Quality in non- infectious serology

Quality in noninfectious serology
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Willy Vandersteen
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
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
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
• Choice of method
• Reagent Lot change
• QC
• First level - IQC
• Second level
• Third level - EQC
• Key Performance Indicators
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Choise of a new method
Martine Vercammen
National Symposium
onSymposium
Non-Infectious
Serology 2015
National
on Non-Infectious
Serology 2015
Performance
Practical considerations
•
EQC
•
Personnel and organisation
•
Literature
•
Available equipment
•
Colleages
•
Reagent stability
•
Sample number
•
Frequency of analysis
•
Turn-around time
•
Finances
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
»
»
»
»
5 most important patterns: 5 sera/pattern (known diagnosis)
Titration
10 negative sera
Reproducibility
homogeneous
speckled
nucleolar
centromere
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
cytoplasmatic
»
»
»
»
3 patterns: 5 sera/pattern (known diagnosis)
10 negative sera
Reproducibility
Titration
C-ANCA
P-ANCA
Atypical ANCA
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Qualitative
Quantitative
•
min 5 sera/antigen
•
≥ 10 patiënts/antigen
•
10 negatives
•
Concentration range
•
Routine in duplo
•
Routine in duplo
•
Reproducibility
•
(Evaluation cut-off)
•
Reproducibility
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Lot change
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
important patterns: 1 serum/pattern
1 negative serum
1 titration
homogeneous
C-ANCA
speckled
P-ANCA
nucleolar
centromere
atypical ANCA
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
cytoplasmic
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
» 1/antigen or 1 pool of antigens
» 1 negative serum
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
IQC jump control
material
comparison
patient samples
if clinically relevant
contact supplier
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Patient DWR
Patient DWR
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Quality control
Martine Vercammen
National Symposium
onSymposium
Non-Infectious
Serology 2015
National
on Non-Infectious
Serology 2015
Control 200+/-30
Patients high
Control 200+/- 30
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
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
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
• Each run: start and end
• Material: commercial or patient
• Acceptance criteria: company or laboratory
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
IQC Automatic microscope:
role for the fluorescence
intensity?
Martine Vercammen
National Symposium
onSymposium
Non-Infectious
Serology 2015
National
on Non-Infectious
Serology 2015
» Reproducibility
˃ Within-run
o Speckled
o Homogeneous
o Centromere
o Nucleolar
o SSA
o Scl-70
1,4%
G-sight
10,9%
16%
5%
15%
2 - 25% company-dependent
Bonroy, Clin Chem Lab Med 2013
Bizarro, JAutoimmunity Rev 2014
G-sight, NovaView, ImageNavigator, EuroPattern, Aklides
Between run
o Homogeneous 16%
o Negative control 48%
G-sight
Bonroy, Clin Chem Lab Med 2013
˃ Between-labs? to be developed
» End-point titers
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Zenit G sight
Maenhout, Bonroy et al. Clin Chem Lab Med 2014; Bossuyt, Cooreman et al Acta Clinica Chimica Acta 2013; 2014
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Nova view
Schouwers Clin Chem Lab Med 2013
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
EUROIMMUN reagents
Peng, Tang et al. J Immunol Methods
Martine Vercammen
National Symposium
onSymposium
Non-Infectious
Serology 2015
National
on Non-Infectious
Serology 2015
» Statistics on parameters that regulate sensitivity of the
camera
» Time to focus related to 1/ fluorescence intensity
Martine Vercammen
National Symposium
onSymposium
Non-Infectious
Serology 2015
National
on Non-Infectious
Serology 2015
nucleolar
Westgard
rules
CV= SD x 100
mean
Warning 1S2
CV 2 %
Rejection
2S2
1S3
R4S
CV 41%
Maenhout, Bonroy et al. Clin Chem Lab Med 2014
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Maenhout, Bonroy et al. Clin Chem Lab Med 2014
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Standardization between instruments
o Intensity
o Cut-off for positivity
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Personnel: tuning and evaluation of competence 1/year
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
» = Proficiency testing
» External QC
» Frequency
» - minimum WIV/IPH programm (RIZIV/INAMI)
» - preferentially 1 programm/test/year
»
(ISO 15189 accreditation)
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
1S3
2S2
2S2
1S3
Maenhout, Bonroy et al. Clin Chem Lab Med 2014
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
2008
2009
2010
2011
2012
2013
2014
39
36
23
23
22
Positive/
total (%)
?
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
?
Homogeneous
Speckled
Centromere
Cytoplasmatic
Nucleolar
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Different Hep2 cells
Different ENA blot
2010
2014
DNA ENA
%
Expected Ag
%
DNA ENA
%
Expected Ag
%
21
7
dna histones
29
12
dna histones
speckled
16
13
SSA SSB Ro52
RNP SmD ribP
Scl70
36
25
SSA SSB Ro52
RNP SmD
PM/Scl Scl70
centromere
85
79
Cenp B
100
100 cenp B
cytoplasmatic
8
1 Jo 1
17
10 Jo1 M2
nucleolar
7
4 RNP Scl70
1
0,25 Scl70
homogeneous
Martine Vercammen
National Symposium on Non-Infectious Serology 2015
Marc Sleen
National Symposium on Non-Infectious Serology 2015
National Symposium on Non-Infectious Serology 2015
Martine Vercammen