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