Quality in noninfectious

Transcription

1 Quality in noninfectious serology

2 Willy Vandersteen

3 o Pre-analysis o Method o Pipetting and incubation station o Reagents o Microscopy/Signal detection o People o Informatics o Post-analysis o Murphy? o Method» Validation/Verification o Training of personnel o Monitoring o Short term o Long term

4 Choice of method Reagent Lot change QC First level - IQC Second level Third level - EQC Key Performance Indicators

5 Choise of a new method National Symposium National on Symposium Non-Infectious Non-Infectious Serology 2015 Serology 2015

6 Performance Practical considerations EQC Literature Colleages Personnel and organisation Available equipment Reagent stability Sample number Frequency of analysis Turn-around time Finances

7 » 5 most important patterns: 5 sera/pattern (known diagnosis)» Titration» 10 negative sera» Reproducibility homogeneous speckled nucleolar centromere cytoplasmatic

8 » 3 patterns: 5 sera/pattern (known diagnosis)» 10 negative sera» Reproducibility» Titration C-ANCA P-ANCA Atypical ANCA

9 Qualitative Quantitative min 5 sera/antigen 10 negatives Routine in duplo Reproducibility 10 patiënts/antigen Concentration range Routine in duplo (Evaluation cut-off) Reproducibility

10 Lot change

11 important patterns: 1 serum/pattern 1 negative serum 1 titration homogeneous speckled nucleolar centromere cytoplasmic C-ANCA P-ANCA atypical ANCA

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14 » 1/antigen or 1 pool of antigens» 1 negative serum

15 IQC jump control material comparison patient samples if clinically relevant contact supplier

16 Patient DWR Patient DWR

17 Quality control National Symposium National on Symposium Non-Infectious Non-Infectious Serology 2015 Serology 2015

18 Control 200+/-30 Patients high Control 200+/- 30

19 1. Positive control - IF: 1 important pattern - immuno-assay: 1/antigen, alterning in case of multiplex - Dilution and handling: exactly equal to patient samples - Origin: patiënt or commercial, preferentially with known pathology stability: 4 C 0.1 g/% Na azide/ frozen aliquots - Titration/Intensity score 2. Negative control if small series 3. Known patients: IF pattern/specificity/intensity

20 Each run: start and end Material: commercial or patient Acceptance criteria: company or laboratory

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22 IQC Automatic microscope: role for the fluorescence intensity? National Symposium National on Symposium Non-Infectious Non-Infectious Serology 2015 Serology 2015

23 » Reproducibility Within-run o Speckled 1,4% G-sight Bonroy, Clin Chem Lab Med 2013 o Homogeneous 10,9% o Centromere 16% o Nucleolar 5% o SSA 15% o Scl % company-dependent Bizarro, JAutoimmunity Rev 2014 G-sight, NovaView, ImageNavigator, EuroPattern, Aklides Between run o Homogeneous 16% G-sight Bonroy, Clin Chem Lab Med 2013 o Negative control 48% Between-labs? to be developed» End-point titers

24 Zenit G sight Maenhout, Bonroy et al. Clin Chem Lab Med 2014; Bossuyt, Cooreman et al Acta Clinica Chimica Acta 2013; 2014

25 Nova view Schouwers Clin Chem Lab Med 2013

26 EUROIMMUN reagents Peng, Tang et al. J Immunol Methods National Symposium National on Symposium Non-Infectious Non-Infectious Serology 2015 Serology 2015

27 » Statistics on parameters that regulate sensitivity of the camera» Time to focus related to 1/ fluorescence intensity National Symposium National on Symposium Non-Infectious Non-Infectious Serology 2015 Serology 2015

28 nucleolar Westgard rules Warning 1 S2 CV= SD x 100 mean CV 2 % Rejection 2 S2 1 S3 R 4S CV 41% Maenhout, Bonroy et al. Clin Chem Lab Med 2014

29 Maenhout, Bonroy et al. Clin Chem Lab Med 2014

30 Standardization between instruments o Intensity o Cut-off for positivity

31 Personnel: tuning and evaluation of competence 1/year

32 » = Proficiency testing» External QC» Frequency» - minimum WIV/IPH programm (RIZIV/INAMI)» - preferentially 1 programm/test/year» (ISO accreditation)

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34 1 S3 2 S2 2 S2 1S3 Maenhout, Bonroy et al. Clin Chem Lab Med 2014

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36 Positive/ total (%) ?

37 ? Homogeneous Speckled Centromere Cytoplasmatic Nucleolar

38 Different Hep2 cells Different ENA blot DNA ENA % Expected Ag % DNA ENA % Expected Ag % homogeneous 21 speckled 16 centromere 85 7 dna histones 13 SSA SSB Ro52 RNP SmD ribp Scl70 79 Cenp B dna histones 25 SSA SSB Ro52 RNP SmD PM/Scl Scl cenp B cytoplasmatic 8 1 Jo Jo1 M2 nucleolar 7 4 RNP Scl70 1 0,25 Scl70

39 Marc Sleen