Serological Testing in

Autoimmune Rheumatologic
Diseases

Dr. I Nyoman Suarjana, SpPD-KR

Division of Rheumatology Department of Internal Medicine
Faculty of Medicine University of Lambung Mangkurat
Ulin General Hospital Banjarmasin
Introduction

Autoimmune disease :
a disease resulting from
a disordered immune
reaction in which
antibodies are produced
against one's own
tissues
 The spectrum of autoimmune diseases

Organ specific

Non organ specific

3
 Systemic autoimmune
rheumatologic diseases
Disease
Sjgren's syndrome
Rheumatoid arthritis
Systemic lupus erythematosus
Subacute cutaneous lupus erythematosus
Mixed connective tissue disease (MCTD)
Systemic sclerosis (scleroderma)
CREST syndrome
4
Characteristic feature
specific way it affects the lacrimal
and salivary glands
specific way it affects the joints
specific way it affects the skin
specific way it affects the skin
combination of clinical symptoms
and anti-RNP
specific way it affects the skin
combination of symptoms
 Role of serological test

History taking : 60% DIAGNOSIS

History Taking + Physical examination:

80-90% DIAGNOSIS

History taking + Physical examination + serological test :

95-100 % DIAGNOSIS
 Role of serological test

Help the clinician Dx/ autoimmune disease

(The broad range and partial overlap of the various clinical symptoms makes
it difficult in establishing a definite diagnosis)

Serological testing is a great value when evaluating a patient

with a suspected autoimmune disease

The serological result :

confirmed a diagnosis, estimate disease severity, assesing
prognosis & useful to follow disease activity and therapy

Asia Pacific Journal of Tropical Disease (2012) 159-162

 Autoantibody Tests

ANA P-ANCA
DS-DNA C-ANCA
RF Anti-GBM
CCP ASCA
Ro/La ASMA
Jo LKM
Scl-70 AMA
RNP ACE
Anti-Histone TTG
 ANA

Serologic hallmark of autoimmune

diseases
May bind DNA, RNA, nuclear
proteins, protein-nucleic acid
complexes
 ANA
Historically, HEp2 cells have been used as the
cell substrate because the result offers the
advantage of detecting a nuclear
fluorescent pattern (ANA IF)

However, because of the time and expense

for testing with HEp2 cells, the assay procedures
are largely done by ELISA methods
 PERIPHERAL

ANA pattern (result of ANA IF test)

 photo by Nyoman (3/4/2015)

Male, 40th yo, skin rash, muscle weakness

ANA-IF : 1/100
Pattern : Cytoplasmic granular dermatomypsitis?
 Female, 38th yo
ANA (ELISA) : 119
photo by Nyoman (2/28/2015)
 ANA Testing

Test for autoantibodies only when a

consistent clinical suspicion of
rheumatic disease is present

Not a good screening test for patients

with vague symptoms

ANA can be positive in sick people (non-

rheumatic) and healthy people
ANA in rheumatologic diseases

SLE 99 %
Scleroderma 97 %
Mixed connective tissue disease 93 %
Polymyositis/dermatomyositis 61 %
Rheumatoid arthritis 52 %
Rheumatoid vasculitis 33 %
Sjgren's syndrome 90 %
Drug-induced lupus 100 %
Discoid lupus 15 %
Pauciarticular juvenile chronic arthritis 71 %
ANA in non-rheumatologic diseases
Hashimotos thyroiditis - 40 50 %
Graves disease - 50 %
Autoimmune hepatitis - 60 90 %
Primary biliary cirrhosis - 10 40 %
Chronic infectious diseases
Mononucleosis
Hepatitis C
Subacute bacterial endocarditis
TB

Normal Population -5%

Higher in women, elderly
 Sensitivity and specificity of ANA tests in
certain connective tissue diseases

Disease Sensitivity Specificity

(%) (%)
Polymyositis/dermatomyositis 61 63
Rheumatoid arthritis 41 56
Scleroderma 85 54
Secondary Raynaud 64 41
Sjgrens syndrome 48 52
Systemic lupus erythematosus 93 57

Habash-Bseiso et al. Serologic testing in connective tissue diseases. Clin Med Res. 2005;3: 190-193
 ANA Test Results
Results typically include positive/negative,
titer and pattern of staining

Titers less than 1:40 should be considered

negative (20-30% of healthy people)

Titers of 1:40 to 1:160 should be

considered positive at low titer (further
workup is not recommended in the absence of
specific symptoms)
 ANA Test Results

Titers equal to or greater than 1:160

should be considered positive and further
workup should be done (only 5% of healthy
people)

If result of ANA test is positive can be

followed by ANA profile test
ANA PROFILE
Anti Associated diseases
1. nRNP/Sm : MCTD, SLE, Polymyositis/dermatomyositis
2. Sm : SLE
3. SS-A : Sjogren syndrome, SLE, Neonatal lupus, Myositis
4. Ro-52 : Sjogren syndrome, SLE
5. SS-B : Sjogren syndrome, SLE, Neonatal lupus
6. Scl-70 : Progressive systemic sclerosis, diffuse form
7. Jo-1 : Polymyositis/dermatomyositis
8. Centromere B : Limited cutaneous systemic sclerosis
9. ds-DNA : SLE
10. Nucleosome : SLE
11. Histones : SLE, Drug-induced lupus erythematosus, RA
12. Rib.P-prot. : SLE
13. PM-Scl : Systemic sclerosis, Polymyositis
14. PCNA : SLE
15. AMA-M2 : Primary biliary liver cirrhosis
 Sensitivity and specificity of specific ANA tests
(ANA Profile)
Antigen Associated condition Sensitivity Specificity
(%) (%)
Anti-centromere Limited cutaneous systemic 55 99,9
sclerosis
Anti-dsDNA SLE 57 97
Anti-SSB/La Sjgrens, subacute 16-40 94
cutaneous lupus
erythematosus, neonatal
lupus syndrome
Anti-SSA/Ro Sjgrens, subacute 8-70 87
cutaneous lupus
erythematosus, neonatal
lupus syndrome

Anti-Smooth muscle SLE 25-30 High

Anti-U3-RNP Scleroderma 12 96
Scl-70 Systemic sclerosis 20 100

Habash-Bseiso et al. Serologic testing in connective tissue diseases. Clin Med Res. 2005;3: 190-193
Sjgrens Syndrome : Oral manifestations

Dry mouth; carry water

bottles, water at bedside
Cracker sign: unable
to eat dry food without
liquid
Poor dentition; unable
to wear dentures
Oral candidiasis
American College of Rheumatology
 RF
RA patients : 75-80% RF positive
RF (+) : autoimmune disease,
inflammatory and chronic
infection
Positive predictive value low
Titer important:
= High titer : RA
= Low titer : non-RA
A negative test does not rule out RA
Up to 30% of RA patients are
seronegative
RF : lack sensitivity & specificity
The need of more
sensitive and
specific especially to
Dx early stages of
the disease

ACPA
(Anti Citrullinated Protein/Peptide Antibody)

anti-CCP1 anti-CCP2 anti CCP3 : Se 78.8%

Gen 2
Gen 1

Gen3
Sp 96.6%
Se 49% Se 70%
anti CCP3.1:
Sp 90% Se 83.3%
Sp 98,3%
Anti-citrullinated protein antibody
(ACPA) :

Anti-CCP (Cyclic Citrullinated Peptide)

Anti-MCV (Mutated Citrullinated Vimentin)

Anti-VCP (Viral Citrullinated Peptide)

 Anti-CCP
Is a very specific marker, 98% for RA

Will be positive in 70% of RA patients in early

disease

Not found in other diseases (contrast to RF)

Should be a one time test, does not need to be

repeated or followed

Indicates pts at high risk of progressive

erosive disease, should be treated aggressively
Anti-CCP antibodies are potentially important
surrogate markers for diagnosis and prognosis in RA,
because they :

Are as sensitive as, and more specific than IgM-RF

in early and fully established disease

May predict the eventual development into RA

when found in undifferentiated arthritis

Are a marker of erosive disease in RA

May be detected in healthy individuals years before

onset of clinical RA
 Evolution of CCP

Sensitivity and specificity of different anti-CCP antibodies

Sensitivity Specificity

74.8 % 95.7 %
CCP 2

78.8 % 96.6 %
CCP 3

CCP 3.1 IgG/IgA 83.0 % 98.3 %

Source: Szabo, Z. et al. Poster CORA. March 2011 Florence, Italy

CCP3 accurately identified 83% of the RF negative
patients
 2010 ACR/EULAR
Classification Criteria for RA
JOINT DISTRIBUTION (0-5)
1 large joint 0
6 = definite RA
2-10 large joints 1
1-3 small joints (large joints not counted) 2
4-10 small joints (large joints not counted) 3
>10 joints (at least one small joint) 5 What if the score is <6?
SEROLOGY (0-3)
Negative RF AND negative ACPA 0 Patient might fulfill the criteria
Low positive RF OR low positive ACPA 2
High positive RF OR high positive ACPA 3
Prospectively over time
SYMPTOM DURATION (0-1) (cumulatively)
<6 weeks 0
6 weeks 1
Retrospectively if data on all
ACUTE PHASE REACTANTS (0-1) four domains have been
Normal CRP AND normal ESR 0
adequately recorded in the past
Abnormal CRP OR abnormal ESR 1
 Photo by Nyoman 13-09-2013

Male, 38 yo.
Arthritis in both of wrist joint , right shoulder & right
MCP II
Duration of disease : 5 mo.
Lab : RF
Anti-CCP3.1 : 372,1 (N < 20)
CRP : 48
 Anti-neutrophil cytoplasmic
autoantibodies (ANCA)

Serologic testing for ANCA is useful diagnostic test, but

should be interpreted in the context of other patient
characteristics

Sensitivity for pauci-immune small vessel vasculitis and

GN is 80-90%
Clinical features of primary systemic
vasculitis

Clinical signs that should raise suspicion of

primary systemic vasculitis :

Fever of unknown origin

Unexplained migratory polyarthritis
Mononeuritis multiplex
Rapidly progressive glomerulonephritis
Palpable purpura
Diffuse alveolar hemorrhage
Unexplained multisystem disease

J Allergy Clin Immunol 2009;123:1226-36

 Skin (vasculitis)

*Purpura most common

in lower extremities,
occurs as recurrent crops.

*Nodular lesions occur

more frequently in
Churg-Strauss and
Wegeners
 Gastrointestinal (vasculitis)

*typically
abdominal pain,
blood in the stool,
mesenteric
ischemia and
rarely perforation

*can also mimic

pancreatitis and
hepatitis
 HLA-B27
70 to 80% of people with the HLA-B27
antigen have no clinical
manifestations related to the presence
of this gene

However, it has been estimated that up

to 20% of people carrying this antigen
have at least one of several
associated conditions
HLA-B27 Associated Diseases
The spondyloarthropathies :
Ankylosing Spondylitis
Reiters Disease and Reactive Arthritis
Psoriatic Arthritis
Undifferentiated Spondyloarthropathies
Enteropathic Synovitis
Inflammatory Bowel Diseases

Isolated acute anterior uveitis

Clinical Associations with HLA-B27

Disorder HLA-B27 (%)

Ankylosing Spondylitis > 90%
Reiters syndrome 80%
Juvenile Spondyloarthritis 70%
Inflammatory bowel dz 50%
Psoriatic arthritis
With Spondylitis 50%
With Peripheral arthritis 15%
Acute Anterior Uveitis 50%

Aortic insuff. w/ heart block 80%

SAPHO 20-30%
 SPA
Characterized by :
Sacroilitis
Inflammatory back pain
Peripheral arthropathy
Absence of rheumatoid factor/CCP
and subcutaneous nodules
Enthesitis
Extra spinal involvement (eye, heart,
lung and skin)
HLA-B27
 Inflammatory Low Back Pain

Assumed to be characterized by
inflammation of SIJ and lumbar spine

Young age of onset

Continuous pain > 3 months

Morning stiffness

Pain improving on activity

Questions
1. Besides a rising anti-dsDNA titer, what
other lab. test can help predict an
upcoming SLE flare?
= Falling C3 & C4 levels

2. What two antibodies are the most

specific for SLE (not ANA)?
= Anti-dsDNA & anti-Smith
Questions
1. What specific autoantibody is
characteristic of drug-induced lupus?
= Anti-histone

2. What is the major autoantibody in diffuse

scleroderma?
= Scl-70
Summary
Serologic tests for rheumatologic diseases are
SUPPORTIVE rather than DIAGNOSTIC

anti-CCP antibody is currently the best test for RA in

a patient with undifferentiated polyarthritis

Before ordering an ANA on a patient with possible

SLE, make sure youve got 3 out of 11 of the ACR
criteria

Most rheumatologic serologies are specific, but not

sensitive. That is, they are useful in CONFIRMING
a likely diagnosis. (remember : SpPIN)
Thanks for listening!