Point-of-care Autoantibody Testing in a Rheumatology Setting

Konstantin N. Konstantinov, MD, PhD.Associate Professor – Medicine / Rheumatology

UNM School of Medicine, New Mexico, USA

Disclosure Statement

Nothing to declare

Evidence-based medicine (EBM) slide

Konstantinov KN et al. Rapid detection of anti-chromatin autoantibodies in human serum using a portable electrochemical biosensor. Biosensors and Bioelectronics, 2009, 24:1949-1954

Renger F et al. Immediate determination of ACPA and rheumatoid factor – a novel point of care test for detection of anti-MCV antibodies and rheumatoid factor using a lateral-flow immunoassay. Arthritis Research & Therapy, 2010, 12:R120

Schlichtiger A et al. Biosensor approaches for the detection of autoantibodies in human serum. Bioanalytical Reviews, March 2012, Springer online publication

American Academy of Microbiology. Bringing the Lab to the patient: developing point of care diagnostics for resource limited settings. June 2012, American Society for Microbiology online publication

Hieronymous Bosch (c 1450-1516), The Cure of Folly (Extraction of the Stone of Madness), 1475-1480, Dutch. Oil on board, 48 × 35 cm, Museo del Prado, Madrid

POCTherapy Diagnostics

The Point of Point of Care Testing

No accepted universal definition, often referred to as near patient, bedside, or extra laboratory testing

Critical elements are:» Rapid turn-around and communication of results to guide clinical

decisions

» Completion of testing and follow-up action in the same clinical encounter

Point of Care Tests: The Times They Are a-Changing

Near real-time monitoring of clinical samples Overcomes disadvantages of centralized, purpose-built laboratories Equipment miniaturization due to technological advances in

materials, microfluidics and computerization Mass fabrication of disposable components Acceptable replacement cost Operational use with minimal instruction

Possibility for instant, result-deduced therapeutic action

Is the ideal POC test only cheap or fast?

The ideal POC test should have different characteristics simultaneously:

A S S U R E D

AffordableSensitiveSpecificUser-friendlyRobust and RapidEquipment-freeDeliverable

Bringing the Lab to the Patient; Report from the American Academy of Microbiology, 2012; www.asm.org

Biosensors: the canary in the coalmine

Science and Economics: the “canary in the coalmine” is healthy and kicking

Biosensors world market generate annual sales of $13 billion, the lion’s share still belongs to glucose home meters

» Cranfield University Study, 2009

Global demand for biosensors in medical diagnostics is forecast to reach $16.5 billion by 2017

» (Global Industry Analysts Report, 2012)

Spawn 6,000 research papers in 2011» Anthony PF Turner, Editor-in-Chief: Biosensors and Bioelectronics

_____________________________

Comparison: NIH budget for 2012 is $ 32 billion

What is a Biosensor?Device with 2 elements in spatial proximity: a biological recognition

unit (interacts specifically with a target) and a transducer (converts the recognition event into a measurable signal}

ElectrochemicalOpticalSurface acousticThermalMagneticSwitch-based, etc.

AntibodiesProtein receptorsNucleic acidsEnzymesWhole cells, etc

Small and light weight

Use small sample sizes

Store results

Transmit them (wirelessly)

Be amenable to quality control checks

“The time taken to reach the correct diagnosis may critically impact on the patient’s chances of survival”

British Medical Journal, Gordon Caldwell

Accurate diagnostics affects health care decisions to a degree wellout of proportion to their cost.

Diagnostics account for 2% of the cost of health care, yet can affect60-70% of treatment decisions

Bufton A. 2002, Business Briefing: Medical Device Manufacturing & TechnologyMedicare laboratory payment policy. 2000, Institute of Medicine, National Academy Press

POC Testing in Rheumatology or Seeing the Forest for the Trees

Getting the big picture means asking a lot of little questions and S E T T I N G P R I O R I T I E S

Which diagnostic tests are most needed? What characteristics do they have to have to succeed? Is there a clear link to diagnosis, treatment or counseling plan? Does it offer convenience to patients and care providers? Can it improve health care in limited resource areas?

Why autoantibodies?

Predict incipient disease Facilitate diagnosis refinement Indicate disease severity or impending flares Prognostic marker for further organ involvement Monitor efficacy of therapy Monitor induction of autoimmunity by drugs

POC Autoantibody Testing: Is It Ready for Primetime?

Support for considerable value of direct monitoring of autoantibodies in the clinic comes from:

1. Diagnostic Process in acute-care settings2. Assessing active disease

3. Other advantages

Autoimmune serology assessment for possible rheumatic disease in emergency settings

Symptom PositiveTestResult DiseaseAirway problems

Hemoptysis Anti-dsDNA, other lupus serologies Alveolar hemorrhage in SLEAirflow obstruction Anti-CCP, RF Crycoarytenoid arthritis in

rheumatoid arthritis (RA)Mucopurulent rhinorrhea; subglottic stenosis; hypo-pharyngeal ulcerations

Anti-neutrophil cytoplasmic antibodies (ANCA, MPO or PR3)

Wegener’s granulomatosis

Stridor, laryngotracheal strictures Anti-type II collagen Relapsing polychondritisAcute pneumonitis Anti-dsDNA, other lupus serologies SLE

Pulmonary-renal problemsPulmonary hemorrhage and acute renal failure Anti-GBM, MPO-ANCA,

PR3-ANCAGoodpasture’s syndrome;systemic vasculitis

Neuropsychiatric problemsEncephalopathy, psychosis, focal central nervous system disease

Anti-N-methyl-D-aspartate receptor (NMDA-R), anti-ribosomal P antibodies,antiphospholipid antibodies

Neuropsychiatric SLE, antiphospholipid syndrome

Weakness, paralysis, bilateral sensory deficit, impaired sphincter control

Lupus serologies Transverse myelitis in SLE

Seizures Anti-dsDNA, other lupus serologies Lupus cerebritisThromboembolic problems

DVT, pulmonary thromboembolism, fetal loss, retinal artery occlusion

Anti-phospholipid antibodies Antiphospholipid syndrome

Neuromuscular problemsProgressive symmetric muscle weakness; dysphagia; dysphonia

Anti-Jo-1, other myositis-specific autoantibodies

Dermatomyositis, polymyositis

Unusual weakness and hypokalemia Anti-SSA; anti-SSB Sjogren’s syndrome hypokalemic paralysis

Autoimmune serology assessment for possible rheumatic disease in emergency settings

Symptom Positive test result Disease

Cardiac problemsPleuritic or positional chest pain, dyspnea, tachycardia

Anti-dsDNA, otherlupus serologies,Anti-phospholipidantibodies

SLE pleuro-pericarditis, pericardial tamponade

Congenital heart block; neonatal carditis

Anti-SSA; anti-SSB Neonatal SLE

Renal problemsRapidly progressive renal failure MPO-ANCA, PR3-ANCA,

anti-dsDNA and other lupus serologies, anti-phospholipid antibodies

Microscopic polyangiitis, WG, lupus nephritis, catastrophic antiphospholipid syndrome

Accelerated hypertension Anti-Scl-70; anti-centromeres, anti-RNA-Polymerase III

Renal crisis in systemic sclerosis

Joint problemsPain, stiffness, swelling with symptoms of systemic disease

Anti-CCP, RF and lupus serologies RA, SLE

Ocular problemsRed, painful, photophobic eye RF, anti-CCP, lupus serologies RA, Behcet’s, juvenile RA, SLE

Gastrointestinal problemsColicky abdominal pain Lupus serologies SLE mesenteric arteritis

Skin problemsPetechiae, palpable purpura, hemorrhagic blisters, ulcerations and gangrene

SLE and RA serologies SLE, rheumatoid vasculitis

Neonatal skin rash Anti-SSA, anti-SSB Neonatal lupusHematological problems

Anemia, thrombocytopenia,leukopenia

Anti-DNA and lupus serologies; anti-erythrocyte, anti-platelet antibodies

SLE, autoimmune hemolytic anemia

Thrombocytopenia Antiphospholipid antibodies Antiphospholipid syndrome

Associations between autoantibody changes and disease activity

Disease/Condition Autoantibody Change Clinical Prediction

Systemic lupus erythematosus

Anti-dsDNA Active flare

Anti-dsDNA Active flare

Anti-nucleosome Active disease/lupus nephritis

Anti-C1q Lupus nephritis /active disease

Anti-NMDA-R Permanent CNS impairment

Anti-NMDA-R Transient CNS symptoms

Anti-CRP Lupus nephritis/response to therapy

Anti-interferon-α Inactive disease

Systemic vasculitis Anti-PR3 Active disease/disease relapse

Anti-MPO Active disease/disease relapse

Anti-GBM Active disease/disease relapse

Scleroderma Anti-topoisomerase I Active scleroderma

Associations between autoantibody changes and disease activity

Disease/Condition Autoantibody Change Clinical Prediction

Rheumatoid arthritis Anti-drug (Adalimumab) Treatment failure

Antiphospholipid syndrome/SLE

Anti-phospholipid Procoagulant state, thrombosis

Necrotizing myopathy Anti-signal recognition particle

Decreased muscle strength, increased creatine kinase activity

Thrombotic thrombocyto-penic purpura

Anti-ADAMTS13 antibodies Disease relapse

Pregnancy in SLE Anti-Ro(SSA)/ anti-Ro52 Congenital heart block

Anti-La(SSB) Neonatal lupus

Autoantibody serum screening

Anti-DFS70 ANA-positive healthy individuals

Possible advantages for POC testing of autoantibodies

1. Isotype determination vs Biosensor analysis of β2-glycoprotein I-reactive autoantibodies: evidence for isotype-specific binding and differentiation of pathogenic from infection-induced antibodies. Metzger et al, Clinical Chemistry, 2007, 56:3

2.Affinity of interactionBiomolecular interaction monitoring ofautoantibodies by scanning surface plasmon resonance microarray imaging. Lokate et al, J Am Chem Soc, 2007, 129:14013

3. Epitope “profiling” during active diseaseDiversity of PR3-ANCA epitope specificity in Wegener’s granulomatosis. Analysis using the biosensor technology. Rarok et al, J Clin Immunol, 2003, 23:460

POC modus operandi(Devices with potential to test

autoantibodies at the point-of-care)

Autoantibody Detection Technology/Assay Platform Assay duration

Anti-dsDNA Electrochemical reduction of redox-tagged probe/Ab Inhibition in single-step cell

Decreased resonance frequency/Piezoelectric quartz crystal microbalance

Refractive index change/Surface plasmon resonance sensor chip

~45 min

<60 min

~5 min

Anti-CCP Formation of visual line by colored nanoparticles/lateral-flow chromatography

Refractive index change/Surface plasmon resonance sensor chip

10 min~5 min

Anti-chromatin Peroxidase-mediated electrochemical amplification/ flow-through cell

20 min

Anti-IgG (RF)Anti-MCV

Formation of visual line by colored nanoparticles/lateral-flow chromatography

15 min

Anti-Ro/SSA,Anti-Ro52,Anti-La/SSB

Luminescence by luciferase-tagged probe/ bead immobilized Ab in two-step cells

25 min

Anti-β2-glyco-protein I Refractive index change/Surface plasmon resonance sensor chip

~5 min

Surface Plasmon Resonance (SPR) Imaging

Lokate AMC et al, JAMC, 2007

anti-GBManti-GADanti-dsDNAanti-CCPanti-MCVanti-2βGPIanti-CEA

Electrochemical Transduction for Antibody Detection

RL Rubin, 2012, unpublished

Flow through device for measuring antibodies in 8 samples

Some autoantibody biosensors have generally equaled or surpassed traditional central laboratory methods in

performance metrics, esp. time to result

A. reactivity of normal and SLE sera (signal output measured in 7 min)B. relationship between biosensor / ELISA results with SLE sera

Anti-Chromatin Autoantibody Electrochemical Assay

Vive la Difference, but…

Surface plasmon resonance-based sensors are the most rapid, but will require adaptation to inexpensive miniaturized devices

Lateral flow based methods will probably be restricted to non-quantitative readouts

Devices requiring specialized antigen tags may have limited practical potential

Electrochemical amplification methods using readily available autoantigens are especially promising.

A Pioneer’s perils

T e c h n i c a l r e q u i r e m e n t s Non-specific binding: any autoantibody specificity is usually a

tiny fraction of total serum immunoglobulin Built-in capacity for data portability, security, and reporting Shelf-life: long-term stability of biological receptor and

transducer in the test environment

R e g u l a t o r y r e q u i r e m e n t s Successful deployment requires long-term commitment Health care managers must invest in POCT programs, rather

than merely purchase rapid tests Regulators and will have to address financial matters and

reimbursement issues

Hold that thought

Autoantibody tests are widely employed by rheumatologists to aiddiagnosis and monitor disease activity and treatment efficacy.

The need for centralized clinical laboratories for measuringautoantibodies impedes ability of physicians to promptly act on thisserological information (ACR position statement on value of de-centralized laboratory testing)

Point-of-care testing for autoantibodies is an emerging technology thatholds promise to enhance the usefulness of these biomarkers forphysicians, resulting in better patient management by providing real-timeautoantibody data while patients remain in the clinic.

William French, 1818–1898, engraver,Die Kranke Frau, Clements C. Fry Collection of Prints and

Drawings, Cushing/Whitney Medical Library, Yale University.

The POC Autoantibody Testingthat Came In From the Cold

Collaborators

Albuquerque, NM, USA

Antonios Tzamaloukas, MD Renal Section, VA Med CenterDavid Wall Voss ScientificKonstantin Konstantinov, MD, PhD. UNM School of MedicineRobert Rubin, PhD UNM, Dept. Mol. Genet & Microbiology