Post on 11-Mar-2018
STATO ATTUALE DELLE TECNOLOGIE DIAGNOSTICHE PER LA MISURA DI AUTOANTICORPI: SOTTO IL SEGNO DELL’AUTOMAZIONE
Renato Tozzoli
Laboratorio di Patologia Clinica
Dipartimento di Medicina di Laboratorio
Azienda Ospedaliera ‘S. Maria degli Angeli’, Pordenone
THE EVOLUTION OF AUTOIMMUNE DIAGNOSTICS
1. The evolution of the role (diagnostic, prognostic, predictive) of circulating autoantibodies
2. The evolution of clinical immunology laboratories: from academic research to autoimmunity laboratories
3. The evolution of immunoassays for autoantibody detection: from 1st, to 2nd and 3rd generation
4. The evolution of immunology laboratory specialists
The evolution of clinical immunology laboratories: from academic research to autoimmunology laboratories
Clinical Immunology Laboratory Autoimmunology Laboratory
Diagnostic, predictive, prognostic roles of
autoantibodies
Consolidation and healthcare
economics
Evolution of diagnostic
technologies
New knowledge in clinical research
New knowledge in basic research
The evolution of autoimmunology labs: consolidation and healthcare economics
Nakamura RM, Bylund DJ.
Factors influencing changes in the Clinical Immunology Laboratory
Clin Chem 1994; 40: 2193-2204
The major factors that will influence future laboratory test are:
A) governmental regulations
B) healtcare economics
C) advances in technology
D) advances in development and use of therapeutic agents
Hoffmann GE.
Concepts for the third generation of laboratory systems
Clin Chim Acta 1988; 278: 203-16
The two key concepts of third generation systems are consolidation and integration….
Consolidation: Combining different analytical technologies or strategies on one instrument or on one group of connected instruments.
Integration: Linking analytical instruments of groups of instruments with pre- and post-analytical devices
The evolution of autoimmunology labs: consolidation and automation
Van Lente F.
Methodology and subspecialty consolidation
in the Clinical Laboratory
Clin Chem 1997; 43: 11-2
“ Automation of sample handling as well as of instrument loading and unloading can then be maximally effective. Consolidating these processes should challenge the inefficiences of the subspecialty approach. The ability to consolidate assays for performance by the fewest number of analytical instruments can only facilitate efficiency of laboratory operations”.
Tomar R.
Total laboratory automation and diagnostic immunology
Clin Diagn Lab Immunol 1999; 6: 293-4
“ The number of total laboratory automation (TLA) installations is growing but TLA has only begun to impact diagnostic immunology…..
The reasons that diagnostic immunology is not more automated, relate to the lack of standardized assays and interpretations, the low volume of such test relative to those in other laboratory sections, and the high risk/benefit ratio of investing in development……”
The evolution of autoimmunology Labs: trend of test requests
(500.000 inhabitants, West Friuli-Venezia Giulia)
No. of autoimmunity tests 1997 a
(%)
2001 b
(%)
2007 c
(%)
2012 d
(%)
<5.000 50.0 45.0 42.0 6.8
5.001-10.000 21.5 23.0 20.0 15.6
10.001-50.000 28.5 31.0 34.0 47,6
50.001-100.000 0 2.0 4.0 18,4
>100.000 0 0 0 11.6
a 1° Survey GdS-AI - 44 labs (Villalta, et al) b 2° Survey GdS-AI - 43 labs (Tozzoli, et al) c 3° Survey GdS-AI - 30 labs (Tampoia, et al) d 4° Survey GdS-AI-325 labs (Bizzaro,et al)
Evolution of immunoassay methods for the detection and measurement of autoantibodies
Tozzoli R, et al. Clin Chem Lab Med 2013;51:129-38
1st generation monoplex
Double immunodiffusion ID
Complement fixation CF
Indirect immunofluorescence IIF
Passive agglutination PHA, LPA
Radio-immunoprecipitation RIPA
Westernblot WB
2nd-3rd generation monoplex
Radioimmunoassay-Immunoradiometric assay RIA-IRMA
Radio-receptor assay RRA
Immunoenzymatic assay-Immunoenzymometric assay ELISA-IEMA
Immunoblot IB
Immunodot DB
Chemiluminescence immunoassay CLIA-ILMA
Fluorescence immunoassay FIA-IFMA
Multiplex
Addressable microbeads immunoassay MBA
Planar (membranes, glass slides) immunoassay PMIA
Automation in autoimmune testing
1. The automation of indirect immunofluorescence: the telepathology (telemedicine)
2. The advent of automated monoplex platforms: the reduction of turnaround time
3. The progressive diffusion of monoplex methods: the autoantibody profiling
The automation of IIF:
the telepathology (telemedicine)
L’AUTOMAZIONE DEI METODI DI
IMMUNOFLUORESCENZA INDIRETTA
• L’automazione della procedura analitica
• L’automazione della lettura/interpretazione
• La telemedicina e la telediagnosi
Automation in slide preparation
82% of Italian Labs in 2012
Courtesy by: N. Bizzaro, 2014
Automation for ANA-IIF reading Bizzaro N. et al, Autoimmun Rev 2014
Automated IIF: PubMed reports (43)
ANA IIF automated/manual Positive/Negative Agreement (PNA)
System Studies (no.) Patients (no) PNA (mean)
Aklides 3 1801 0.95
Zenit G-Sight 3 830 0.92
Nova View 2 842 0.95
Europattern 2 565 0.96
Image Navigator 1 3185 0.99
Helios 1 1005 0.98
All systems 1 126 0.94-0.99
Total 13 8377 0.97
ANCA IIF automated/manual positive/negative agreement (PNA)
Year Author System Patients (no) PNA
2012 Damoiseaux Aklides 79 0,80
2012 Knutter Aklides 342 0,94
2012 Melegari Aklides 46 0.89
2014 Sowa Aklides 90 0,83
Total/mean 467 0.87
DNAAb IIF automated/manual positive/negative agreement (PNA)
Year Author System Patients (no) PNA
2012 Melegari Aklides 44 0,91
ANA-IIF: Riconoscimento del pattern Bizzaro N. et al, Autoimmun Rev 2014
Sistema Pattern (no.)
Tipo
Aklides 6 Omogeneo, granulare, nucleolare, centromerico, citoplasmatico, nuclear dots
Nova-view 6 Omogeneo, granulare, nucleolare, centromerico, citoplasmatico, nuclear dots
EuroPattern 6 Omogeneo, granulare, nucleolare, centromerico, citoplasmatico, nuclear dots
G-Sight 5 Omogeneo, granulare, nucleolare, centromerico, citoplasmatico
Helios - -
Image Navigator - -
Considerazioni sull’impiego dell’automazione in IIF Bizzaro N. et al. Autoimmun Rev 2014
19
Automated interpretation systems: the role of computer-aided diagnosis (CAD) and artificial neural network (ANN).
It’s the time of telediagnosis?
It’
• Pathology –Direct Immunofluorescence
• Radiology – Mammography
• Nuclear Medicine – Scintigraphy
• Cardiology – Electrocardiography
• Opthalmology – Digital photography
• Thyroidology – Ultrasonography
The advent of automated monoplex
platforms: the reduction of TAT
I livelli di automazione dei metodi immunologici commerciali per la misura di autoanticorpi
Manuali A tubi sensibilizzati
A micropiastre
Ad automazione parziale (a moduli) A micropiastre
Ad automazione totale Dedicati ad analiti eterogenei
Dedicati ad analiti omogenei (solo autoanticorpi)
L’AUTOMAZIONE DEI METODI IMMUNOMETRICI MONOPLEX
• La sostituzione dei metodi radioisotopici
• L’avvento dei metodi a tracciante ‘ecologico’
• L’automazione della procedura analitica
EIA/ELISA vs RIA: the rise and fall of monoplex IMA
Lequin RM. Clin Chem 2005;51:2415-8
Chemiluminescence immunoassay: main advantages
Large linear response (up to 6 orders of magnitude)
High sensitivity
Fast light emission in a single flash/no need for time resolved detection
High stability of reagents
Low consumption of expensive reagents
Short incubation times
Full compatibility with automated homogeneous, heterogeneous, competitive or non-competitive immunoassay
Absence of toxicity
A selected hystory of automated immunossays (1996-2014)
Year Instrument Company Immunoassay
1996 Architect Abbott Laboratories CLIA
1997 ACS: Centaur Chiron Diagnostics CLIA
1997 Elecsys Boehringer Mannheim ECLIA
1998 Kryptor Cis-Bio TRACE
1999 Vitros ECi Ortho Clinical Diagnostics CLIA
1999 Liaison Byk-Sangtec Diagnostica CLIA
2008 Dimension Vista Siemens LOCI
2009 iSYS Immunodiagnostic Systems CLIA
2012 Maglumi Snibe CLIA
Automated CLIA Instruments and Autoimmunity Bizzaro N, Tozzoli R, Villalta D. Autoimmune Diagnostics. CRAI 2014 (in press)
Instrument Company Tracers Tests menu Refs (no.)
Immulite 1000/2000/ XPI
Cirrhus, DPC, Bayer, Siemens
Alkaline Phosphatase Adamantyl 1,2-Dioxetane phosphate (AMPPD)
TgAb, TPOAb, Anti-CCP, TRAb 7
Access/Access 2/ Unicel Dxi 600/800
Sanofi, Beckman-Coulter
Horseradish Peroxidase Dioxetane phosphate (Lumi-Phos 530)
IFAb,TPOAb TgAb -
Architect i1000/2000/4000
Abbott Acridinium esters CCPAb, TgAb, TPOAb 2
ADVIA Centaur/CP/XP
Chiron, Bayer, Siemens
Acridinium esters TgAb, TPOAb, CCPAb 2
Automated CLIA Instruments and Autoimmunity Bizzaro N, Tozzoli R, Villalta D. Autoimmune Diagnostics. CRAI 2014 (in press)
Instrument Company Tracers Tests menu Refs (no.)
Liaison/Liaison XL DiaSorin Isoluminol derivatives ANA, dsDNAAb, tTG-IgA, ENAAb, aCL-IgG,IgM, TgAb, TPOAb
4
Elecsys/Cobas Modular
Boehringer Mannhein, Roche
Rutenium tris-bipyridyl TgAb, TPOAb, TRAb, CCPAb 10
Maglumi 600/1000/2000/2000 Plus/4000
SNIBE ABEI-FITC TgAb, TPOAb, TRAb, ICA, IAA, GADAb
2
Zenit RA A. Menarini Acridinium Esters ANA, ENA, tTG-IgG,IgA, DGP-IgG, IgA, aCL-IgG,IgM, B2GPI-IgG-IgM, CCPAb, MPOAb, PR3 Ab
5
Instrument Company Tracers Tests menu Refs (no.)
BioFlash Inova/IL Isoluminol aCL-IgG,IgA,IgM tTG- IgGA, IgG DGP-IgA,IgG MPOAb, PR3Ab, GBMAb B2GPI-IgG, IgA, IgM ENA, ANA DFS70Ab ASCA-IgG,IgA F-ActinAb LKMAb, SLAAb, SP100Ab, gp210Ab, M2Ab
12 Lakos G, et al 2014 Webb T, et al, 2014 Mondejar R, et al. 2014 Infantino M, et al 2013 Miyara M, et al 2013 Noel N, et al.2013 Mahler M, et al. 2013 Bentow C, et al. 2013 Mahler M, et al. 2013 Mahler M, et al. 2012 Mahler M, et al. 2012 Mahler M, et al. 2012
Automated CLIA Instruments and Autoimmunity Bizzaro N, Tozzoli R, Villalta D. Autoimmune Diagnostics. CRAI 2014 (in press)
The role of automated platforms:
• increased workload in the autoimmunity lab
• higher throughput
• rapid testing and response (reduction of TAT)
• standardization of analytical processes
• complete traceability
• cost containement
Cortesy by: N. Bizzaro
Autoimmune diagnostics in 2014. New technologies have the potential to change
the way we do autoimmune testing
The progressive diffusion of multiplex methods: the autoantibody profiling
L’AUTOMAZIONE DEI METODI IMMUNOMETRICI MULTIPLEX
• La sostituzione dei metodi monoplex
• L’avvento dei metodi a tracciante ‘ecologico’
• L’automazione della procedura analitica
From monoplex to multiplex IMA Ekins RP. Clin Chem 1991-1998
Planar and Nonplanar Microarrays
for Autoantibodies (Tozzoli R, et al. CCLM 2013)
Support Format Detection
system AutoAbs
tested Estimated
throughput Reference
Microbeads Wells FL 9 1.000 Rouquette, 2003
Al-barcoded microparticles
Wells FL 10 10.000 Pang, 2005
NC membranes Microspots CL 30 1.000 Hentschel, 2004
NC membranes Nanodots CL 10 1.000 McBride, 2008
Polystyrene chips Microspots FL 20 10.000 Klause, 2007
Glass slides Microspots FL 225 10.000 Hueber, 2005
Glass-slides gold-coated
Microspots SPR 24 10.000 Segbers-Lokate,
2007
Multiplex bead array (MBA): le soluzioni commerciali
Multiplexing: analytical systems and diffusion in Italy (2004-2013).
Tozzoli R, et al. CCLM 2013 (modified)
Society System Autoantigenic profiles (no.) Evolution Diffusion in Italy
BMD, France
FIDIS (Luminex)
ANA (10), Vasculitis (3) Celiac (2), Thyroid (2), APS IgG, IgM (3), Gastritis (2), Liver (3)
++++ +/-
IL (INOVA), Spain-USA
QuantaPlex (Luminex)
ANA (8), Celiac (2) ? -
ZEUS, USA
Athena Multi-Lyte (Luminex)
ANA (10), Vasculitis (3) Celiac (2), Thyroid (2)
++ +/-
Origene, USA
TruePlex (Luminex)
ANA (5), OSADS (5), Customizable ++ -
BIO-RAD, USA
Bioplex 2200 (Luminex)
ANA (13), Vasculitis (3), RA (1), APS IgG, IgA, IgM (6), Celiac IgA, IgG (5)
+++ +
Pronostics, UK
UltraPlex ANA (2), Thyroid (2) ? -
ImmunoConcept, USA
Microzyme ANA (10) ? -
SQI Diagnostics Canada
Ig-Plex (Luminex)
ANA (11), RA (4), Vasculitis (3), Celiac IgA, IgG (4)
++ -
Most relevant studies of ANA/ENA detection in SARDs with Bio-Plex 2200
Tozzoli R, Villalta D. Autoimmun Rev 2014 (modified)
Aabs Author Year Journal Article
ANA/ENA Desplat-Jego S, et al 2007 Ann N Y Acad Sci 1
ANA/ENA Moder KG, et al 2007 J Rheumatol 2
DNA/ANuA Bardin N, et al 2009 Autoimmunity 3
ANA/ENA Hanly JG, et al 2010 J Immunol Methods 4
ANA/ENA Shanmugam VK, et al 2011 Clin Rheumatol 5
ANA/ENA Kim Y, et al 2012 Clin Chim Acta 6
ANA/ENA Op De Beèck K, et al 2012 Autoimmun Rev 7
ANuA Bose N, et al 2012 Int J Clin Exp Med 8
ANA/ENA Bruner BF, et al 2012 Arthritis Rheum 9
DNA Venner AA, et al 2013 Clin Biochem 10
ENA Infantino M, et al 2013 Clin Dev Immunol 11
ANA/ENA Tozzoli R, et al 2014 Submitted 12
ANA/ENA detection in SARDs with BioPlex 2200: Cohen’s kappa agreement with different
monoplex and multiplex IMAs Study Patients AIDs Comparator dsDNA ENA (mean)
1 1104 ND ELISA DID
0.66 ND
ND 0.61
2 952 SARDs ELISA 0.73 0.70
3 105 SLE Farr 0.59 ND
4 192 SLE Multiplex IB
0.56 0.58
0.69 0.63
5 238 SSc ELISA ND 0.76
6 127 SARDs ELISA ND 0.58
10 105 SLE Farr 0.48 ND
11 241 SARDs CLIA ND 0.83
12 Our study
261 SARDs CLIFT ELISA
0.35 ND
ND 0.91
Total 3365 0.35-0.73 0.58-0.91
The evolution of diagnostic technologies and ANA/ENA two-step testing strategy
Time
Throughput
Manual
Monoplex methods(IIF, DID, WB, RIA)
1970
Full automated
Multiplex methods(microarray)
2005
Low Automated
Monoplex methods(EIA, FIA, CLIA)
1995
Low automated
Multiplex methods(line blot, array)
2003
Full automated
IIF2009
Full Automated monoplex
methods(EIA, FIA, CLIA)
2000
Manual methods ANA Reflective Era
Manual IIF-Automated ENA ANA Reflective Era
Automated methods ANA Reflex Era
Building effective algorithms of ANA/ENA testing: the two-step approach
(ANA IIF 1st step, ENA IMA 2nd step) Era Years Methods
Manual ANA IIF-Reflective Manual ENA
1970-2000 Manual IIF Manual Monoplex Methods Low Automated Monoplex Methods
Manual ANA IIF- Reflective Automated ENA
2000-2009 Manual IIF Full Automated Monoplex Methods Low Automated Multiplex Methods
Automated ANA IIF – Reflex Automated ENA
2009-2014 Automated IIF Full Automated Monoplex Methods Full Automated Multiplex Methods
Terminology Meaning
Manual, Low and Full Automated Methods Different levels of procedure automation
Reflecting and Reflex testing Neural or computerized adding on laboratory test (from ANA to ENA)
Monoplex and Multiplex Methods Single or multiple antibody detection in the same analytical reaction