PR3/MPO-ANCA is as effective as immunofluorescence as a first-line test in AAV screening Stephen Holding PhD FRCPath Consultant Clinical Scientist Hull & East Yorkshire Hospitals steve.holding@hey.nhs.uk +44 (0)1482 607710
Declaration of interest Biorad Laboratories have contributed to costs to enable SH to attend The 17 th International Vasculitis & ANCA Workshop.
Consensus Statement Consensus statement (1999) Addendum to Consensus Statement (2003) 1. Patient selection 2. Structured testing protocol 3. Defined methods and quality control 4. Appropriate report comments Savige J et al. Am J Clin Pathol 1999;111:507-513 Savige J et al. Am J Clin Pathol 2003;120:312-318
Consensus Statement Minimum: Test all samples by IFT on ethanol fixed neutrophils. All new positive IFT must have PR3-ANCA and MPO-ANCA performed. Savige J et al. Am J Clin Pathol 1999;111:507-513
Test Performance Proportion of patients with AAV IFT Pos Neg Total EIA Pos 72% 8% 80%* Neg 9% 11% 20% Total 81% 19% 100% *between 76% and 84% Based on data extracted from: Hagen EC et al. Kidney International 1998;53:743-753 Savige J et al. Am J Clin Pathol 1999;111:507-513
Changes Since the Consensus Documents IFT is essentially unchanged Microscopes have improved More use of standardised reagents and commercial slides PR3/MPO-ANCA methods Improved comparability between assays though quantitative results are not comparable. Direct EIA (antigen coated solid phase) Capture EIA (typically solid phase antibody capture of a soluble antigen) Anchor or hseia (using a bridging molecule to attach the antigen to the solid phase) Improvements in automation Multiplex assays may provide additional antigens in the future Revised Chapel Hill consensus on nomenclature of Vasculitides (Arthritis Rheum 2013;65:1-11)
Hull ANCA testing protocol Screen using IFT. Euroimmun ANCA slides (ethanol fixed neutrophils and separate HEp2 cells). PR3-ANCA and MPO-ANCA on all positive IIF-ANCA (C-ANCA, P-ANCA, atypical ANCAs and ANA positive). Biorad BioPlex 2200 multiplex assay
Assessing the effect of changing to EIA 1 st (i) Retrospective review of outcomes from IFT positive, EIA negative samples. The patients that would be missed in an EIA 1 st approach. (ii) Retrospective review of clinical outcomes for all ANCA test requests. Enables accurate assessment of sensitivity in a specific clinical setting. (iii) Assessment of specificity in routine use.
EIA Sensitivity for AAV - Hull Study (i) Retrospective review of outcomes from IFT positive, EIA negative samples All IFT positive, EIA (PR3/MPO) negative samples: All screens from current protocol (02/2011 to 02/2014): 8985 consecutive IFT ANCA screens 4060 consecutive PR3/MPO-ANCA tests 101 C-ANCA (69 EIA +ve) & 36 P-ANCA (14 EIA +ve) Findings: 32 C-ANCA +ve, EIA ve. 3 known AAV (3 patients), 29 no evidence of AAV. 22 P-ANCA +ve, EIA ve. 3 known AAV (2 patients), 19 no evidence of AAV. All 5 of the known patients had positive EIA at presentation. 452 atypical ANCA, EIA ve (random selection of 89 reviewed). 36 known AAV (16 patients), 53 no evidence of AAV. All 16 known AAV patients had positive EIA at presentation. Conclusion: No missed new diagnoses if EIA 1 st approach used.
Sensitivity for AAV - Hull Study (ii) Retrospective review of outcomes for all ANCA tests Sample source mainly rheumatology, renal, chest medicine, ENT, dermatology (review of diagnosis for all ANCA requests): Prospective testing of samples, diagnosis from casenotes. 16 new patient presentations with AAV (10 GPA; 3 MPA; 3 EGPA) n IFT Clinical Sensitivity PR3- ANCA PR3/MPO-ANCA MPO- ANCA PR3 or MPO ANCA GPA 10 90% (100%) 70% 30% 100% MPA 3 0% (100%) 0% 100% 100% EGPA 3 0% (33%) 0% 33% 33% Any AAV 16 56% (88%) 44% 44% 88%
Summary Sensitivities IFT 1st EIA 1st n GPA 100% 100% 10 MPA 100% 100% 3 EGPA 33% 33% 3 All AAV 88% 88% 16 Conclusion: IFT 1 st & EIA 1 st protocols have equivalent sensitivity.
Specificity (iii) Assessment of specificity for EIA 1 st in routine use 343 patient samples tested for PR3/MPO-ANCA by Biorad BioPlex 2200 Luminex based Multiplex assay. 17 positive by EIA 8 with a diagnosis of AAV 9 with no evidence of AAV Specificity = (343-8)-9 (343-8) = 97% (95% confidence interval = 95% - 99%) Conclusion: Specificity of an EIA 1 st protocol is very good
PR3/MPO-ANCA is as effective as immunofluorescence as a first-line test in AAV screening. Summary Studies informing the Consensus Statement showed similar efficacy for IFT 1 st and EIA 1 st approaches. EIA method reliability, sensitivity and comparability have improved since the consensus statement was agreed (i) Retrospective review of outcomes from IFT positive, EIA negative samples showed no clinically significant false negative EIA results. (ii) Retrospective review of outcomes for all ANCA tests Showed sensitivity of EIA 1 st & IFT first protocols to have similar sensitivity for AAV (88%). (iii) Assessment of specificity in routine use Specificity for AAV was excellent (97%).
Acknowledgements Dr Mohamed Abuzakouk Immunology & HYMS, Hull Dr Sathish Kallankara Rheumatology, Hull Dr Tim Rowland Chest Medicine, Hull Dr Vicky Fisher Vascular Surgery, Hull Dr Simon Hart Chest Medicine & HYMS, Hull Hull Hospitals Patient Coding Team Hull Hospitals/HYMS Vasculitis Research Group