDNA antibody and 7 for other specific antibodies (1 anti-Rib-P, 3 anti-PM/Scl, 1 anti-Fibrillarin, 2 anti RNA Pol III).

Figure 1: EliA CTD Screen and Hep-2 in CTD, non-CTD and UCTD Figure 2: ROC analysissamples

Calculated Parameters Hep-2 EliA CTD Screen

1:80 1:160 Pos > 10 μg/l Pos > 15 μg/l

Sensitivity 72.5% 60.0% 80.0% 70.0%

Specificity 69.2% 81.3% 86.8% 90.1%

PPV 50.9% 58.5% 72.7% 75.7%

NPV 85.1% 82.2% 90.8% 87.2%

Table 1: Performance data

Conclusions: The new EliA CTD Screen showed a good correlation with ANA and a perfect agreement with ENA EliA Symphony. Moreover, the wider antibodies profile offered greater CTD diagnostic performances with a higher negative predictive value.

Allowing the detection of minor antigen antibodies with high clinic relevance, EliA CTD Screen is a great companion of ANA IFI determinations for Connective Tissue Disease diagnosis.

4 JournAL

CTD SCREEn _________________________________________

Performance of a new screening test for connective tissue disease specific antibodies compared to HEp2 screeningI. Baptista-Fernandes, A. Matoso-Ferreira, A. Torrão-Mendes, J. Faro-VianaImunologia, Centro Hospitalar de Lisboa Ocidental - Portugal

Objective: To compare the classical ANA test on HEp2 cells by indirect immunofluorescence and a new CTD specific screening.

Patients and Methods: 167 patients (87 with various CTD (sensitivity estimations), 80 patients with a variety of other diseases as controls (specificity estimations)), treated and untreated patients included. Two different screening tests were used: HEp 2 (NOVA Lite HEp-2 ANA Kit, Inova, USA) and EliA ANA CTD Screen (ECS) on ImmunoCAP (Phadia GmbH, Germany).

Results: The performance of ECS is very similar to the 1:320 HEp2 cut-off (ECS / HEp2 Specificities: 87.3% / 88.6% and Sensitivities: 55.7% / 53.4%). The relatively high positive likelihood ratios at these cut-off values indicate the impact of a positive test result with respect to support a diagnosis, while the relatively poor negative likelihood ratios (>0.1) suggest that a negative test result does not exclude the presence of a CTD.

34 of 78 (39%) CTD patients HEp2 (1:160) positive were ECS negative (including 4 equivocal): 25 SLE, 2 MCTD, 4 PM/DM and 3 UCTD. Within the 25 SLE only 2 tested positive for dsDNA antibodies. Within the group of the HEp2 and ECS positive results (44 patients / 26.3%) there were 29 SLE and out of these 11 tested positive anti-dsDNA. One Sjogren Syndrome positive by ECS was missed by HEp2.

HEp2 ECS

1:160 cut-off 1:320 cut-off Ratio 0.7 cut-off Ratio 1.0 cut-off

Sensitivity (%) 88,6 53,4 55,7 51,1

Specificity (%) 63,6 88,6 87,3 92,4

Positive LHR* 2,41 4,69 4,40 6,73

Negative LHR* 0,18 0,53 0,51 0,53

* Likehood Ratio

Table 1: performance data

CTD EliA ANA CTD Screen Controls EliA ANA CTD Screen

neg equiv pos Total neg equiv pos Total

IIF; pos (1/160) neg 9 0 1 10 IIF; pos (1/160) neg 46 1 3 50

pos 30 4 44 78 pos 23 3 3 29

IIF; pos (1/320) neg 30 2 9 41 IIF; pos (1/320) neg 62 3 5 70

pos 9 2 36 41 pos 7 1 1 9

Total 39 4 45 88 Total 69 4 6 79

Table 2: agreement of methods in CTD and Control patients

Conclusions: HEp2 (1:160) is more sensitive than ECS, at the cost of a higher False Positive Rate. HEp2 (1:320) has a similar performance to ECS. When ECS is used alone, its negative values should be confirmed with a more sensitive test if this is clinically indicated. ECS positive tests are highly predictive of CTD.

Figure 1: The receiver operator characteristics (ROC) curves were very similar: HEp2 AUC=0.82, ECS AUC= 0.78.

5JournAL

_________________________________________ CTD SCREEn

A comparison between two enzyme-linked immuno-sorbent assays and an indirect immunofluorescence test for Antinuclear Antibodies screeningM.J. Martínez, C. Serrano, M.J. Rodriguez, P. Tramón, P. PalominoDepartment of Immunology, Fundación Jiménez Diaz-CAPIO, Madrid. Spain

Objective: To compare two different ELISA with the IIF test in sera of patients with different pathologies in order to identify the best according to sensitivity and specificity.

Patients and Methods: 142 sera were prospectively evaluated. Each serum was tested by two different ELISA: the EliA ANA CTD Screen (Phadia Diagnostics, Uppsala, Sweden) based on human recombinant antigens: Sm, U1RNP, Ro, La, Centromere B, Scl-70, Fibrillarin, PCNA, PM-Scl, RNA Pol III, Mi-2, Jo-1, Ribosomal-P and purified dsDNA; and the QUANTA Lite ANA (INOVA Diagnostics, San Diego, CA) including: dsDNA, histones, Sm/RNP, Ro, La, Scl-70, Centromere, PCNA, Jo1, M2 and ribosomal-P plus extracts from HEp-2 nuclei and nucleoli. We also tested these sera with IIF (two different microscope slides, based on HEp2 cell lines and a combination of three different rat tissues -liver, kidney and stomach- both from INOVA). The association with clinical features was examined by the systematical review of the clinical histories. The antigen specificities were studied by Inmunoblot (BlueDot. D-tek) and EliA IgG (Phadia).

Results: Compared to the results of IIF, the EliA ANA CTD Screen showed a sensitivity of 79.31% and a specificity of 77.38% (PPV: 0.71; NPV: 0.84); whereas QUANTA Lite had a sensitivity of 96.55% with a specificity of 58.33% (PPV: 0.62; NPV: 0.96).

Compared to the clinical features, the IIF showed a sensitivity of 77.42% and a specificity of 87.50% (PPV: 0.83; NPV: 0.83), the EliA ANA CTD Screen showed a sensitivity of 88.71% and a specificity of 87.50% (PPV: 0.85; NPV: 0.91) whereas QUANTA Lite had a sensitivity of 95.16% with a specificity of 60.00% (PPV: 0.65; NPV: 0.94).

Both ELISA detected all the specificities except sp-100 that was not detected by any of the ELISA studied.

A PL-7 case has not been detected neither by IIF, nor by any of the ELISA studied.

QUANTA Lite detected isolated anti-mitochondrial antibodies in two cases (data not shown).

Conclusions: Taking into consideration the clinical features, similar specificities were observed with EliA ANA CTD Screen and IIF, con-sidered the gold-standard method for ANA screening. On the other hand QUANTA Lite shows a higher sensitivity.

The inclusion of Hep-2 cell extracts could explain the higher sensitiv-ity obtained with QUANTA Lite, as well as the ability to detect anti-mitochondrial antibodies.

The limitations of IIF to detect some antigens (as many cytoplasmic aminoacyl-tRNAsynthetase) could be overcome by introducing them on an improved ELISA.

Sp-100

1%

Ro26%

Sm1%

Centromere4%

PL-71%

RNP4%

Jo-12%

DNAds8%

La9%

Others (Scl-70, Fibrilarina, PCNA, PM-Scl,

RNA Pol III, Mi-2)<0,1%

Ribosomal1%

Negatives43%

IIF

97%

58%

79% 77%

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

QUANTA Lite® ANA 96.55% 58.33%

ELiA ANA CTD® Screen 79.31% 77.38%

Sensitivity Specificity

Figure 1: Sensitivity and Specificity of QUANTA Lite® ANA and EliA ANA CTD Screen compared to the results of the IIF.

CLINICAL FEATURES

77%88%

95%

60%

89% 88%

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

IFI 77.42% 87.50%

QUANTA Lite® ANA 95.16% 60.00%

ELiA ANA CTD® Screen 88.71% 87.50%

Sensitivity Specificity

Figure 2: Sensitivity and Specificity of IIF, QUANTA Lite ANA and EliA ANA CTD Screen.

Figure 3: Antigen specificities of the sera.

6 JournAL

CTD SCREEn _________________________________________

Comparison of performance of HEp-2 and an automated CTD Screening test in a routine settingM. Montes-Cano, I. Wichmann, A. García, A. Núñez-RoldánServicio de Inmunología. HH.UU. Virgen del Rocío, Sevilla, Spain

Objective: To compare two different antinuclear antibodies screening approaches, one using classical Hep-2 cell and a screening test based on CTD specific antibodies.

Patients and Methods: A total of 1998 consecutive patients with different autoimmune diseases were analysed. Two different methods were performed: Indirect immunofluorescence using Hep-2 substrate (Labodia, ANA Orkit, Switzerland) and EliA ANA CTD Screen (Phadia GmbH, Germany).

Results:

Evaluation of a novel automated CTD Screen for Connec-tive Tissue DiseasesL.F. Pereira, J.A. Garcia-Trujillo, S. Romero-Chala, M. Timon, J. Galindo, C. CamaraImmunology Laboratory. Hospital San Pedro de Alcántara. Cáceres. Spain

Objective: To evaluate and compare Immunofluorescence ANAs and a new ELISA developed to screen connective tissue disease re-lated ANAs (EliA CTD Screen).

Patients and Methods: 213 consecutive sera sent for ANA testing (from primary care and hospital care) were analysed in parallel by IFA and EliA CTD Screen (Phadia). Positive sera were confirmed by immunoblot. Analysis of discrepant results with respect to clinical background (if possible) was carried out.

0

5

10

15

20

25

30

35

40

ANA Hep-2 positive (1/160) ANA Hep-2 positive (1/320) CTD Screen

%

IFI vs. ELISA N= 1998 (%)

% of concordance 77.3

% of disconcordance 22.6

Conclusions: The different screening approaches result in a large portion of overlapping results, but also in a number of discrepancies.

A significant proportion of Hep-2 results are found positive in non CTD and negative by the CTD specific screening test. Detailed investigation of the discrepancies is required and ongoing.

Table 1: ROC analysis

Test Area 95% CI

ANA (Hep-2); invers Titer 0,94 0,90 to 0,98

EliA CTD Screen in Ratio 0,98 0,96 to 1,00

7JournAL

_________________________________________ CTD SCREEnResults:

Table 2: agreement of ANA (Hep-2) and the CTD Screen.

Conclusions: In this preliminary study 144 sera negative in both tests were assumed with high probability NOT to be from CTD patients (most were from primary care and post test diagnosis couldn’t be achieved).Sensibility and specificity were better for EliA CTD than for ANA IFA. EliA CTD Screen could be a good screening for detecting a specific CTD, in particular in patients from primary care where PPV is very low for IFA.

Performance of a New One-Step Diagnostic Test for Accurate Screening of Connective Tissue DiseasesJ. G. Ruiz de Morales, S. Calleja, J. Martín. Immunology, Complejo Hospitalario Universitario de León. 24080-León. Spain

Objective: To evaluate a new diagnostic antigen-expanded screening test for the most common specificities associated with CTD in ANA positive sera.

Patients and Methods: 135 consecutive IFI ANA positive samples (≥1/40) referred to the Laboratory of Immunology were selected and prospectively analysed by means of a new immunoassay EliA (ImmunoCAP, ANA CTD Screen, Phadia, Sweden) containing 17 of the most common antigens so far identified in CTD. In addition, all sera were studied by our routinely approach consisting of IFI + classical ENA screen (EliA Symphony, 10 antigens) + anti-DNAn. In either the case, when screening was positive, a further antigen identification by 17 single antigen assays was pursued. After immu-nological work-up completion, clinical records were obtained from all the patients. Statistically significant differences among tests were calculated by means of Fisher´s exact test (p<0.05, two-tailed).

Results: The diagnosis of the 135 ANA+ blind-selected serum samples analysed were grouped in Group A: 48 with a previously established CTD diag-nosis (18 SLE, 6 subacute LE, 7 Sjögren (primary), 5 Mixed Connective Tissue Disease, 3 Scleroderma, 4 CREST, 3 Polymyositis, 2 Undifferentiated CTD); Group B: 28 new patients with a highly suspicion but yet undefined CTD diagnosis and Group C: 59 patients with autoimmune diseases other than CTD or autoimmune phenomena. In every single group of patients, CTD Screen yielded more positive results than our routinely used screening strategy (EliA Symphony): 14.5% (p =0.015) in group A, 28.57% (p= 0.036) in group B and 11.86% (p= ns) in group C. Most of the differences were due to anti-DNAn, absent within the Symphony. However 5 PCNA, 2 Fibrillarin, 1 Pm-Scl and 1 Rib-P additional positive sera were observed, that might contribute to a better patient´s disease characterisation.

Conclusions: A significant amount of patients with low IFI ANA titers positive in CTD Screen and some CTD-associated specificities could be further identified in individual assays. This finding suggests ANA 1/40 as the recommended cut-off value if false negatives are not desired.

By increasing the number of antigen specificities recognized in a single assay, CTD Screen in IFI positive sera allowed the identification of more patients with CTD-associated autoantibodies, thus requiring further analysis.

When clinical information is not available in ANA + samples from confirmed CTD and highly suspicious CTD patients, CTD Screen significantly im-proved the patient antibody profiling and the final clinical diagnosis, especially in the autoimmune myositis and scleroderma groups.

Some positive samples, mostly PCNA and low titer DNAn, were identified within the presumed non-CTD disease group.

CTD Screen

ANA (Hep-2) neg equivocal pos Total

neg 144 4 4 152

pos 41 0 20 61

Total 185 4 24 213

Hep-2 positive at CTD Screen positive >

1/40 1/80 1/160 0.7 1

Sensitivity 100.0% 91.7% 91.7% 100.0% 100.0%

Specificity 78.8% 78.8% 88.1% 92.2% 94.3%

LHR + 4.71 4.32 7.69 12.87 17.55

LHR - 0.00 0.11 0.09 0.00 0.00

PPV 22.6% 21.2% 32.4% 44.4% 52.2%

NPV 100.0% 99.3% 99.4% 100.0% 100.0%

Table 3: performance overview of ANA (Hep-2) and the CTD Screen.

8 JournAL

Evaluation of CTD Screen on the ImmunoCAP 100 System: Confirmed Diagnosis Samples and Consecutive Unknown Routine SamplesM. Sánchez-Castañón1, V. Martinez-Taboada2, M.J. Novo-Fernández1, M. González1, M. San Martín1, C. Santa Cruz1, M. Lopez-Hoyos1

1 Immunology and 2 Rheumatology Sections, Hospital Universitario Marqués de Valdecilla-IFIMAV, Santander, Spain

Objective: to evaluate the performance of a new automated fluorescence assay developed to screen for CTD related ANA and to compare it with the indirect immunofluorescence (IIF).

Patients and Methods: 472 diagnosed patients (103 systemic lupus erythematosus (SLE), 28 scleroderma, 98 rheumatoid arthritis (RA), 43 Sjögren Syndrome (SS), 40 antiphospholipid syndrome (APS), 35 inflammatory bowel disease (IBD), 64 osteoarthritis and 61 chronic CHV hepatitis). In parallel 478 consecutive serum samples received at the laboratory for routine determination of ANA were studied.

ANA screening was performed by IIF on Hep-2 cells (Medica) at a 1/160 serum dilution. ANA is considered positive at serum dilutions >1/320. CTD Screening was performed following the manufacturer’s protocol on ImmunoCAP 100. CTD Screen is considered positive at a ratio >1.0. Discordances between both methods were assessed.

Results:

Figure 1: Left panel: Descriptive data of IIF data on Hep-2 cells on patients with diagnosis. Data are expressed as inverse of the titer obtained. Patients were divided in CTD and non-CTD groups. Right panel: Descriptive data of CTD Screen results on patients with diagnosis. Patients were divided into CTD and non-CTD groups.

10

100

1000

10000

NonCTD CTD

HEp

-2; i

nver

s Ti

ter

0.1

1

10

100

NonCTD CTD

EliA

CTD

in R

atio

Hep-2 CTD Screen

Sensitivity 81.9% 63.3%

Specificity 71.8% 86.3%

LR (+) 2.9 4.6

LR (-) 0.25 0.4

PPV 66.8% 76.2%

NPV 85.1% 77.2%

Table 1: Test performance of IIF (at >1/320) and CTD Screen (ratio >1) in a sample prevalence of CTD of 0.410.

CTD SCREEn _________________________________________

9JournAL

Conclusions: The CTD Screen automated methods showed a lower sensitivity to screen CTD in the diagnosed samples, although the specificity was pretty nice with a nice PPV and NPV.

The positive results in the non-CTD patients were mostly due to the hepatitis C virus infected patients.

Both methods missed patients with CTD in the routine cohort what highlights that clinical findings remains as the gold standard for diagnosing and serology is not the holy grail.

The high proportion of negative results with both methods reflects the lack of criteria to order CTD related ANA.

Disease IIF+ CTD- IIF- CTD+

rheumatoid arthritis 12 0

discoid lupus 2 1

psoriatic arthritis 1 4

systemic lupus erythematosus 3 3

antiphospholipid syndrome 3 1

scleroderma/CREST 1 0

Raynaud phenomenon 2 0

Sjögren syndrome 1 0

osteoarthritis 4 2

arthralgia/conectivopathy 4 1

autoimmune thyroiditis 7 0

others 7 4

Figure 2: ROC plots using data on patients with defined diagnosis. IIF data are shown in green and CTD Screen data in red. .

Table 2: Number of discordances between IIF and CTD on routine samples analysed according to the clinical diagnosis reached.

Figure 1: ROC plots using data on patients with defined diagnosis. IIF data are shown in green and CTD screen data in red.

Figure 1: ROC decision plots for IIF (two upper plots) and CTD screen (two lower plots).

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True

pos

itive

rate

(Sen

sitiv

ity)

No discrimination

HEp-2; invers Titer

EliA CTD in Ratio

ROC Decision Plot

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Figure 3: ROC decision plots for IIF (two upper plots) and CTD Screen (two lower plots).

_________________________________________ CTD SCREEn

10 JournAL

CTD SCREEn _________________________________________

Evaluation of a new enzyme fluoroimmunoassay for ANA detection in systemic sclerosisD. Villalta1, G. Morozzi2, M. Tampoia3, C. Alpini4, I. Brusca5, M. Da Re1, N. Bizzaro6.1 Allergy and Clinical Immunology, A.O. “S. Maria degli Angeli, Pordenone, Italy, 2 Rheumatology Unit, University of Siena, Italy, 3 Clinical Pathology, Policlinico Consorziale, Bari, Italy, 4 Clinical Chemistry, IRCCS Policlinico S. Matteo, Pavia, Italy, 5 Clinical Pathology, Buccheri-La Ferla Hospital, Palermo, Italy, 6 Clinical Pathology, Civic Hospital, Tolmezzo (UD), Italy

Objective: to evaluated the clinical accuracy of a new enzyme fluoroimmunoassay (EliA CTD Screen, Phadia, Freiburg, Germany) for ANA detection in SSc patients.

Patients and Methods: Sera of 50 SSc ACA- and anti-topo I-negative patients (SSc-neg), 42 SSc ACA- or anti-topo I-positive patients (SSc-pos), and 80 non-autoimmune controls (30 HCV with cryoglobulinemia, 20 with other infectious diseases, and 30 healthy subjects) were tested with the new EliA CTD Screen and the results compared to those observed with EliA Symphony. In addition, the sera were tested for single autoantibody specificities by EliA (Sm, U1RNP, SSA/Ro, SSB/La, CENP-B, topo-I, Jo1, dsDNA) and by new automated enzyme immunoassays developed by Phadia, but not yet released in the market (AFA, anti-RNAP III, anti-PM-Scl).

Results: Using a cutoff corresponding to 95% specificity (15 μg/L), all 42 SSc-pos and 22/50 (44%) SSc-neg sera were positive with the EliA CTD Screen assay; of the latter, only 5/50 (10%) were positive with EliA Symphony. Of the 42 SSc-pos, 32 were ACA-positive and 10 anti-topo I-positive. Of the 22 SSc-neg sera that were positive with the EliA CTD-screen assay, five were positive for SSA/Ro, one for SSB/La, three for dsDNA, five for PM-Scl, five for RNAP III and one for AFA, whereas all five ANA-Symphony positive sera were anti-SSA positive.

Table 1: Agreement between qualitative results obtained with the EliA CTD Screen and with the methods for detection of single autoantibody specificities.

Figure 1: Values of anti-nuclear autoantibodies obtained using the EliA CTD Screen assay in scleroderma patients who were negative (SSc-neg) or positive (SSc-pos) for ACA and anti-topo I and in the control group.

Conclusions: In SSc patients the EliA CTD Screen assay shows a good diagnostic accuracy, comparable to that obtained with the assays for single au-toantibody detection. This screening test may be very useful to identify a subset of SSc patients who are positive for autoantibodies other than anti-topo I and ACA (AFA, anti-RNAP III and anti-PM-Scl).

Assa

ys f

or a

ntib

ody

spec

ificit

ies

EliA CTD Screen

pos neg total Overall agreement 90.2% (95% CI: 82.2-95.4%)

pos 60 5 65 Positive agreement 93.2% (95% CI: 88.4-97.5%)

neg 4 23 27 Negative agreement 83.6% (95% CI: 73.0-94.1%)

total 64 38 38 Cohen’s k 0.766 (95% CI: 0.622-0.910)

11JournAL

_____________________________________________ RIB-p

Detection of Ribosomal P (Rib-P) autoantibodies in SLEM. Kast, C. Löbke, G. Fürle, W. PapischPhadia GmbH, Munzinger Str. 7, 79111 Freiburg, Germany

Objective: Systemic lupus erythematosus (SLE) is a chronic autoimmune connective tissue disease characterized by acute and chronic inflammation of various tissues of the body. Certain autoantibodies in SLE patients are targeted to Ribosomal P (Rib-P) proteins and react with three specific ribosomal proteins (P0, P1 and P2). Antibodies to Ribosomal P proteins can be detected in approx. 15 to 20% of patients with SLE and are highly specific for this disease. According to the literature these autoantibodies occur during active SLE and are associated with neuropsychiatric, renal and hepatic involve-ment. The new and fully automated assay presented here uses certain molar ratios of the three ribosomal peptides P0, P1 and P2, described to increase the assay’s sensitivity.

Patients and Methods: A clinical correlation study was performed using samples from more than 400 patients (250 SLE cases, 173 disease controls) and several independent anti-Rib-P assays.

Results:

Figure 1: Performance of the EliA Rib-P assay using clinically defined samples.

Test name / cut-off Sensi-tivity

Speci-ficity

True posi-tives

True nega-tives

False posi-tives

EliA Rib-P / 10 U/ml 28,4 % 99,5 % 71 183 1

Competitor Rib-P / 20 U/ml 23,2 % 99,5 % 58 183 1

Table 1: Performance comparison of EliA Rib-P and a competitor test.

Conclusions: The fully automated EliA Rib-P assay presented here shows the highest sensitivity (28.4%) of all tests compared, gives precise results (3 CV% intra-run, 4 CV% inter-run), performs linear over the measuring range and exhibits an excellent specificity of 99.5% using disease control samples and healthy blood donors. Therefore EliA Rib-P is a new clinical relevant serological tool to specifically support the diagnosis of SLE.

Figure 2: Sensitivity / Specificity compared to best competitor.

Figure 3: EliA Rib-P signal distribution on 400 blood donors.

12 JournAL

RIB-p / SCL-70 ________________________________________

Diagnostic value of antibodies against ribosomal protein P in SLETorsten Witte Klinik für Immunologie und Rheumatologie, Medical School Hannover, for the German EASI group

Objective: Antibodies against ribosomal protein P (RibP) have been shown to be very specific for SLE. The prevalence of RibP antibodies in SLE varied however between 3 and 40 %. Recently, it has been discussed to use antibodies against RibP as another classification criterion for SLE. We now wanted to study, whether RibP antibodies are useful in the differential diagnosis of connective tissue diseases in a rheumatological clinic.

Patients and Methods: 105 patients (90 female, average age: 35 years) with the following diagnosis established by the first rheumatologist: 26 SLE, 18 primary Sjögren`s syndrome, 17 Undifferentiated CTD, 6 Primary vasculitis, 4 SCLE/DLE, 3 Autoimmune thyroiditis, 31 Miscellanous. Arthritis was the most common possible classification criterion of SLE (60 %).

All the 105 patients that were referred by general practitioners to our rheumatological clinic (both as in- and outpatients) for the first time for diagnostic evaluation of SLE in 2009 and had at least one clinical ACR classification criterion of SLE were included in the study. Antibodies against RibP were measured using an automated test system (EliA Rib P, Phadia, Freiburg, Germany), antibodies against dsDNA by Farr assay. The diagnosis was estab-lished by a rheumatologist knowing all the laboratory results with the exception of the RibP antibodies.

Afterwards, the sensitivity and specificity of RibP antibodies for SLE was evaluated by a second rheumatologist, who was not involved in establishing the diagnosis.

Results: Antibodies against RibP were present in 5/26 patients with SLE using the cut off 7 U/ml as suggested by Phadia, but in none of the other pa-tients. Using a cut off of 5 U/ml, antibodies against RibP would have been detected in 7/26 SLE patients.

Figure 1: Concentration of RibP antibodies in all 105 patients. All of the 5 patients with antibodies against RibP had active disease (SLEDAI score at least 7). 1 of the 5 SLE patients with antibodies against RibP did not have antibodies against dsDNA.

Conclusions: Antibodies against ribosomal protein P are specific for SLE.

They are helpful in the evaluation of patients with active disease, in particular in the absence of antibodies against dsDNA

Quantitative evaluation of anti-topoisomerase I antibodies in systemic sclerosisM. Lotzniker1, G. Re1, S. Finazzi1, C. Alpini2, P. Faggioli3, A. Mazzone3

1 Clinical Chemistry Laboratory A.O. Ospedale Civile di Legnano, Italy , 2 Clinical Chemistry Laboratory IRCCS San Matteo, Pavia, Italy, 3 Department of Medicine A.O. Ospedale Civile di Legnano, Italy

Objective: Anti-topo I was serially measured in a large number of patients in order to define prognostic impact of different concentrations of this pa-rameter and for evaluation of its utility in follow-up of patients.

Patients and Methods: 265 sera from 30 anti-topo I positive patients (dcSSc 16, lcSSc 14, F/M 25/5, mean age 58 + 15, mean observation n = 9, follow-up 2-6 years) were tested by quantitative EliA technology on Unicap 250 analyzer (Phadia, Freiburg, Germany); CV intra/inter assay was below 5%. Patients classification was performed by ACR and Le Roy criteria and by EUSTAR and MEDSGER scale for disease activity and severity.

Figure 2: RibP antibodies are associated with active SLE.

13JournAL

_____________________________________________ SCL-70Results: For each patient we calculated median value of anti-topo I; this value showed positive correlation with EUSTAR score (r = 0.36). Me-dian values were categorized into three groups: group I (lower tertile) < 52 U/ml, group II (middle tertile) = 61-124 U/ml, group III (upper ter-tile) > 164 U/ml. LeRoy disease pattern was similar in the three groups. Patients in the lower tertile, in comparison with upper tertile, showed a better EUSTAR score (p = 0.049).

Medgser evalution showed absence of renal and muscular involvement and less articular and gastrointestinal involvement in the lower tertile; no differ-ence was found for other organ involvement.

Kidney GI Muscle Joint/tendon General

Group 1 (lower tertilte) 0% 20% 0% 10% 20%

Group 2 (middle tertile) 19% 50% 40% 30% 50%

Group 3 (upper tertile) 30% 50% 20% 60% 40%

Table 1: Medsger evaluation: different organ system involvement in selected groups.

Follow-up: for 93% of patients anti-topo I levels remained in the tertile of the first visit. Most patients showed stable levels or variations without clinical correlation. Change at last visit: using an arbitrary cut-off of 50% increase or decrease of anti-topo I levels, only 6 patients showed this variation, 4 with increase (+74, + 92, +155, +326%) and 2 with decrease (- 68, -69%). Only in one patient anti-topo I increased (+ 326%) with disease progression, in the others no clinical correlation was found.

Figure 2: Anti-topo I levels during follow-up of single patients (values near the upper detection limit are shown as 500 U/ml) • anti-topo I decrease after Rituximab treatment for overlap with Rheumatoid Arthritis• disease progression and anti-topo I increase from lower to upper tertile.

Follow-up studies after one year observation: one interesting finding is that, if the anti-topo I decreases after one year, such patients generally have a good outcome. There was only 1 non-stable patient among those whose value decreased and no death.

Conclusions: Detected signal strength of anti-topo I at diagnosis cor-relates with prognostic indices: patients with low anti-topo I levels show favorable clinical outcome.

Longitudinal analysis of anti-topo I seems of limited help for continuous patients monitoring, but observation after one year of treatment could identify a setting of patients with better prognosis.

In spite of lack of anti-topo I standardisation, routinely quantitative ex-pression of this autoantibody seems to be a useful tool for risk assess-ment at diagnosis and for the re-evaluation after one year.

Figure 1: Eustar score in patients of selected groups

Figure 3: One year variation of anti-topo I in non stable (A) and stable (B) patients (4 patients with anti-topo I levels near the upper detection limits are not shown).

14 JournAL

CCp _______________________________________________

A comparison of anti-CCP and rheumatoid factor in the diagnosis of rheumatoid arthritisM.L.V. Watkins1, F. Moosajee2, E. Kotze1, D. Hawarden3, A. A. Kalla2

1 National Heath Laboratory Service, Groote Schuur Hospital, Cape Town and Dept of Medicine, 2 Rheumatology Division and 3Allergology Division, University of Cape Town, South Africa.

Objective: In 2000 anti-cyclic citrullinated peptide (ACCP) antibodies were described for diagnosis of RA. ACCP is an early marker of RA and has been measured in the serum of patients up to 10 years prior to devel-opment of symptoms.

Patients and Methods: This study was a retrospective study of patients from the arthritis clinic at Groote Schuur Hospital from May 2008 until June 2009 in whom both RF and ACCP assays were requested by the cli-nician. Patient folders were studied and patients divided into three groups based on the number of ACR criteria present at the time of the folder review: definite RA (>4 ACR criteria present), probable RA (<4 ACR criteria present) or not RA (no ACR criteria present). Of the 303 patients, folders were only available on 200 patients. Thirteen patients <18 years of age were excluded. This study was therefore carried out on 187 patients. Second generation ACCP (IgG) was performed on the ImmunoCAP 100 (Phadia Uppsala Sweden) and compared to RF (IgG) conducted on the Modular (Roche, Basel, Switzerland).

Results:

Figure 1: RF (A) and ACCP (B) results in RA, probable RA and not RA patients. Statistically significant results are indicated when two-tailed Mann Whitney test for nonparametric data is used.

Conclusions: 75% and 85% of patients were female in the definite RA and probable RA patients group, respectively. This confirms published reports indicating that women are two to four times more likely to be affected with RA than men.

In our group of patients, ACCP is highly specific for the diagnosis of RA (specificity 92%).

This study found that the use of both RF and ACCP increases the sensitivity for the diagnosis of RA from 75% to 77%.

As Groote Schur Hospital is a tertiary institute, patients are usually seen when RA is already established. This could account for the lower sensitivity of ACCP (65%) in our group of patients. Alternatively the difference in genetics of our population group could account for the decreased sensitivity.

As ACCP is documented as being an early marker for RA, predating the onset of symptoms, patients positive for ACCP will be monitored who do not have symptoms to determine whether they develop RA over time.

Figure 2: Percentage of patients positive for ACCP and/or RF in RA (A) probable RA (B) and not RA patients (C).

  Correlation p value Spearman rRA Yes p <0.0001 0.5758

Probable RA Yes 0.0028 0.6312Not RA No

Table 1: Correlation between ACCP and RF in the different patient groups using Spearman correlation.

  Sensitivity SpecificityRF 75% 78%

ACCP 65% 92%RF and ACCP 77% 76%

Table 2: Sensitivity and specificity of tests in determining RA.

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_______________________________________________ CCp

Preliminary evaluation of anti-cyclic citrullinated peptide antibodies of IgA and IgM isotypes using a fully automated fluoroenzymeimmunoassay (EliA) in Rheumatoid Arthritis1 Alpini C, 2 Bobbio-Pallavicini F, 3 Valaperta S, 1 Avalle S., 4 Finazzi S, 4 Lotzniker M, 2 Caporali R, 2 Montecucco C, 1 Moratti R1 Clinical Chemistry Laboratories, University of Pavia, IRCCS Policlinico S. Matteo Foundation, Pavia, Italy, 2 Chair and Division of Rheumatology, University of Pavia, IRCCS Policlinico S. Matteo Foundation, Pavia, Italy, 3 Clinical Investigation Laboratory, IRCCS Istituto Clinico Humanitas, Rozzano (MI), Italy, 4 Laboratory of Biochemical-Clinic Analysis, Legnano, Italy

Objective: To define the IgA and IgM ACPA cut-off values for a diagnosis of RA through a new fully automated fluoroenzyme-immunoassay (EliA) test already validated for the assessment of IgG ACPA.

Patients and Methods: Serum samples from 134 RA patients (33 with long-standing and 101 with early disease) and 179 internal controls (79 connec-tive tissue diseases: 33 Sjögren’s Syndrome, 15 SLE, 6 Connective tissue disease, 22 Systemic Sclerosis, 1 PBC, 2 PM/DM

and 100 healthy blood donors) were preliminarly tested for total IgA and Rheumatoid Factor (immunoturbidimetric methods on Abbott Ci16200 instru-ment). IgA, IgG and IgM ACPA antibodies isotypes were then measured using an EliA method on the ImmunoCAP 250 instrument (Phadia, Freiburg, Germany).

Results: Achieved data were investigated by means of ROC analysis for establishment of best cut-off:Proposed cut-offs: IgA Anti-CCP: 2.2 U/ml IgM Anti-CCP: 100 U/ml

Long Standing RA Early RA

  IgA Anti-CCP IgM Anti-CCP IgA Anti-CCP IgM Anti-CCP

Sensitivity 51.5% 7.0% 55.4% 22.8%

Specificity 93.7% 92.4% 93.7% 92.4%

Conclusions: The diagnostic sensitivity of IgA and IgM ACPA isotypes is lower in long-standing RA than in early-RA compared to the IgG ACPA assay.

Future studies are needed to explore whether different ACPA isotypes might provide additional clinical value in predicting disease’s course and response to therapy.

Figure 1: left panel: CCP IgA in early RA; right panel: CCP IgA in long standing RA

Figure 2: left panel: CCP IgM in early RA; right panel: CCP IgM in long standing RA

16 JournAL

CELIAC _____________________________________________

High sensitivity of anti-deamidated gliadin peptide IgG antibodies for diagnosis of celiac disease in childrenL. Mozo, E. Escanlar, C. GutiérrezImmunology Department, Hospital Universitario Central de Asturias, Oviedo, Spain

Objective: To evaluate the influence of age at onset on the diagnostic accuracy of anti-deamidated gliadin peptide (anti-dGp) antibodies (Ab) for the diagnosis of celiac disease (CD).

Patients and Methods: CD group: 100 newly diagnosed CD patients: 37 children aged £5 and 63 patients aged >5 years, all positive for anti-tissue transglutaminase and with a Marsh I-IV proven biopsy. Control group: 100 patients (32 aged < 5 and 68 aged >5 years) with negative duodenal biopsy (23 cases), other digestive autoimmune diseases (40cases) or food allergy (37 cases).

Anti-dGp Ab IgG and IgA and anti-gliadin (Glia) IgA were measured by EliA (Phadia, Freiburg, Germany). The diagnostic performance of each test was measured as the area under the ROC curve (AUC). Sensitivity, specificity, positive and negative predictive value (PPV and NPV) were calculated using optimal cut-off points obtained on the basis of the ROC associated Youden Index.

Results: When analysing the diagnostic performance in all CD patients and controls, anti-dGp of both isotypes and anti-Glia IgA displayed very high and significantly different AUC, the highest being obtained with anti-dGp IgG. Sensitivity and NPV of both deamidated assays were similar and sig-nificantly higher than those obtained with anti-Glia IgA. Regarding specificity and PPV, no statistically differences were found, anti-dGp IgG reaching the highest values.

AUC (%) Sensitivity (%) Specificity (%) PPV (%) NPV (%)

anti-Glia IgA 97.8 88.0 94.0 93.5 89.5

anti-dGP IgA 98.8 96.0 96.0 96.0 96.0

anti-dGp IgG 99.5 95.0 99.0 98.9 95.2

p=0.003 p=0.04 ns ns p=0.035

Table 1: Anti-dGp IgG showed the highest diagnostic performance for CD.

AUC values in both, in children < 5 years and in older patients were similar to those obtained when both groups were analyzed together, the highest being also displayed by anti-dGp Ig. AUC were significantly different among the older group whereas those in children were almost statistically significant.

Patients ≤5 years Patients >5 years

AUC (%) Sensitivity (%) Specificity (%) PPV (%) NPV (%) AUC (%) Sensitivity (%) Specificity (%) PPV (%) NPV (%)

anti-Glia IgA 97.8 94.6 93.7 94.6 93.7 97.6 85.7 94.1 93.1 87.7

anti-dGP IgA 99.2 97.3 96.9 97.3 96.9 98.4 95.2 95.6 95.2 95.6

anti-dGp IgG 100 100 100 100 100 99.5 92.1 98.5 98.3 93.0

p=0.053 p=0.037 p=0.020

Table 2: Anti-dGp IgG performed better in CD children aged < 5 years than in older patients.

Figure 1: Levels of anti-Glia IgA and anti-dGp are inversely correlated with age.

17JournAL

_____________________________________________ CELIACWe also retrospectively analyzed 6 CD patients with IgA deficiency. All patients were anti-dGp IgG positive whereas anti-Glia IgG were detected only in five.

Conclusions: Our results stress the high usefulness of anti-dGp in the diagnosis of CD in children and indicated that its routine determination can im-prove diagnosis of patients with IgA deficiency.

Evaluation of a new fluorescent enzyme immunoassay in celiac disease diagnosis based on the use of deamidated gliadin peptidesE. Vergara Prieto, M.L. Vargas Pérez, I. Alcalá Peña, S. Gordillo Vázquez, N. Román Pinto, J. Melero RuizImmunology and Genetic Service, University Hospital of Badajoz, Spain

Objective: To evaluate the diagnostic utility of antibodies to synthetic deamidated gliadin peptides (GliadinDP) in celiac disease diagnosis.

Patients and Methods: 115 samples were collected from patients referred for celiac disease screening. After serological markers were determined through standard techniques (IgA gliadin antibodies, IgA tissue transglutaminase antibodies, IgA endomysial antibodies), the presence of IgA deami-dated gliadin peptides antibodies were determined using EliA GliadinDP on ImmunoCAP 250 (Phadia GmbH, Germany). Celiac disease diagnosis was made following a confirmatory intestinal biopsy.

Results: Of the patients studied, 38 were diagnosed with celiac disease and 77 were diagnosed as non-celiac condition.

Figure 1: ROC Curve analysis and test performance for deamidated and native gliadin peptide antibodies.

Conclusions: The diagnostic utility of antibodies against deamidated gliadin peptides is very high. This marker is more sensitive and specific than antibodies against native gliadin. It is therefore recommendable to include it in the celiac disease diagnostic panel as a replacement for native gliadin antibodies.

AUC 0.966

Cut-off 4 μg/l Sens 100% Spec 92.2%

AUC 0.892

Cut-off 10 U/ml Sens 92.9% Spec 68.6%

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CELIAC ______________________________________________

Good sensitivity of tests for antibodies to deamidated gliadin peptides in children – correlation with jejunal morphologyM. Viander1, P. Arikoski2, A. Lammi3, A. Hinkkanen4, J. Ilonen5

1 Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland, 2 Department of Paediatrics, Kuopio University Hospital, Kuopio, Finland, 3 Department of Clinical Microbiology, University of Eastern Finland, Kuopio, Finland, 4 A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland, 5 Department of Clinical Microbiology, University of Eastern Finland, Kuopio, Finland and Immunogenetics Laboratory, University of Turku, Turku, Finland

Objective: to compare the efficacy of a new test for deamidated gliadin peptides (DGP) antibodies, EliA GliadinDP, with Phadia’s tests for tissue trans-glutaminase (tTG) and anti-gliadin antibodies (AGA), and with in-house tests for endomysium (EmA) for the screening of celiac disease (CD) in small children.

Patients and Methods: A total of 62 children were included, 39 children biopsied for suspected CD (aged from 1 year 2 months to 14 years), and 23 healthy infants (aged from 10 months to 10 years). During follow-up, serum samples were taken at 6 month intervals and celiac antibodies were screened for EmA (in-house test on human umbilical cord) and DGP antibodies (University of Turku, Finland) and anti-tTG with EliA Celikey IgA/IgG (Phadia 100, Phadia AB).

Those with positive antibodies and/or clinical signs of celiac disease were subjected to intestinal biopsy graded according to Marsh (1992).

Aliquots of the samples taken at time of biopsy were studied using EliA GliadinDP IgA/IgG, EliA Celikey IgA/IgG and EliA Gliadin IgA/IgG, and with Anti-DGP IgA/IgG using TR-FIA (University of Turku, Finland; Ankelo et al. 2007).

Results:

Test Cut-off Sensitivity

EmA 5 100%

tTG IgA 10 100%

DGP IgA 150 100%

DGP IgG 150 93.6%

EliA Celikey IgA 10 100%

EliA Celikey IgG 10 48.5%

EliA Gliadin IgA 10 69.7%

EliA Gliadin IgG 10 97.0%

EliA GliadinDP IgA 10 87.5%

EliA GliadinDP IgG 10 97.0%

Table 1: Sensitivity of different tests in 33 children with celiac disease.

Sample Age at blood with-drawal

EmA tTG IgA DGP IgA DGP IgG EliA Celikey IgA

EliA Celikey IgG

EliA Gliadin IgA

EliA Gliadin IgG

EliA GliadinDP IgA

EliAGliadinDP IgG

1 10,5 neg 7.5 46.0 86.0 9.3 1.3 1.3 0.7 2.0 0.6

2 3,7 neg 9.0 54.0 55.0 15.6 4.0 2.2 13.1 2.9 4.7

3 1,75 neg 12.0 62.0 166.0 23.5 9.8 1.9 29.9 5.1 28.4

4 11,5 neg 3.7 142.0 340.0 21.3 4.8 3.2 18.4 6.2 27.8

5 6 neg 1.0 47.0 18,0 1.5 1.4 1.5 2.0 1.3 2.0

6 3,7 5 5.5 88.0 266.0 6.5 4.2 2.8 28.0 2.9 28.3

Σ 1 1 0 3 3 0 0 4 0 3

Table 2: Different antibodies in six children with suspected CD but negative biopsy – suggesting a latent CD?

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_____________________________________________ CELIAC

Figure 1: A significant difference in the levels of antibodies in children with severe villous atrophy (n=18) compared to those with only partial or moderate lesions (n=15) in gliadin and EMA tests but not in tTG tests.

Conclusions: The novel EliA tests for IgA and IgG class antibodies to deamidated gliadin peptides proved, in small children, as sensitive and as specific as the established tests for celiac screening, endomysial and tissue transglutaminase antibodies. Good correlation of the antibody levels with the intesti-nal biopsy morphology suggests that the test may be useful in follow-up of the gluten-free diet.

20 JournAL

Diagnostic performance of IgG anti-DGP assays is comparable to IgA anti-tTG in celiac diseaseVermeersch P Laboratory Medicine, University Hospitals Leuven, Belgium

Objective: To compare the performance of IgG anti-DGP assays from Euroimmun, Inova, Phadia and The Binding Site to other serologic assays for celiac disease including 3 IgA anti-tTG assays.

Patients and Methods: 86 consecutive celiac disease patients and 741 consecutive disease control patients. The study population consisted of 599 adults and 228 children. All CD and 98 control patients had a duodenal biopsy. Serologic assays for CD (anti-tTG, anti-DGP and anti-Gliadin) were obtained from the following companies: Euroimmun (Lübeck, Germany), Genesis (Cambridgeshire, United Kingdom), Inova Diagnostics (San Diego, CA), Phadia (Uppsala, Sweden) and The Binding Site (Birmingham, United Kingdom).

Results: Of the 4 IgG anti-DGP assays, the Inova assay had the highest area under the curve and the best sensitivity (together with the Phadia assay) and specificity.

The diagnostic performance of the IgG anti-DGP assays and the IgA anti-tTG assays in pediatric patients was better than the diagnostic performance of the 2 IgG anti-tTG assays, the IgA anti-gliadin assay and the IgG anti-gliadin assay.

All patientsPediatric patients

Proposed cut-off Cut-off specificity 98%

AUC Cut-off Sensitivity Specificity LR Cut-off Sensitivity Sensitivity Specificity LR

IgA anti-tTG

BioRad 0.936 >15 88.40% 94.90% 17 (13–24) 31.6 81.40% 96.40% 94.50% 18 (10-31)

Phadia 0.927 >7 83.70% 98.40% 52 (29–91) 6.4 84.90% 96.40% 98.50% 64 (21-198)

Genesis 0.913 7 84.90% 92.00% 11 (8–14) 14 80.20% 96.40% 89.00% 9 (6-13)

IgG anti-tTG

BioRad 0.937 >15 60.50% 98.10% 32 (19–55) 11.4 68.60% 75.00% 95.00% 15 (8-28)

Phadia 0.767 >7 38.40% 98.50% 26 (14–49) 6.5 40.70% 57.10% 99.00% 57 (14-235)

IgA anti-DGP

Phadia 0.931 >7 65.10% 99.10% 69 (32–146) 5.3 72.10% 92.90% 98.50% 62 (20-191)

IgG anti-DGP

Euroimmun 0.929 25 76.70% 99.20% 95 (42–212) 15.2 80.20% 92.90% 98.50% 62 (20-191)

Inova 0.961 20 83.70% 99.30% 124 (52–299) 17.1 84.90% 92.90% 98.50% 62 (20-191)

Phadia 0.926 >7 83.70% 97.30% 32 (21–50) 7.8 83.50% 92.90% 97.00% 31 (14-69)

The Binding Site 0.943 10 79.10% 97.60% 33 (20–52) 12.9 74.40% 96.40% 95.50% 21 (11-41)

IgA anti-Gli

Phadia 0.898 >7 74.40% 95.10% 15 (11–22) 9.6 66.30% 75.00% 95.00% 15 (8-28)

IgG anti-Gli

Phadia 0.882 >7 67.40% 93.30% 10 (7–14) 18.7 44.70% 82.10% 85.00% 5 (4-8)

Conclusions: The diagnostic accuracy of the four IgG anti-DGP assays tested was comparable to the three IgA anti-tTG assays tested and better than the IgG anti-tTG, IgA anti-gliadin and IgG anti-gliadin assays.

When the IgG anti-DGP assay from Inova would be performed in all patients in addition to the IgA anti-tTG assay from Phadia (the IgA anti-tTG and IgG anti-DGP assay with the highest LR in this study), the sensitivity would increase from 83.7% to 89.5%, while the specificity would only decrease from 98.4% to 98.0%.

CELIAC _____________________________________________

21JournAL

Comparison of two cardiolipin antibodies tests and two β2 glycoprotein 1 antibodies testsS. Brito, V. Domingues, A. Mendes, L. Correia, H. Pereira, Medicina Laboratorial, Centro Hospitalar de Coimbra, EPE, Coimbra, Portugal

Objective: To compare tests to Cardiolipin antibodies (CL) and β2 Glycoprotein 1 (β2) antibodies as part of the classification criteria of the antiphos-pholipid syndrome (APS)

Patients and Methods: 220 patients were examined: 49 APS, 12 questionable APS (qAPS), 60 SLE, 100 various infections. Two automated CL tests were used (Liaison Cardiolipin, Diasorin, Italy; EliA Cardiolipin, Phadia, Germany) and two β2 tests – one automated (EliA β2 Glycoprotein I, Phadia, Germany) and one Elisa (β2 Glycoprotein I, Diamedx, USA). The serological APS diagnosis was based on the Liaison/Diamedx test results.

Results: Of 49 APS patients there was positivity in Cl IgG/IgM in 42.9%/38.3% (Liaison) and 46.9%/38.8% (EliA). β2 IgG/IgM was positive in 51%/36.7% (Diamedx) and 34.7%/34.7% (EliA), respectively. Combining the results using the ´Sydney cut off´ for Cardiolipin (40 GPL-, MPL-U/ml) together with the results of β2 (at kit cut off = DFU) sensitivities of 67.3% (Liaison) and 65.3% (EliA) at still high specificity (93%/92%) resulted. Table 1 lists the positivity rates (in %) if the cut off values for cardiolipin IgG and IgM according to the package inserts are used (DFU). Also the positivity found by combining all data is shown. In Figure 1 the same information is listed but the positivity for cardiolipin IgG and IgM was calculated based on the´Sydney´ recommendation of 40 GPL-/MPL-U/ml. Taking into account that the serological part of the diagnosis was based on the combination of Liaison/Diamedx test results, the effect coming from this non-avoidable bias is realtively low, but still may account for some discrepancies (Figure 2). The positivity rates are on very comparable levels for cardiolipin IgG, IgM and β2 IgM. Interestingly, the Diamedx β2 IgG is significantly more positive in the APS than the Liaison cardiolipin IgG or EliA cardiolipin IgG (51% vs 42.9% / 46.9%). This is contradictory to the expectations from literature as well as theoretical considerations: the cofactor β2 glycoprotein is implemented as antigenic target in the classical anti-cardiolipin test. Thus cardiolipin antibodies are said to be per se more sensitive than β2 glycoprotein I antibodies and only a limited amount of exclusively β2 positive and cardiolipin negative results is expected. This finding maybe due to the usage of 2 different assay systems (different reagents / solid phase) and consequently also different antigen preparation in Liaison / Diamedx. Comparing the APS with the qAPS higher antibody levels are found in the cardiolipin IgG testing for the APS group (Figure 3). This difference is more expressed in the β2 testing.

Figure 1: Positives in % in different groups of all tests combined at DFU cut-off.Table 1: Perfomance data.

Figure 3: Graph of CL IgG/IgM in APS and qAPS.Figure 2: Graph of discrepant results in CL IgG with clinical background.

_______________________________________________ ApS

22 JournAL

Conclusions: The agreement of the different methods was found to be reasonably good. As the patients have been diagnosed based on the serological findings of the Liaison/Diamedix assays it was an unexpected finding that the sensitivity of EliA CL IgG is higher than the sensitivity of Liaison CL. Overall, the agreements are moderate/good and positivity rates are on a comparable levels. The usage of the ´Sydney´ cut off recommendation for car-diolipin may increase the comparability of results.

Comparative evaluation of the fully automated new PR3S sensitive test (EliA PR3S), for the diagnosis of the ANCA-associated systemic vasculitis (AASV)1 A. Radice, 2L. Bianchi, A. 3Palumbo, 2RA Sinico. 1 Microbiology Institute & 2 Nephrology and Clinical Immunology Unit, Osp S. Carlo Borromeo, Milano, Italy, 3 Procrea SA, Lugano, CH.

Objective: To evaluate the diagnostic performance of a new, fully automated PR3-ANCA sensitive test for the diagnosis of Wegener’s granulomatosis. This 3rd generation test uses an “anchor” technology, coupling the purified human PR3 to the surface of the wells by means of little spacer molecules, both assuring the best epitope accessibility and avoiding epitope competition between the “capturing” anti-PR3 MoAb and the autoantibodies in pa-tient’s sera.

Patients and Methods: Out of 270 patients’ samples, 127 belong to the group of ANCA associated vasculitides (55 Wegener’s granulomatosis; 52 microscopic polyangiitis; 20 Churg-Strauss syndrome) and 142 belong to the control patients group (21 RCU, MDC, IBD; 30 SLE; 28 CTDs, AR; 32 Infectious diseases; 12 other diseases).

The new PR3-ANCA sensitive test (EliA PR3S, Phadia) was compared to direct PR3-EliA (Phadia), capture PR3-ANCA (Wieslab Euro-diagnostica), anti-PR3 hs (Orgentec) and ANCA-IIF (Granulocyten-Mosaik, 2 Euroimmun). For comparison of the immunometric assays, cut-off values reported by the manufacturers were used.

Results:

Figure 1: ROC curves comparative analysis, left: all WG patients (55); right: WG patients, P-ANCA/MPO positive excluded (48)

% EliA PR3S EliA PR3 Capture PR3 anti-PR3 hs

sens 69.1 61.8 72.7 72.7

spec 99.1 95.8 99.1 98.1

effic 93.0 88.9 93.7 93.0

PPV 95.0 79.1 95.2 90.9

NPV 92.6 90.7 93.4 93.4

Table 1: Perfomance

pR3 AnCA ___________________________________________

23JournAL

EliA PR3S

neg = 0, pos = 1, equ =neg

IFI PR3, pos=1, neg=0

neg pos Total

neg 16 1* 17

pos 1** 37 38

Total 17 38 55

Table 2: Comparison of EliA PR3S and IFI results. * P-ANCA/PR3-ANCA confirmed with different assays, ** C-ANCA/PR3-ANCA low pos with different assays.

Figure 2: Usefulness of four different PR3-ANCA tests, shown with positive and negative likelyhood ratio. LR(+) above 10 and LR(-) below 0.1 indicate a very useful test result, while LR(+) between 5 and 10 and LR(-) between 0.1 and 0.2 indicate a moderate usefulness.

Noteworthy 7/55 WG patients were historically P-ANCA/MPO+ve. They are actually “true analytical negative”, excluding them from the calculation would improve the sensitivity of the assays.Combining IIF + EliA PR3S, improves the sensitivity of the ANCA detection.All 2nd and 3rd generation anti-PR3 immunometric assays tested showed very good and comparable clinical & analytical performances, far better than the PR3 direct test.

Conclusions: Antigen coated to the solid-phase by means of spacer molecules should assure the best epitope accessibility, and enhance the sensitivity of the test, without any loss in specificity. Indeed, the overall diagnostic performance of the EliA PR3S was much better than the direct EliA, and equivalent to the compared non-direct assays (capture PR3-ANCA & anti-PR3 hs).

Therefore, our results confirm that the novel formats to detect PR3-ANCA have better performances than the classic direct assays. Among the novel assays, EliA PR3S is the only fully automated method available until now. The advantages of the EliA PR3S, based on Phadia 100 and Phadia 250 in-struments, combining high quality reagents with an outstanding automation technology, fits most laboratory needs, and should accomplish diagnostic and monitoring necessities of ANCA-associated vasculitis. The wide range of the reference curve should allow autoantibody measuring without further dilutions.

Finally, its performance is very close to that of the IIF test, suggesting the possibility of replacing IIF with antigen-specific high-sensitive assays in the diagnostic algorythm of the AAV. However, further studies are needed to confirm the data, both in diagnostic phase and in monitoring patients before applying a different algorythm in the clinical practice.

EliA PR314.77

EliA PR3 sensitive 74.27

Eurodiagnostica cPR378.18

Orgentec hsPR339.09

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Likelihood ratio (+)

EliA PR30.40

EliA PR3 sensitive 0.31 Eurodiagnostica cPR3

0.28Orgentec hsPR3

0.28

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

Likelihood ratio (-)

______________________________________________ pR3S

Orde

r no.

525

5060

1 Fr

eibu

rg 0

6/20

10

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