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1 ! How does methodology influence autoantibody test results? Johan Rönnelid MD, PhD Unit of Clinical Immunology and Transfusion medicine Uppsala University Hospital Department of Immunology, Genetics and Pathology Uppsala University Sweden!""#$%&'(#)'%$*+!,-*!./&*0123$'4#1*!.51$*031*0123$'4#1*#)1&*0%* )3%6*031*7))%2'78%$* $*'$&':'&#7/*&')17)1)* 7$&*);12'<2*7#0%7$89%&'1)*! B%6*)1$)'8:'0C*! D'>3*);12'<2'0C* 1!

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3 Addressable Laser Bead ImmunoAssay ALBIA ( Bead array, Luminex ) Some piyalls in immunoassays: Heterophilic an8bodies/rheumatoid factor (in sandwich assays) Background reac8vity Rheumatoid factor false IgM reac8vity 3

4 Heterophilic an8bodies (including RF) 24-plex for cytokines in RA sera (unpublished): 17/24 showed strong correla8on to RF No correla8on to an8- CCP Mostly a problem im sandwich assays (not simple EIAs for autoantibodies) (one) solution: buffer composition Reac8vity to the background matrix: (one) solution: use individual blank wells Åhlin et al, Scand J Immunol

5 IgM RF interference in solid phase assays (one) solution: deplete IgG before measuring IgM antibodies High sensitivity (and low specificity) tests should not be used as diagnostic tests, but can be used: * For screening (and later follow-up with more specific tests) * For monitoring of autoantibody levels High specificity tests are preferable in a diagnostic setting, and for confirmatory tests. 5

6 Soluble antigens in native state Surface bound and at least partly denatured antigens 6

7 EQUALIS 2001: anti-ssa positive with double immune diffusion (DID), IIF ANA negative Number of labs using the technique Only anti-ssa Anti-SSA and anti-ssb ELISA DID Line blot All laboratories using ELISA and reporting anti-ssb above reported negative ANA by IIF. Conclusion: the old screening technique (IIF ANA) is less sensitive than the follow-up ELISA. External control results from NEQAS 2008 (sample 0821) CIE n=4 DID n=3 ELISA N=278 Line blot N=78 ALBIA N=17 SSA SSB Sm RNP Scl70 Jo-1 Neg. 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 4(100) 0 (0) 0 (0) 0 (0) 1 (33) 0 (0) 0 (0) 2 (67) 42 (15) 14 (18) 2 (1) 5 (2) 272 (98) 1 (1) 3 (4) 68 (87) 2 (12) 0 (0) 0 (0) 17 (100) 4 (1) 1 (0) 6 (2) 0 (0) 0 (0) 7 (9) 0 (0) 0 (0) 0 (0) Target response: anti- RNP (14%, 96%) 7

8 External control results from NEQAS January 2011 (sample 1112) CIE n=4 DID n=1 ELISA N=243 Line blot N=71 ALBIA N=27 SSA SSB Sm RNP Scl70 Jo-1 Neg. 0 (0) 0 (0) 1 (25) 1 (25) 0 (0) 0 (0) 3(75) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1(100) 7(3) 3(1) 236 (97) 2(3) 1(1) 71 (100) 1(4) 1(4) 27 (100) 219 (90) 49 (69) 17 (63) 1(0) 0(0) 7(3) 1(1) 0(0) 0(0) 0(0) 0(0) 0(0) Target response: anti-sm (20%, 98%) External control results from NEQAS July 2011 (sample 1142) CIE n=7 DID n=0 ELISA N=293 Line blot N=97 ALBIA n=32 SSA SSB Sm RNP Scl70 Jo-1 Neg. 7(100) 7(100) 0(0) 0(0) 0(0) 0(0) 0(0) _ 293 (100) 97 (100) 32 (100) 293 (100) 95 (98) 32 (100) 1(0) 12(4) 1(0) 2(1) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(3) Target response: anti-ssa + anti-ssb 8

9 ELISA/EIA responses against ENA in UK NEQUAS samples during one year! Sample! Ro/SSA! La/SSB! Sm! RNP! Scl70! Jo1! negative! 135<1!(Aug!2013)! 296$ 285$ 4! 3! 1! 3! 0! 135<2!(Aug!2013)! 297$ 40$ 5! 2! 0! 1! 0! 136<1!(Oct!2013)! 299$ 8! 2! 2! 1! 1! 0! 136<2!(Oct!2013)! 5! 3! 3! 3! 2! 4! 94$ 141<1!(Jan!2014)! 289$ 9! 0! 0! 0! 2! 0! 141<2!(Jan!2014)! 285$ 284$ 0! 1! 0! 3! 2! 142<1!(March!2014)! 12! 4! 287$ 277$ 3! 4! 3! 142<2!(March!2014)! 289$ 172$ 280$ 284$ 2! 5! 0! 143<2!(May!2014)! 8! 1! 0! 0! 0! 0! 178$ 143<2!(May!2014)! 296$ 1! 0! 0! 0! 1! 1! 144<1!(July!2014)! 0! 0! 0! 0! 269$ 0! 12! 144<2!(July!2014)! 2! 0! 17! 273$ 0! 0! 3!! Conclusions The older techniques have high diagnostic specificity but low sensitivity. Newer techniques favour sensitivity over specificity. Old techniques used native autoantigens. New solid phase techniques have a risk for antigen denaturation. Newer techniques are better for autoantibody quantification. QA programmes tend to use strong monospecific samples yielding comparable (qualitative) results irrespective of technique. Results cannot be used to compare techniques. There is a need for comparative programmes not aiming for the mandatory pass or fail - judgment - Focus on quality aspects. - Interpret the samples in a clinical context. - Not to avoid ambiguous polyspecific samples. 9

10 ! Four IgG anti-dsdna assays in relation to SLE disease specificity and activity (Enocsson H et al. J Rheumatol. 2015;42:817)! 178 SLE patients from a single centre (ACR 1982 and/or SLICC-12)! 11 of them followed consecutively! Controls: healthy (n=100), RA (n=95), primary Sjögren s syndrome (n=54)! Four different techniques compared:!!!! Bead-assay/ALBIA Luminex (FIDIS, Theradiag) EIA (EliA/Phadia-Thermofisher) Indirect IF (Crithidia luciliae/immunoconcept) Lineblot (Euroline/Euroimmun)! SLICC 2012! (2x ELISA cutoff)! IIF (C luciliae)! ALBIA luminex! Rec. by manufacturer! Enzyme immunoassay! Line blot! 10!

11 The European Consensus Finding Study Group on Autoantibodies (ECFSG). Associated to the European League Against Rheumatism (EULAR) 42 participating European laboratories Yearly themes (eg dsdna, myositis, scleroderma) Ten samples dispatched to all labs in December Results collected electronically in January Yearly meeting in March in conjunction to European Workshop for Rheumatology Research (EWRR). Results discussed in a clinical context No involvement of diagnostic companies 11

12 AIMS of the ECFSG aims at investigating the interlaboratory agreements concerning autoantibody findings in sera containing unspecified antibodies, and to get insight into the variability of autoantibody detection in routine clinical practice. Courtesy Dörte Hamann, Amsterdam 12

13 Courtesy Dörte Hamann, Amsterdam Courtesy Dörte Hamann, Amsterdam 13

14 Courtesy Dörte Hamann, Amsterdam 26 year old female, catastrophic APS Suspicion of SLE Courtesy Dörte Hamann, Amsterdam 14

15 Yearly themes: 2004: anti-dsdna antibodies 2005: anti-phospholipid antibodies 2006: myositis autoantibodies 2007: vaskulitis autoantibodies 2008: scleroderma-associated autoantibodies : consistency testing 2012: cytoplasmatic ANA patterns 2013: anti-dsdna antibodies 2014: tentative new anti-dsdna reference standard 2015: tentative new ACPA reference standard 2016: tentative new reference standards for MPO, PR3, beta2-gp1 European consensus study 2013/2014 Sample 10 Plasmapheresis from SLE patient Putative new anti-dsdna standard 15

16 ANA (IIFT) n=42 Positive Hep2 pattern Sample 10 % TN Evaluation Criteria Nr a1 b1 b2 b5 Fluorescence Pattern a1 smoothe membranous b1 homogenous/pos nucleoli b2 homogenous/neg nucleoli b5 fine speckled Sample 10 dsdna n=42 Final result Strongly positive dsdna method details ELISA n=30, Crithidia n=31 Farr n=7, other n=2 % TN % Evaluation Criteria % TN ELISA Farr 100 Crithidia other Evaluation Criteria 0 Negative, 1 Borderline, 2 low positive, 3 positive, 4 high positive 16

17 Histones n=25 Positive Sample 10 Nucleosomes n=19 Positive % TN Evaluation Criteria % TN Evaluation Criteria 0 Negative, 1 Borderline, 2 low positive, 3 positive, 4 high positive Ku n=18 (all IB) Positive Sample 10 % TN Evaluation Criteria 0 Negative, 1 Borderline, 2 low positive, 3 positive, 4 high positive Ku 70 n=1 positive Ku 80 n=2 positive 17

18 Summary sample 10 ANA strongly positive, homogenous Consensus on anti-dsdna, no difference between techniques Consensus on anti-histone and anti-nucleosome Consensus on anti-ku Material seems commutable Non WHO Reference Material Candidate International Standard for anti-dsdna and collaborative study samples NIBSC code: 15/174 Instructions for use (Version 1.00, Dated ) This material is not for in vitro diagnostic use. 1. INTENDED USE Preparation 15/174 is the candidate 2 nd WHO International Standard for anti-dsdna. It is intended to be evaluated in an international collaborative study. The aim of the study is to assign a value in IU, and assess its performance in a range of anti-dsdna assays. Three patient serum samples are also included in the study: Sample 1, Sample 2 and Sample CAUTION This preparation is not for administration to humans. The preparation contains material of human origin, and either the final product or the source materials, from which it is derived, have been tested and found negative for HBsAg, anti-hiv and HCV RNA. As with all materials of biological origin, this preparation should be regarded as potentially hazardous to health. It should be used and discarded according to your own laboratory's safety procedures. Such safety procedures should include the wearing of protective gloves and avoiding the generation of aerosols. Care should be exercised in opening ampoules or vials, to avoid cuts. 3. UNITAGE Previous assessments have indicated that the potency of 15/174 is broadly around IU/ml. This is intended as a guide only for the preparation of appropriate dilution series for testing. Samples 1 and 2 are approximately 1-2x as potent as 15/174; Sample 3 is approximately 10-15x as potent as 15/ CONTENTS Country of origin of biological material: United Kingdom. Each ampoule contains the lyophilised residue of ~0.5 ml defibrinated plasma i.e., serum, containing anti-dsdna. 5. STORAGE Please store unopened ampoules at -20 o C or below. Please note: because of the inherent stability of lyophilized material, NIBSC may ship these materials at ambient temperature. 6. DIRECTIONS FOR OPENING DIN ampoules have an easy-open coloured stress point, where the narrow ampoule stem joins the wider ampoule body. Tap the ampoule gently to collect the material at the bottom (labeled) end. Ensure that the disposable ampoule safety breaker provided is pushed down on the stem of the ampoule and against the shoulder of the ampoule body. Hold the body of the ampoule in one hand and the disposable ampoule breaker covering the ampoule stem between the thumb and first finger of the other hand. Apply a bending force to open the ampoule at the coloured stress point, primarily using the hand holding the plastic collar. Care should be taken to avoid cuts and projectile glass fragments that might enter the eyes, for example, by the use of suitable gloves and an eye shield. Take care that no material is lost from the ampoule and no glass falls into the ampoule. Within the ampoule is dry nitrogen gas at slightly less than atmospheric pressure. A new disposable ampoule breaker is provided with each DIN ampoule. 7. USE OF MATERIAL No attempt should be made to weigh out any portion of the freeze-dried material prior to reconstitution Ensure the lyophilised contents are at the bottom of the ampoule. Reconstitute the ampoule contents with 0.50 ml distilled or deionised water, using a calibrated pipette. Vortex VERY gently and inspect contents to ensure complete dissolution. Transfer contents to a clean tube. The reconstitution volume of 15/174 and Samples 1, 2 and 3, is 0.50 ml. 8. STABILITY Reference materials are held at NIBSC within assured, temperaturecontrolled storage facilities. Reference Materials should be stored on receipt as indicated on the label. NIBSC follows the policy of WHO with respect to its reference materials. 9. REFERENCES N/A 10. ACKNOWLEDGEMENTS N/A 11. FURTHER INFORMATION Further information can be obtained as follows; This material: enquiries@nibsc.org WHO Biological Standards: JCTLM Higher order reference materials: Derivation of International Units: asked_questions/how_are_international_units.aspx Ordering standards from NIBSC: estions.aspx NIBSC Terms & Conditions: CUSTOMER FEEDBACK Customers are encouraged to provide feedback on the suitability or use of the material provided or other aspects of our service. Please send any comments to enquiries@nibsc.org 13. CITATION In all publications, including data sheets, in which this material is referenced, it is important that the preparation's title, its status, the NIBSC code number, and the name and address of NIBSC are cited and cited correctly. 14. MATERIAL SAFETY SHEET Physical and Chemical properties Physical appearance: Corrosive: No lyophilisate Stable: Yes Oxidising: No Hygroscopic: No Irritant: Unknown Flammable: No Handling:See caution, Section 2 Other (specify): Consists of lyophilised human serum Toxicological properties Effects of inhalation: Not established, avoid inhalation Effects of ingestion: Not established, avoid ingestion Effects of skin absorption: Not established, avoid contact with skin National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, EN6 3QG T +44 (0) WHO International Laboratory for Biological Standards, UK Official Medicines Control Laboratory nibsc.org Page 1 of 2 18

19 ! National Institute for Biological Standards and Control Blanche Lane South Mimms Potters Bar Herts EN6 3QG nibsc.org 8 th February 2016 Dear Colleague, Re: Collaborative study to evaluate the candidate 2 nd WHO International Standard for anti-dsdna The National Institute for Biological Standards and Control (NIBSC) is collaborating with Professor Johan Rönnelid of Uppsala University Hospital and Chairman of the European Consensus Finding Study Group on Autoantibodies (ECFSG) and Prof. Pier Luigi Meroni MD, MaACR, Rheumatology Dept. Clinical Sciences and Community Health, University of Milan on the production of the 2 nd World Health Organisation International Standard for antidsdna (preparation 15/174). We would like to invite you to participate in an international collaborative study to evaluate the candidate International Standard along with three patient serum samples using a suitable method. The evaluation will consist of assaying three ampoules of each preparation according to a detailed protocol. If you are able to take part, please complete and return the accompanying reply form by 12 th February 2016 (even if you have already expressed an interest in participating). The study samples and protocol will be sent out soon afterwards. Yours sincerely, Dr Carl Dolman (Study Co-ordinator) Parenterals Section Biotherapeutics Group Tel: +44 (0) Carl.Dolman@nibsc.org.031J*01)01&*2%"'$>*J1I1J1$21* J17>1$0)L*! O$8GK_.*+\c]d-*! O$8G_ZF*+\c]d-*! O$8G9107\*>/C2%;J%01'$*]*+\c]d-*! O$8GeTK*+\c]^-*! H%"'$>L*7$8G,E?^cW*7$8G0J7$)>/#07"'$7)1N* 19!

20 New interna8onal nomenclature for IF ANA paferns Need for a uniform nomenclature for IIF ANA paferns Two interna8onal ICAP mee8ngs: Sao Paulo 2014 and Dresden 2015, next mee8ng in Kyoto October Nomenclature freely available at hfp:// 20

21 21

22 (2010) 11 clinical criteria 6 immunologic criteria SLE: 4, at least one clinical and one immunologic criterium (2012) 22