Best Practice & Research Clinical Rheumatology 28 (2014) 907e920

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Best Practice & Research Clinical

Rheumatology
journal homepage: www.elsevierhealth.com/berh

Role of autoantibody testing

Amita Aggarwal*
Department of Clinical Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences,
Lucknow, India

a b s t r a c t
Keywords:
ELISA Autoantibodies are the serological hallmark of autoimmune dis-
Autoantibody ease. Though their pathogenic role is debatable, they play an
Anti-dsDNA antibodies important role in the management of a patient with rheumatic
Automation disease. However, due to their presence in the general population
Protein arrays as well as in multiple autoimmune diseases, the presence of an
autoantibody alone does not make a diagnosis; the result has to be
interpreted along with clinical ndings. Similarly, the absence of
autoantibody does not exclude a disease.
The common autoantibodies used in clinical practice include
rheumatoid factor, anti-CCP antibodies, antinuclear antibodies
(ANAs), anti-neutrophil cytoplasmic antibodies (ANCA) and anti-
phospholipid antibodies. Once an autoantibody to a broad anti-
gen is detected in a patient, sub-specicity analysis can improve
the utility of the antibody. Autoantibodies are detected in the
serum using different assays and results of which can vary; thus, it
is important for a clinician to know the method used, its sensitivity
and specicity to help in the proper interpretation of the labora-
tory results. This review will address these issues.
2015 Elsevier Ltd. All rights reserved.

Introduction

During the development of lymphocytes in the primary lymphoid organs, the body tries to avoid
generation of cells directed against self-antigens; however, the deletion process is not perfect, and a
small frequency of self-reactive T and B lymphocytes escape to the periphery. In the periphery, they are

* Tel.: 91 522 2494296.

E-mail addresses: aa.amita@gmail.com, amita@sgpgi.ac.in.

http://dx.doi.org/10.1016/j.berh.2015.04.010
1521-6942/ 2015 Elsevier Ltd. All rights reserved.
908 A. Aggarwal / Best Practice & Research Clinical Rheumatology 28 (2014) 907e920

kept under check by different mechanisms involved in tolerance induction. However, in autoimmune
disease due to the failure of these regulatory mechanisms or to an increased load of autoantigens, there
is a generation of a signicant amount of autoantibodies. The pathological signicance of autoanti-
bodies in the causation of disease is limited, but they serve as good serological markers of
autoimmunity.
Autoantibodies are useful in diagnosis, prognosis and follow-up of patients with rheumatic disease.
However, as is true for any laboratory test, the interpretation of the test is dependent on the clinical
situation (pre-test probability), the characteristics of the test (sensitivity, specicity and the likelihood
ratios) and the reason for doing the test (conrmation or exclusion of a diagnosis). Autoantibodies can
be directed against any component of a cell, that is, nuclear, cytoplasmic or membrane. In addition,
antibodies against cytokines, hormones, coagulation proteins, phospholipids, etc. are also known.
Most autoantibodies for clinical usage are detected in the serum, but they can be detected in the
cerebrospinal uid and other body uids. The various tests used for their detection include indirect
immunouorescence assay (IIF), enzyme-linked immunosorbent assay (ELISA), nephelometry and
immunoblotting. In recent years, newer techniques such as bead array and chip array are being
explored for the ease of automation and a high throughput.
For each test, kits from various companies are available; however, the results are usually not
comparable due to differences in the various reagents used. Though there has been an attempt to
standardize kits and express results in international units, it has not been possible for all antibodies.
Kits certied by a regulatory body such as Food and Drug Administration (FDA) or European Medicines
Agency (EMA) should be used.
The autoantibody testing is different from other laboratory tests because of the presence of
multitude of antibodies in one disease, the same antibody in many diseases, occurrence prior to the
onset of symptoms and uctuation in levels during the course of a disease. Autoantibodies are also
found in healthy individuals, and their prevalence increases with age especially so in the elderly
population, which further makes their interpretation difcult. Thus, their interpretation requires
training of physicians so that they are used in the right clinical context.
In this review, commonly used autoantibodies and their clinical signicance are discussed.
Rheumatoid factor: Rheumatoid factor (RF) is an autoantibody directed against aggregated
immunoglobulin G (IgG). They are produced as a result of polyclonal B-cell activation as well as in
response to the modied antigen. Though all subtypes of RF are generated, we usually test for IgM RF.
IgG and IgA are present in a proportion of patients with RF. RF is present in many conditions besides RA
(Table 1).
The presence of RF in a patient with arthritis increases the probability of a diagnosis of rheumatoid
arthritis (RA). In a patient with RA, it increases the risk of the development of erosions, extra-articular
features and poor outcome. In RA, the presence of RF also increases response rate to B-cell depletion
therapy.
In addition to RA, RF is present in many autoimmune diseases such as Sjo gren's syndrome (SS),
systemic lupus erythematosus (SLE), juvenile idiopathic arthritis, all of which may present with pol-
yarthritis. Patients with chronic infections such as subacute endocarditis, tuberculosis (TB) and leprosy

Table 1
Prevalence of RF in different clinical situation.

Disease Prevalence Disease Prevalence

Rheumatoid arthritis 60e70 Chronic infections

Early undifferentiated arthritis 45e55 Endocarditis 15e20
Juvenile idiopathic arthritis 10e12 Hepatitis B/C 15e40
Other autoimmune diseases Leprosy 10e50
SLE 15e30 Tuberculosis 10e20
gren's syndrome
Sjo 75e85
Systemic sclerosis 20e30
Inammatory myositis 5e10
Interstitial lung disease 10e40 Healthy control 5e20a
a
Elderly persons.
 A. Aggarwal / Best Practice & Research Clinical Rheumatology 28 (2014) 907e920 909

also have a higher prevalence of RF, and some of them can present with polyarthritis-like features. RF
production in them occurs due to chronic B-cell stimulation.
RF should be ordered in a patient with

 Early inammatory arthritis without any obvious cause

 Clinical diagnosis of RA
 Prior to considering B-cell depletion therapy in a patient with RA
 Patient with Sicca symptoms
 Child with chronic polyarthritis

Indiscriminate use of RF in the primary and secondary health-care setting results in the wastage of
money as well as in a large number of false-positive results [1].

Methods

RF can be measured using latex agglutination, ELISA and nephelometry. Among these, nephelom-
etry is the most commonly used method as it is quick and reproducible, provides quantitative result
and can be fully automated for a high throughput. Though latex agglutination is a cheap and easy assay
and can be performed quickly and at the point of care in a community, it provides qualitative data and
has about 20% coefcient of variation due to problems in setting the assay [2]. ELISA is a good method
but it is too sensitive, and samples need to be processed in a batch and thus there is a delay in reports.
ELISA can help detect other isotypes of RF such as IgG and IgA RF. IgG RF levels are high in patients with
RA vasculitis, and IgA RF at onset heralds a poor prognosis but that is rarely required in clinical practice.
A World Health Organization (WHO) standard is available, and all kits should report the result as
international unit (IU). A value above 20 IU is taken as signicant, but values above 50 IU have a higher
diagnostic value.

Anti-citrullinated peptide antibodies

Due to the low specicity of RF, there has been a quest to have a more specic test for RA. In fact, it is
being considered that RF may no longer be needed [3]. Over the last 25 years, anti-citrullinated peptide
antibodies (ACPAs) have emerged as a major diagnostic test. Though anti-perinuclear factor was
described in 1964, it was only in 2000 that the rst ELISA for antibodies to cyclic citrullinated peptide
was described. Over the years, many new targets have been identied; thus, now they are named as
ACPAs. ACPAs are a group of antibodies directed against citrullinated self-proteins such as laggrin,
vimentin, enolase A, brinogen, etc. [4] Due to the high specicity of ACPA, they are being used more
often for the diagnosis of RA, and they are included in the new classication criteria for RA [5].
Like RF, their presence increases the probability of RA in a patient with undifferentiated arthritis.
Negative ACPA does not help much in a patient with undifferentiated arthritis. Accumulated data from
164 studies calculated between 2002 and 2019 involving >18,000 patients and 20,000 controls showed
that it had a sensitivity of 61.4% and a specicity of 99% as compared to healthy controls and 94% as
compared to disease control for a diagnosis of early RA4. Similarly, their presence increases the risk of
erosions, cardiovascular risk and mortality [7e10]. ACPAs are most useful in patients with undiffer-
entiated arthritis where a diagnosis of RA is being considered. The presence of ACPA increases the
probability of progression to RA by 16-fold [11]. Even the risk of arthritis is higher in patients with
arthralgia if they are ACPA positive [12]. It should not be ordered in other conditions as rarely high-titre
antibodies may be seen in reactive arthritis, psoriatic arthritis, myositis, etc. [6] Serial monitoring or
ACPA is not recommended as the levels do not correlate with disease activity or with response to
therapy and change in only a small proportion of patients [13].

Methods

ACPA is detected by ELISA. Over time, different generations of ELISA have come in the market. The
rst-generation kits used synthetic peptides derived from human laggrin, whereas the second-
910 A. Aggarwal / Best Practice & Research Clinical Rheumatology 28 (2014) 907e920

generation kit anti-cyclic citrullinated peptide (CCP2) used epitopes selected from libraries of cit-
rullinated peptides as antigens. The exact nature of antigen for anti-CCP3 is not known. Anti-CCP2
ELISA had a higher sensitivity as compared to anti-CCP1, but there is no difference in the perfor-
mance characteristics of anti-CCP3 as compared to anti-CCP2; thus, anti-CCP2 is the most widely used
ELISA for the detection of these antibodies [14]. Recently, an international reference standard has been
generated and is being evaluated [15]. In view of multiple assays available, the use of a generic term
such as ACPA was suggested by the panel on multinational evidence-based recommendations on how
to investigate and follow up undifferentiated peripheral inammatory arthritis 2011 [16].

Practice points

 ACPA is more specific than RF for rheumatoid arthritis

 CCP2-based ELISA is currently the most widely used assay for ACPA detection
 No need to repeat if once positive
 Titres do not correlate with disease activity
 The presence of ACPA in undifferentiated arthritis increases the probability of development
of RA to 70e90%

Antibodies to nuclear antigens

These are one of the most widely used autoantibody tests. The screening test, that is, ANA, looks at
the presence of antibodies to all nuclear antigens by using cells as substrate. These include DNA- and
RNA-associated proteins, centromere, nuclear membrane as well as nucleoli. On dividing, cells
proliferating cell nuclear antigen, etc. are expressed, and antibodies against them can also be detected
when using cell-line substrates [17]. Once ANA is found to be positive, the sub-specicities are iden-
tied using different assays [18].
The indications of ordering ANA includes clinical situation where we suspect SLE or other con-
nective tissue disease as follows:

 Arthritis with fever

 Glomerulonephritis
 Unexplained multisystem disease
 Immune cytopaenias
 Unexplained neurological disease
 Polyserositis
 Sicca syndrome
 Raynaud's phenomenon
 Systemic sclerosis
 Palpable purpura

Methods

Traditionally, ANA has been detected by IIF for the last 65 years, but in the recent past, solid-phase
assays such as ELISA and laser bead assays are competing with IIF. Most laboratories use Hep2 cells or
its variants such as Hep2000, which has a higher amount of Ro antigen for IIF assay [19]. The major
drawback of IIF assay is that it is a labour-intensive test with multiple steps of incubation and washing,
the need for a trained reader and difcult to automate. Recently, attempts to use automated systems for
staining slides and reading them have been developed; they are yet to reach a wide applicability for
clinical usage [20,21].
 A. Aggarwal / Best Practice & Research Clinical Rheumatology 28 (2014) 907e920 911

By contrast, solid-phase assays can be automated for a high throughput, and they are less la-
bour intensive. The major drawback of newer assays is the variability in antigens used, which
affects their utility and comparability [22]. The American College of Rheumatology (ACR) still
recommends IIF to be the gold standard for the determination of ANA [23]. However, the recent
international recommendation suggests that IIF is the preferred method, but alternative methods
can be used mentioning their sensitivity and specicity. If the alternative method is negative and
the clinical suspicion of autoimmune rheumatic disease is high, then it is mandatory to perform IIF
assay [24].
For IIF, screening dilution should be determined locally, and not more than 5% of healthy controls
should be positive at that dilution. As a rough estimate, a screening dilution of 1:100 is good. All test
reports should mention the pattern (Fig. 1), the titre (highest dilution at which the test is positive) and
substrate used (Hep2 or Hep2000 cells). A positive ANA test should be followed by other tests to dene
the sub-specicities. The pattern of ANA can give some clue to the antigenic specicity as stated in
Table 2.

Anti-double-stranded DNA antibodies

In a patient suspected to have SLE, once the ANA is positive, the sera should be tested for antibodies
to double-stranded DNA (dsDNA). Anti-dsDNA antibodies are present in two-thirds of patients with
SLE, and they have a good association with disease activity and lupus nephritis [25]. Serial monitoring
of anti-dsDNA antibodies has modest correlation with disease activity, and a rising titre may herald the
development of relapse [26]. Thus, anti-dsDNA test should be ordered for

Fig. 1. Hep2 cells showing different ANA patterns: A, homogeneous; B, centromere; C, nucleolar; and D, speckled.
912 A. Aggarwal / Best Practice & Research Clinical Rheumatology 28 (2014) 907e920

Table 2
Staining pattern of ANA and likely antigenic targets.

Pattern of staining on cell line Likely antigens

Nuclear
Homogeneous Histone, dsDNA, nucleosome
Rim Lamin, nuclear-pore complex
Speckled
Coarse Sm, nRNP
Fine Ro, La, topoisomerase-I
Nucleolar RNA polymerase 1, PM-Scl, brillarin
Centromere Centromeric proteins A, B, C
Cytoplasmic Ribosomal P protein, t-RNA synthase including
Jo-1, mitochondrial, actin, golgi

 Suspected diagnosis of SLE

 Follow-up of patients with SLE

Methods

Anti-dsDNA antibodies are measured using IIF assay with Crithidia lucillae as the substrate, Farr
radioimmunoassay or ELISA. IIF assay using C. lucillae has a very high specicity but a lower sensitivity;
thus, it is good to conrm a diagnosis of SLE. However, it is at best semi-quantitative and hence not
good for follow-up. Farr assay and ELISA give quantitative results, are easy to perform and are used for
monitoring of patients. Among these, the Farr assay detects high-avidity antibodies, and it is the
method of choice as suggested by the international panel [24]. ELISA detects both low-afnity and

Table 3
Prevalence as well as pattern of ANA varies in different diseases.

Disease ANA pattern Antigenic Prevalence (%) Clinical signicance

specicity

SLE Homogeneous/ dsDNA 50e75 SLE, correlate with

rim renal activity
Homogeneous Histones 50e70 Drug-induced SLE (>95%)
Speckled Sm 15e30 Specic for SLE
UIRNP 30e40 Raynaud's phenomenon
La 10e30 Neonatal lupus (75%)
Ro 25e50 More common in SCLE,
neonatal lupus
Systemic Speckled Scl-70 20e60 Diffuse SSc
sclerosis (70e76%)
Centromere Centromere 30e35 Limited SSc
proteins (50e80%)
A, B and C
Nucleolar Fibrillarin, <10 e
NOR-90,
RNA-Pol 1
Rheumatoid Homogeneous Histones 5e10 e
arthritis Speckled Ro 3e10 Associated with
secondary Sjo gren's
syndrome
MCTD Speckled UIRNP 100 Diagnostic criterion
gren's
Primary Sjo Speckled Ro 40e70 e
syndrome La 30e60 e
Polymyositis Speckled UIRNP 4e15 Overlap with SLE
of MCTD
Nucleolar/ Ku 5e12 Overlap with
homogeneous systemic
sclerosis
 A. Aggarwal / Best Practice & Research Clinical Rheumatology 28 (2014) 907e920 913

high-afnity antibodies and has a low specicity, but it is easy to do and has no radiation hazard; thus,
it is used widely. The international panel recommends that the method used should always be
mentioned when reporting a test [24].

Anti-nucleosomal antibodies

During apoptosis, nucleosomes are generated due to DNA cleavage, and the defective clearance of
apoptotic bodies in SLE helps in the generation of anti-nucleosomal antibodies. Nucleosome consists of
dsDNA wrapped around a core of histones, and anti-nucleosomal antibodies have a slightly higher
sensitivity and specicity than anti-dsDNA antibodies for the diagnosis of SLE [27]. Further, they can be
found in 10% of patients with lupus nephritis who are anti-dsDNA antibody negative. They are useful in
the follow-up of lupus patients, and they have shown to be even better than anti-dsDNA antibodies
[28]. However, they are not widely available; thus, their use in clinical practice is limited.

Practice points

 The presence of ANA increases the likelihood of a connective tissue disease

 Higher tires have a better predictive value as low titres can occur in healthy individuals
 No need to repeat if once positive
 IIF is the preferred method, but ELISA may be used for ANA detection
 Use C. lucillae assay if using anti-dsDNA antibodies for the diagnosis of SLE
 Serial anti-dsDNA antibodies are useful in disease monitoring in SLE

Antibodies to extractable nuclear antigens

Antibodies to extractable nuclear antigens (ENAs) are used to dene sub-specicities of ANA as
different ENAs have an association with different connective tissue diseases. They are called anti-ENA
antibodies as in the past they were detected using saline extract of nuclei as the antigen. Antibodies to
ENA may be ordered in patients with

 Suspected systemic autoimmune rheumatic disease with ANA positivity

 Special situations with ANA negativity may include
B Mother of a child with congenital complete heart block for anti-Ro antibodies

B SS for anti-Ro and anti-La antibodies

B Inammatory myositis for anti-synthetase antibodies

Methods

Antibodies to ENA can be determined using double immunodiffusion, immunoblotting, ELISA or

bead-based assay. Most of the assays use recombinant or afnity-puried antigens. ELISA is highly
sensitive; thus, the cut-off needs to be carefully determined. In a recent study involving >16,000
samples, ELISA was found to give signicant false-positive results, and the positive predictive
value was 66% as compared to 96% for the standard double immunodiffusion assay [29].
Immunoblots or line assays have good specicity and sensitivity, and they have an advantage of
allowing multiple specicities to be tested at the same time as well as allowing exibility to run
one or multiple sample thus reducing the turnaround time. The best algorithm for ENA analysis is
ANA positivity and pattern followed by the ENA requested by the clinician rather than a wide
screen [30].
914 A. Aggarwal / Best Practice & Research Clinical Rheumatology 28 (2014) 907e920

Anti-Ro antibodies

Anti-Ro antibodies are antibodies directed against 52- and 60-kD RNA-associated proteins. It is also
called anti-Sjo gren's-syndrome-related antigen A (SSA) antibody. Anti-Ro antibodies are present in
multiple autoimmune diseases including SS, SLE, inammatory myositis and RA. In SS, their prevalence
is 40e60%, and it is primarily against the 52-kD protein. In SLE, anti-Ro antibodies are seen in 25e30%
of patients, and they correlate with sub-acute cutaneous lupus, Sicca syndrome, immune cytopaenias
and nephritis [31]. In RA, their presence increases the risk of Sicca syndrome.
Anti-Ro antibodies are also associated with congenital complete heart block (CHB) and neonatal lupus.
This occurs because of transplacental transfer of antibodies during pregnancy. The conducting system of
foetal heart is most vulnerable to attack by anti-Ro antibodies between 14 and 20 weeks, and this can
result in atrio-ventricular block in the foetus. In addition, circulating maternal antibodies in the newborn
can result in facial rash, mild hepatitis and cytopaenias, which last for 4e6 weeks and spontaneously
resolve with a decrease in maternal antibodies over time. Most of these mothers do not have evident
autoimmune rheumatic disease. Thus, mothers of all babies or foetus diagnosed with a heart block should
be tested for anti-Ro antibodies. The risk of CHB is 2e3% in mothers having anti-Ro antibodies [32].

Anti-La antibodies

Anti-La antibodies mostly coexist with anti-Ro antibodies, and they occur due to epitope spreading.
They recognize a 45-kD protein involved with the termination of RNA polymerase III. Anti-La anti-
bodies are predominantly seen in primary SS (50%) and SLE (15e20%). Their prevalence is lower than
anti-Ro antibodies. There is a debate as to whether all samples sent for ANA be tested for anti-Ro and
anti-La antibodies [33].

Anti-RNP antibodies

Anti-ribonucleoprotein (RNP) antibodies are antibodies directed against components of spliceo-

some. These antibodies lead to a speckled pattern of ANA. Anti-RNP antibodies are seen across a wide
array of connective tissue diseases such as RA, SLE, myositis, systemic sclerosis, etc. However, the
presence of high-titre anti-RNP antibodies in isolation is the hallmark of mixed connective tissue
disease [34]. The presence of anti-RNP antibodies in SLE has an association with Raynaud's phenom-
enon and less risk of nephritis [35].

Anti-Sm antibodies

Anti-Sm antibodies are the most specic antibodies seen in SLE, and they are included in the Sys-
temic Lupus International Collaborating Clinics (SLICC) as well as 1987 ACR criteria. They are present in
15e30% of patients with SLE with a higher prevalence in Asians, and they are commonly associated with
nephritis, vasculitis and rash [36]. Anti-Sm antibodies are rarely seen in other autoimmune diseases.
Anti-Sm antibodies are directed against small nuclear RNA-associated proteins (snRNP) that form part of
the spliceosome. Most sera that have anti-Sm antibodies have antibodies against U1 snRNP.

Anti-topoisomerase I antibodies

Anti-topoisomerase I antibodies are also called anti-Scl 70 antibodies as they are directed against
70-kD protein on Western blotting, and they are present in patients with diffuse systemic sclerosis. The
presence of anti-topo 1 antibodies is associated with a higher mortality and a higher prevalence of
interstitial lung disease [37].

Anti-Jo1 antibodies

Anti-Jo1 antibodies are directed against histidyl-transfer RNA (tRNA) synthase. Anti-Jo1 and other
anti-tRNA synthase antibodies are seen mainly in patients with inammatory myositis, and they dene
 A. Aggarwal / Best Practice & Research Clinical Rheumatology 28 (2014) 907e920 915

a clinical subset characterized by the presence of fever, arthritis, Raynaud's phenomenon, interstitial
lung disease and frequent relapses [38]. Recently, some patients with isolated interstitial lung disease
were also found to have anti-tRNA synthase antibody [39].

Practice points
The practice points include the following:

1. Order ENA only when ANA is positive

2. Immunoblotting or ELISA can be used for their detection
3. Anti-Sm antibody is highly specific for SLE
4. Titres have a poor correlation with disease activity, so there is no need to repeat them

Anti-neutrophil cytoplasmic antibodies

ANCA are autoantibodies directed against antigens of primary granules of neutrophil cytoplasm.
Though multiple antigenic targets have been identied like ANA, only proteinase 3 and myeloperox-
idase (MPO) have clinical utility. ANCA is commonly associated with small vessel vasculitis. ANCA
testing has a good positive-predictive value only when the pre-test probability is high as the preva-
lence of primary vasculitis is very low. It has been suggested that ANCA screening should be done only
in select situations such as [40].

 Pulmonary renal syndrome

 Rapidly progressive renal failure
 Sub-glottic stenosis
 Pulmonary haemorrhage
 Retro-orbital mass
 Skin vasculitis with systemic features
 Mononeuritis multiplex
 Chronic non-resolving sinusitis
 Multiple pulmonary nodules

ANCA positivity in granulomatosis with polyangiitis (GPA) is nearly 100% in active systemic disease,
while in limited disease it varies from 60% to 70% [41]. It also entails a worse prognosis, whereas the
presence of ANCA in microscopic polyangiitis (MPA) has no prognostic signicance.
Like anti-dsDNA antibodies, longitudinal measurement of ANCA is useful in monitoring disease
activity as the titres fall after 3e4 months of treatment. Persistently elevated ANCA despite treatment
predicts a higher chance of relapse [42]. In addition, a recent modest rise in ANCA titre either by IIF or
by ELISA in a stable patient is followed by relapse in two-thirds of such patients [43]. However, a mere
increase in the antibody level does not warrant an increase in immune suppression, and it only pro-
vides a clue for a close follow-up for relapse.

Methods

ANCAs were initially detected using human neutrophils as substrate with IIF. On alcohol-xed
neutrophils as substrate, three patterns can be recognized: (i) cANCA pattern: diffuse cytoplasmic
staining with central accentuation; (ii) pANCA pattern: perinuclear linear staining around the nucleus
916 A. Aggarwal / Best Practice & Research Clinical Rheumatology 28 (2014) 907e920

(Fig. 2); and (iii) atypical pattern: diffuse cytoplasmic staining or both cytoplasmic and nuclear
staining. All IIF-positive samples should be tested by ELISA to determine the sub-specicity [44].
The indirect ELISA developed in the beginning had a low sensitivity due to loss of the antigenic
epitopes in the process of adsorption on to the ELISA plates. Capture ELISAs capture PR3 antigen using
antibodies. The most recent technique is highly sensitive (hs) ELISA, in which PR3 is immobilized via a
bridging molecule to the plastic plate. This process preserves most epitopes for ANCA binding. Capture
and hs ELISA were found to be comparable but superior to direct ELISA for the detection of PR3
antibody. For antibodies to MPO, all ELISAs perform equally well [45].
The international consensus statement on the testing and reporting for ANCA, 2003, recommends
the use of both ELISA and IIF for the reporting of ANCA. Whereas there is 85% concordance between IIF
and ELISA, 10% of patients are positive by IIF and 5% are positive only by ELISA. Combination gives
almost 99% specicity and around 70% sensitivity for vasculitis [46]. Automated readers for ANCA are
being explored, but they have not reached a stage to be used clinically [47].

Anti-phospholipid antibodies

Anti-phospholipid antibodies are again a group of antibodies directed against different phospho-
lipids such as cardiolipin (Cl), beta2 glycoprotein 1 (b2GP1), prothrombin, phosphotidyl serine, etho-
nalamine, etc. Out of these antibodies, only antibodies against Cl and b2GP1 are done in routine clinical
practice. Anti-phospholipid antibodies should be ordered in patients with

 Recurrent foetal loss

 Unexplained venous thrombosis
 Unexplained arterial thrombosis
 SLE

Methods

Both anti-Cl and anti-b2GP1 antibodies are detected using ELISA. The 2012 international recom-
mendations on the standardization of testing for anti-phospholipid antibodies [48] recommend that if
plasma is being tested, then it should be centrifuged properly to remove platelets as they interfere with
the assay, and for serum heat inactivation is not recommended as it can result in false-positive IgM
anti-Cl antibodies. High-afnity or gamma-irradiated ELISA plates are preferred for the assay. Due to
the lack of international standard, the polyclonal or monoclonal standards available should be used to
prepare calibrators. A standard-curve generation helps in better estimation of the antibody level rather
than the use of a single calibrator.

Fig. 2. Human neutrophils substrate showing ANCA: A, c-ANCA pattern and B, p-ANCA pattern.
 A. Aggarwal / Best Practice & Research Clinical Rheumatology 28 (2014) 907e920 917

Due to high inter-assay and intra-assay variability and as the results do not follow normal distri-
bution, percentiles should be used for cut-off. The results are reported as mild, moderate and marked
elevation. Traditionally, values <40 GPL/MPL are considered as low positive, 40e80 GPL/MPL as
moderate positive and >80 GPL/MPL as high positive. Values above 99 percentile of healthy control
population are taken as signicant and values between 95 and 99 percentiles should be considered as
indeterminate. Anti-b2GPI should be reported as positive or negative along with the numerical units.
Transient elevation of anti-phospholipid antibodies can occur due to multiple stimuli such as
infection and drugs. For the diagnosis of anti-phospholipid syndrome (APS), the persistent elevation of
antibodies, that is, moderate to high levels present on two or more occasions, is required for both anti-
Cl and anti-b2GPI antibodies [49].
IgA anti-Cl antibody testing is recommended when there is a high index of clinical suspicion, but
IgM and IgG antibodies are negative [50]. Similarly, testing for antibodies to phosphotidyl serine/
prothrombin complex in seronegative APS patients may pick up a proportion of patients with APS [51].
About 88% patients with APS have anti-Cl antibodies of which 32% had both IgG and IgM antibodies and
44% had IgG alone and 12% had only IgM antibodies. About 12% of patients have no anti-Cl antibodies
but have lupus anticoagulant [52]. International effort is on to reduce the inter-assay and intra-assay
variability as well as to have a uniformity of reporting across different laboratories [53].
Anti-Cl antibodies like other autoantibodies are seen in a variety of conditions (Table 4).

Miscellaneous antibodies

Antibodies to tissue transglutaminase are highly specic for the diagnosis of celiac disease, which
can also present with anaemia and joint pains. Anti-endothelial antibodies, though present in many
vasculitis and connective tissue diseases with vasculopathy, do not have much diagnostic value. An-
tibodies to smooth muscle can be detected by IIF using rat stomach as substrate, and they may help
differentiate SLE-related hepatitis and autoimmune chronic active hepatitis as in both ANA can be
positive.

Future of autoantibody in rheumatology

With sophistication in instrumentation, improvement in computing abilities and a quest for

personalized medicine, the patients will be better characterized to have accurate diagnosis and assess
response to therapy and prognosis.
In addition, standardization of methods and reporting are being improved with international
collaborative efforts, and this may pave way for a better diagnostic performance of tests. The Auto-
antibody Standardization Committee, European Autoimmune Standardization Initiative and Working
Group on Harmonization of Autoantibody Tests are working together to generate standards especially
afnity-puried antibodies or monoclonal antibodies that can be used to standardize the kits world-
wide [54].
Platform technologies are being explored to reduce dependence on observer for immunouores-
cence assays for ANA and ANCA. The AKLIDES system is one of them, which has been tested, and it

Table 4
Prevalence of anti-cardiolipin antibodies.

Group Prevalence

SLE 30e40%
RA 10e30%
SSc 10e20%
Drugs (chlorpromazine, procainamide, hydralazine, propylthiouracil) 5e15%
HIV infection 25e40%
Tuberculosis 20e30%
Normal population 2e5%
918 A. Aggarwal / Best Practice & Research Clinical Rheumatology 28 (2014) 907e920

utilizes pattern recognition software to report IIF patterns after automated image acquisition using a
camera. They have achieved about 90e95% concordance with manual interpretation [55].
Multiplex bead array utilizes uorescent beads coated with different antigens and a uorochrome
labelled anti-human IgG as detection antibody. The beads are analysed using a ow cytometer or a
Luminex platform. Multiple autoantibodies can be analysed in a single sample simultaneously. Alter-
natively, protein arrays can be used to analyse multiple autoantibodies. In a 96-well format, four au-
toantibodies in RA (three isotypes of RF and anti-CCP) were analysed and found to give a sensitivity of
82.5% and a specicity of 97.7% [56]. Similar studies using microarray have been explored in SLE. It is
still early to know if these multiplex assays offer any advantage in diagnosis or prognosis prediction in
systemic autoimmune rheumatic diseases [57].

Research agenda

 More automation and high throughput methods to meet increasing demands. This will mean
better antigens and instrumentation.
 Image analysis algorithms for the interpretation of IIF slides.
 Development of array of antigens for each disease such as citrullinated protein arrays for RA,
nuclear antigen array for disease associated with ANA, neutrophil antigen array for ANCA
associated diseases.
 Validation of autoantibodies in different clinical situations during disease (pre-disease, active
disease and inactive disease).

Summary

Autoantibodies are useful markers in systemic autoimmune rheumatic diseases. In suspected

rheumatoid arthritis, RF and ACPA are useful in diagnosis and both are included in the latest classi-
cation criteria, but in future ACPA may alone be used due to its high specicity. In patients with sus-
pected connective tissue diseases, ANA is a good screening test and the pattern can give an idea about
antigenic specicity. Further sub-specicity analysis should be dictated by the clinical condition, and it
can be done by ELISA or blotting. Anti-Sm has good specicity for SLE, anti-topoisomerase 1 for diffuse
systemic sclerosis and anti-Jo1 for inammatory myositis. ANCA should be tested both by IIF and by
ELISA for PR-3 and MPO for clinical utility. While testing for anti-Cl and anti-b2GP1 antibodies, a cut-off
above the 99th percentile of normal should be taken as signicant, and it should be retested 12 weeks
later to look for the persistence of antibodies.
In future, multiple autoantibody arrays for each disease such as RA, SLE, myositis, etc. may help in
the better denition of disease subsets for personalized treatment and prognosis. Further automation
and computing may increase the output and decrease the turnaround time for better patient
management.

Conicts of interest declaration

No conicts of interest to declare.

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