immunofluorescence tests in routine laboratories Dr. Dirk Roggenbuck

Transcription

1 Standardization di ti of cellbased immunofluorescence tests in routine laboratories Dr. Dirk Roggenbuck

2 Overview Automatic interpretation system AKLIDES - basis for standardization Cell-based immunofluorescence tests Microbead-based immunofluorescence tests Outlook

3 Cell-based IFT

4 Cell-based IFT Detection of anti-nuclear antibodies (ANA) on substrate: HEp-2 Detection of anti-dsdna antibodies substrate: Crithidia luciliae Detection of anti-granulocyte antibodies (ANCA) substrate: humane granulocytes

5 Benefits of cell-based IFT Assay yp procedure simple, partially adaptable to automation Assay time Multiple staining <2 h simple or slightly laborious Sensitivity high Availability Costs since 1978 commercially available small (murine organs, cell lines, native blood cells) Multiplex Assay

6 HEp-2 cell as multiplex substrate

7 Drawbacks of cell-based IFT Specificity age-related Quantification Reproducibility Standardisation Antigens Automation Data processing semi-quantitativ (titre) one titre step insufficient variable antigen expression, variable fixation insufficient interpretation manual

8 Interlaboratory comparison ANA

9 AKLIDES First automatic reading system for the evaluation of cell-based IFT (ANA, ndna, ANCA) Qualitative evaluation + pattern recognition of ANA and Crithidia luciliae Titre prediction Standardisation, calibration Electronic data processing, storage Connection to LIS

10 AKLIDES

11 AKLIDES Modular system configuration Automatic image acquisition with simultaneous data processing Fast evaluation (less than one minute per well) Four slides per 16 wells or microtitre plate (five slides holder coming soon)

12 AKLIDES modules

13 AKLIDES work flow 1. positive/negative basis: fluorescence signal in RU 2. pattern recognition basis: defined mathematical a algorithms

14 AKLIDES pattern recognition Pattern assessment by comparing DAPI and FITC signals: DAPI FITC cytoplasm nucleus

15 AKLIDES pattern recognition DAPI FITC homogeneous nucleolar speckled centromere dots cytoplasmic

16 AKLIDES pattern recognition speckled fluorescence peripheral fluorescence 200 image-based, abstract characteristics shape area texture intensity topology

17 AKLIDES DAPI staining

18 AKLIDES FITC staining

19 AKLIDES DAPI + FITC

20 AKLIDES pattern recognition homogeneous nucleolar mitotic pattern speckled centromer nuclear dots Sack U et al. for the German EASI (European Autoimmunity Standardization Initiative). Autoantibody detection using indirect immunofluorescence on HEp-2 cells. Ann N Y Acad Sci 2009;1173: Dtsch Med Wochenschr :

21 AKLIDES references

22 AKLIDES man vs. computer Chess remains the most visible and public benchmark of the relentless increase in computer speed over the last 50 years.

23 AKLIDES evaluation distribution of cones on the retina spectral sensitivity of the human eye (Department of Psychology, North Dakota State University)

24 1:80 1:320 1:640 1:1280 1:2560 1:5120 Centromere Centromere + dsdna Sp100 Sp100 + dsdna dsdna

25 AKLIDES evaluation Comparison: human vs. machine AKLIDES expert ,6% 4,1% + 2,2% 9,0% AKLIDES agreement of negative sera agreement of positive sera expert A 97,59% 95,73% expert B 90,36% 94,87% expert tc 86,75% 94,02%

26 AKLIDES evaluation Serum reactivity number AKLIDES positive % Anti-dsDNA Anti-CENP CENP-B Anti-histone Anti-nuclesome nuclesome Anti-Ro Anti-Ro Anti-Scl Scl Anti-U1 U1-RNP/Sm Anti-PDH PDH-E

27 AKLIDES evaluation Serum reactivity number AKLIDES positive % Anti-Jo Jo Anti-CENP CENP-F Anti-Golgi Anti-NuMA Anti-Mi Anti-rib rib-p Anti-Sp Anti-SRP SRP Anti-MSA 1 100

28 AKLIDES evaluation

29 AKLIDES Evaluation 924 samples - Universitätsmedizin Charité, Rheumatology laboratory, Prof. Burmester 298 samples privates referral lab Münster Comparison of automated and visual interpretation of ANA detection by IIF in routine laboratories

30 AKLIDES Evaluation Münster A utomated interpret tation positive Visual interpretation weak positive negative n 57 positive (19.1%) 1%) weak positive negative (5.4%) 225 (75.5%) n 44 (14.8%) 47 (15.8%) 207 (69.4%) 298

31 AKLIDES Evaluation Charité Visual interpretation Aut tomated in nterpretat tion weak positive positive negative n positive weak positive (59.1%) 140 (15.1%) 238 negative (25.8%) n 140 (15.1%) 556 (60.2%) 228 (24.7%) 924

32 AKLIDES Evaluation Charité/Münster Visual interpretation Automatic reading by AKLIDES

33 AKLIDES Evaluation Very good agreement, kappa=0.828 Only 8% (98) differing results out of 1222 samples for positive/negative differentiation 111 (15.3%) samples demonstrated differing results for pattern recognition

34 AKLIDES references Hiemann R et al. Objective quality evaluation of fluorescence images to optimize automatic image acquisition, Cytometry y A 2006;69A: Hiemann R et al. Automatic analysis of immunofluorescence patterns of HEp-2 cells. Ann N Y Acad Sci 2007;1109: Hiemann R et al. Challenges of automated screening and differentiation of non-organ specific autoantibodies on HEp-2 cells. Autoimmun Rev 2009;9:17-22 Egerer K et al. Automated evaluation of autoantibodies on human epithelial-2 cells as an approach to standardize cell-based immunofluorescence tests. Arthr Res Ther 2010;12:R40 Kivity et al. A novel automated indirect immunofluorescence autoantibody evaluation. Clin Rheumatol 2011; DOI /s

35 AKLIDES evaluation assessment of Intraassay variability: CV%=SD/MW??? AKLIDES Resultat

36 AKLIDES evaluation 1:80 1:160 1:320 1:1280 Serum: ACA CV%

37 AKLIDES evaluation assessment of Interassay variability: CV%=SD/MW n x??? AKLIDES Resultat 603

38 AKLIDES evaluation 80 Inter-assay Variablity 60 % CV Intensity Value (Light Units)

39 AKLIDES summary Interpretation of cell-based IFT can be run automatically Pattern recognition of ANA on HEp-2 can be standardized Overcoming of critical drawbacks of IFT: Automation Standardisation Data processing

40 AKLIDES outlook Platform technology for cell- based immunofluoresecence and multiplex tests

41 Pattern recognition technology Camera Pattern recognition software Camera Bead-Assay Integral Ligand Cell-Assay 2,5E+06 AO 1:400 Bb-gekoppelt AO 1:200 HSA-gekoppelt AO 1:100 La/SS-B-gekoppelt AO 1:100 Ye-gekoppelt 2,0E+06 1,5E+06 Inte egral 1,0E+06 5,0E+05 0,0E+00 L527 (Bb-pos) L1188 (Bb-neg) L1314 (HSA) L510 (HSA) L7 (Ye-pos) L2 (Ye-neg) Serum Diagnosis

42 AKLIDES encoded mircrobeads Diameter: 5-25 µm +/- 1 µm Backbone: e.g. PMMA, thermostable Ati Activation: Carboxy, Amino, Epoxy Populationen: 20 available Encoding: 2 dyes, 3 dyes possible rel. Fluoresc cence 100 : 1 rel. Fluoresc cence 50 : 50 rel. Fluoresc cence 1 : 100 wave length [nm] wave length [nm] wave length [nm]

43 AKLIDES antibody bead assay rel. fluo orescence rel. fluo rescence wave length [nm] wave length [nm]

44 AKLIDES bead assay

45 AKLIDES bead assay

46 AKLIDES bead-based assay Application example: ANA-Bead Assay Antigen CENP-B La/SS-B Ro60 Ro52 Scl-70 dsdna Jo1 RNP/S m Correct positive 14/15 12/14 16/17 11/11 10/13 13/13 3/3 10/14 Relative 93% 86% 94% 100% 77% 100% 100% 71% sensitivity Correct negative 39/39 38/39 39/39 39/39 39/39 36/38 39/39 39/39 Relative sensitivity 100% 97% 100% 100% 100% 95% 100% 100% [1] Großmann K, Hiemann R, Berger I, Böhm A, Nitschke J, Tanneberger B, Conrad K, Roggenbuck D, Sack U, Lehmann W. Die universelle Plattform BioResponse für die kombinierte zelluläre und partikelbasierte Analytik. In: Conrad K, Lehmann W, Sack U, Schedler U (Hrsg.). Multiparameteranalytik-Methoden, Applikationen, Perspektiven. Lengerich. Pabst Science Publishers [2] Hiemann R, Hilger N, Michel J, Nitschke J, Böhm A, Anderer U, Weigert M, Sack U. Automatic analysis of immunofluorescence patterns of HEp-2 cells Ann. N.Y. Acad. Sci. 1109:

47 AKLIDES summary First all-in-one solution for multi-colour fluorescence detection: 1) multiparameter assays (microbead or/and cell array) for diverse application areas 2) image based analyses of fluorescence signals 3) one equipment unit for fluorescence detection using standard components and formats

48 AKLIDES outlook free radicals radiation chemotherapy double strand break (DSB) biosensors Proteinkinase ATM cell death repair

49 AKLIDES outlook Immunofluorescence microscopy of PC Cl3-cells after radiation with 188 Re (60 x).

50 AKLIDES outlook P P

51 AKLIDES outlook Detection of genetic damage Biological dosage measurement for X-ray investigations Tissue specific biomarker for the susceptibility of the DNA Pharmacological research

52 Thank you for your attetntion!