Clin Chem Lab Med 2014; 52(12): 17291737

Review
Daniela P. Foti, Marta Greco, Eleonora Palella and Elio Gulletta*

New laboratory markers for the management

ofrheumatoid arthritis patients
Abstract: Rheumatoid arthritis, the most prominent of
systemic autoimmune rheumatic diseases, represents
an important social health problem. Recent insights into
the immunopathogenic mechanism of this complex and
multiform illness might open new perspectives for a more
appropriate laboratory approach. In this review we focus
on the most relevant pathogenetic mechanism; indicating the laboratory biomarkers specifically linked to early
diagnosis, prognosis, evolutive aspects of the disease, and
therapeutic efficacy. Evidence based on laboratory medicine could provide the best outcome for patients.
Keywords: evidence-based laboratory medicine (EBLM);
immunopathogenesis; laboratory assay; rheumatoid
arthritis.
DOI 10.1515/cclm-2014-0383
Received April 7, 2014; accepted May 22, 2014; previously published
online June 14, 2014

Introduction
Rheumatoid arthritis (RA) is the most prominent of systemic autoimmune rheumatic diseases that may affect
different tissues and organs, although the synovial joints
represent the major target. The onset of RA is mainly
observed at the end of adolescence or between the 4th and
5th decades of life, while a second peak of incidence is
reported between 60 and 70 years. In general, the number
of cases increases with age, with a prevalence of 0.5%1%
in industrialized countries, and with rates of 5% in women
over 55years [1]. An early variant of RA is also described in
*Corresponding author: Elio Gulletta, MD, Department of Health
Sciences, Viale S. Venuta, 88100 Catanzaro, Italy,
Phone: +39 0961 3697243, Fax: +39 0961 3697415,
E-mail: gulletta@unicz.it
Daniela P. Foti, Marta Greco and Eleonora Palella: Clinical Pathology
Unit, Department of Health Sciences, University Magna Graecia,
Catanzaro, Italy

childhood [2]. This disease represents an important social

health problem because of its high incidence, invalidating progress and important disabilities. The involvement
of the synovial joints, that results in severe dysfunction,
leading to limitation of motion, may be accompanied and
complicated by several extra-articular manifestations
[35], such as pleural and pericardial diseases, rheumatoid nodules, Caplans syndrome, bronchiectasis [6, 7].
All these clinical conditions can lead to major reduction
in functional and work abilities and health-related quality
of life, while the RA-associated increased risk for cardiovascular disease may reduce life expectancy by 318years
[8]. Today, RA is considered a multifactorial disease, in
which environmental factors, such as several subsequent
viral infections, seem to play a role in the onset and persistence of the disease. Smoking is also considered a predisposing condition [9]. Genetic factors are, however, also
implicated in the susceptibility and the evolution of RA.
A genetic association with antigens of the major hystocompatibility complex (MHC)-II, and in particular human
leukocytes antigens (HLA)-DRB1, has been well established, although non-HLA associated loci have also been
linked to the disease [10, 11]. Although the cause of RA
is still unknown, autoimmunity plays an important role.
It has become increasingly evident that autoantibodies
mainly target proteins that undergo citrullination, a posttranslational modification frequently occurring during
inflammation, apoptosis, and keratinization. Pro-inflammatory substances released by immune cells are involved
in the activation and maintenance of inflammation that
causes swelling and subsequent damage of the cartilage
and bone inside the joint. It is hypothesized that superantigens may be also implicated, while self-antigens (collagen, proteoglycans, rheumatoid factor and citrullinated
proteins) probably play a role in the chronic evolution of
the process. At present, there is no universal cure able to
ultimately eradicate RA. The therapeutic approach in the
management of RA has witnessed revolutionary changes
over the last 15 years, thanks to the wider understanding of the molecular mechanisms involved in the pathogenesis of the disease. Glucocorticoids, non-steroidal

Unauthenticated
Download Date | 11/18/14 9:05 PM

1730Foti etal.: Laboratory approach to RA patients

anti-inflammatory drugs, and few disease modifying
anti-rheumatic drugs (DMARDs), including methotrexate,
sulphasalazine, hydroxychloroquine, and gold have been
the only treatment options until two decades ago. Since
the discovery of new therapeutic targets, a significant
breakthrough has been achieved in rheumatology with
the development of biological agents. The first class of
biologic agents include tumor necrosis factor (TNF) inhibitors, followed by many others with different mechanisms
of action, such as rituximab, abatacept, and tocilizumab.
These agents have changed the fate of patients with RA
due to their good efficacy and safety features. Most importantly, the role of B cells in the pathophysiology of RA has
been highlighted by depleting peripheral B cells using
anti-CD20 agents or inducing apoptosis by the inhibition
of B cell receptors and depleting this lymphocyte population from the germinal center [12]. This novel therapeutic
approach can target intracellular B cell signaling and regulatory B cells. Moreover, new cytokine-directed biological drugs targeting important proinflammatory mediators,
such as GM-CSF, interleukin (IL)-1, IL-6 and its receptor,
IL-17, IL-20, IL-21, IL-23, have been or will be soon available, hopefully enhancing our therapeutic choices [13].
All recent knowledge on pathogenetic mechanisms has
opened a new window upon the efficacious approach of
laboratory performances both in the choice of biomarkers,
and best practice regarding the patient outcome [1416].

Pathogenesis
Presently, a three-step model has been proposed for ACPApositive RA. In the first step, an environmental risk factor
can induce protein citrullination, while the new epitopes
may elicit the ACPA production in susceptible individuals,
bearing a peculiar haplotype. HLA DRB1 encoding the
most widespread MHC class II HLA-DR subunit paralog
is the genetic locus most specifically linked to the risk of
developing RA in the Caucasian population. Several other
genes have been considered as probands for the disease,
such as protein tyrosine phosphatase non-receptor type
22 (PTPN22) Arg620Trp, signal transducer and activator of transcription 4 (STAT4), TNF receptor-associated
factors (TRAF)1/C5, all of which are involved in specific
protein citrullination induced by environmental factors.
The DRB1 allele is responsible for peptide binding specificities and codes for a conserved sequence of amino acids
at residues 6774 of the DR chain, a region involved in
the accommodation of the antigenic peptide and recognition of the T cell receptor [17]. Interestingly, DRB1*0404

or *0401 antigen binding grooves fit better citrullinated,

rather than the corresponding arginine-bearing peptides.
Genome-wide single nucleotide polymorphism (SNP)
analyses of RA patients, compared with healthy controls, confirmed the genetic contribution of two of these
residues, 71 and 74, and of three other amino acids, all of
which are located in the peptide-binding groove, to RA
susceptibility [18]. These data classify high-, intermediate-, low-risk and non-susceptibility alleles, depending
upon the amino acid sequence between 70 and 74 residues within the groove. Instead, the variant PTPN22 is a
gain of function mutation that enhances the ability of its
gene product to inhibit T cell activation [19]. PTPN22, in
fact, codifies an intracellular tyrosine phosphatase (LyP),
which negatively regulates T lymphocyte activity, by modifying the intracellular signal of the T cell receptor (TCR). A
study on the PTPN22 polymorphism 1858T, anti-cyclic citrullinated peptides (CCP) antibodies, rheumatoid factors
of all different classes (IgM, IgA, IgG) and shared epitopes
genes (HLA-DRB1*0404 or *0401) has established their
strong relationship and the high positive predictive value
for RA. The presence of PTPN22 1858T polymorphism and
anti-CCP antibodies shows 100% specificity for RA [20].
In genetically predisposed individuals, several different
environmental factors, depending on the studied population, can trigger autoimmunity response to citrullinated
proteins, impairing tolerance. Among these, smoking and
Porphyromonas gingivalis, a self-producing citrullinated
protein bacterium involved in periodontitis, are attractive
etiological agents playing a role in gene/environment/
autoimmunity interactions [21].
In the second step, an inflammatory response can
develop into the articular synovial layer, with the recruitment of ACPA and the deposition of immunocomplexes. In
RA, ACPA are pathogenic, and specific for this disease, and
have a strong association with HLA-DRB1 shared epitopes
alleles. In rheumatoid synovium, peptidylarginine deiminase isoforms peptidylarginine deiminase (PAD)2 and
PAD4 are abundant, thereby determining an active local
citrullination of proteins, such as fibrin. Citrullinated
epitopes appear longer before the clinical symptoms and
they are associated with more severe joint erosions and
an evolution of clinical course. Citrullination, a modification of arginine side chains catalyzed by PAD enzymes
(deimination) increases the affinity of several peptides for
shared epitopes, thus expressing HLA-DR molecules and
improving their presentation to T cells. The most wellcharacterized epitopes are filaggrin, fibrinogen, vimentin,
type II collagen, and -enolase. Cellular survival response
can be triggered by misfolded proteins in the endoplasmic reticulum (ER). ER-stress associated genes are highly

Unauthenticated
Download Date | 11/18/14 9:05 PM

Foti etal.: Laboratory approach to RA patients1731

expressed in the rheumatoid synovium and synovial cells.

ER chaperone glucose-related protein 78 (GRP78), in vitro,
is crucial for the proliferation of synoviocytes and angiogenesis, and strongly contributes to RA pathogenesis [22].
In summary, autoimmunity to citrullinated protein antigens has specificity for RA and defines a clinically and
genetically distinct form of the disease.
In the final step, the presence of immunocomplexes
induces the increasing presence of immunity cells and
cytokine production, which can act together to perpetuate,
in RA, the chronic inflammation at articular sites. Dysregulated expression of cytokines drives both inflammation
and tissue destruction, thereby implicating the evaluation
of cytokines pattern for a complete and effective understanding of patients immunoresponse [23]. Recently, the
role of IL-36a, which is up-regulated in RA synovium, has
been pointed out. This interleukin, expressed by plasma
cells, and leading to IL-6 and IL-8 production by synovial
fibroblasts, may represent the biological link between
autoimmunity and the gradual development of synovitis [24]. Hypoxia and Th1 lymphocytes-driven inflammation contribute to the pathogenesis of RA, although Th1
cytokines are not sufficient to induce angiogenesis, even
together with hypoxia. Instead, Th2 cytokines induce
angiogenic activity in normoxic and hypoxic conditions,
suggesting a complex and not yet clarified interaction
between hypoxia, angiogenesis and inflammation [25].
The presence of IL-17 in synovial fluid and tissue of RA
patients may suggest an important pathogenic role of this
cytokine, as supported by several mouse models. IL-17A
induces angiogenesis, cell migration and cell invasion, all
key processes in the pathogenesis of RA, by stimulating
GRO and monocyte chemoattractant protein-1 (MCP-1)
expression in RA synovial fibroblasts [26]. Interestingly,
MCP-1 and regulated on activation normal T cell expressed
and secreted, (RANTES) polymorphisms do not contribute to individual RA susceptibility or severity in the Caucasian population [27]. Involvement of MCP-1, IL-1, IL-6,
IL-8, TNF- in RA synovial alterations has been described
for many years. In each single patient, the integration
of cytokine profiles with autoantibody response may be
extremely important for prognosis, for the choice of the
most appropriate therapeutic treatment, and the evaluation of its efficacy [28]. Moreover, there are more CD4+
CD25high Treg cells present in synovial fluid than in peripheral blood, where they have shown a functional defect
impairing their ability to suppress CD4+ CD25 T cells. This
defect can be reversed by anti-TNF- therapy [29]. Furthermore, NK cells seems to play an exclusive role in the
pathogenesis of autoimmune disease, and in the case of
RA, these specialized cells represent a significant fraction

of the lymphocytes (8%25%) in the synovial fluid of RA

patients and could be detected in the joint early during
the disease course [30]. The majority of the NK cells in the
synovial fluid of RA patients are CD56bright (approx. 60%
of NK cells), a subphenotype also found in the blood of
RA patients, but at much lower frequencies (approx. 10%
of NK cells). The synovial NK show an elevated expression of CD94/NKG2A and decreased expression of killer
immunoglobulin-like receptor (KIR)s and CD16. Due to
an upregulated expression of several chemokine receptors and adhesion molecules, they may participate in the
preferential recruitment into the synovium [31]. The synovial CD56bright NK cells express higher levels of activation
markers (CD69 and NKp44) and produce more TNF- and
IFN- than those from the peripheral blood of the same
patients. [32]. Synovial NK cells could induce monocytes
to differentiate into DCs, and, as reported, produce IL-22,
a cytokine that promotes proliferation of synovial fibroblasts, thereby representing a target for molecular therapy
[33]. Aberrant expression of MHC class I polypeptiderelated sequence A in the inflamed synovium may augment
CD56bright NK cell activation, resulting in dysregulated
production of proinflammatory cytokines rather than in
immunoregulation. Taken together, these findings suggest
that the enrichment of CD56bright NK cells may contribute
to the initiation and/or perpetuation of dysregulated production of proinflammatory cytokines in the synovium of
RA patients [34]. The genetic associations between inflammatory arthritis and KIR haplotypes support the hypothesis that dysregulation of cytokine production by CD56bright
NK cells in the synovium and/or decreased cytotoxicity by peripheral CD56dim NK cells may contribute to the
pathogenesis of RA. Distinct antibody profiles have been
associated with subgroups of patients who exhibited high
serum levels of TNF-, IL-1, IL-6, IL-13, IL-15 and GM-CSF.
Significantly increased autoantibody reactivity against
citrullinated epitopes was observed in patients within the
cytokine high subgroup [35, 36]. Increased levels of TNF, IL-1, IL-12p40 and IL-13, and the chemokines eotaxin/
chemokine ligand (CCL)11, MCP-1 and interferon-inducible
protein 10, were present in early RA as compared with the
controls [37]. Between chemokines, only IL8/cysteine x
cysteine (CXC)L8 concentrations were higher in patients
with psoriatic arthritis and ankylosing spondylitis than in
patients with RA [38, 39]. Increased blood levels of proinflammatory cytokines are associated with autoantibodies targeting citrullinated antigens and surrogate markers
of disease activity in patients with early RA [40]. Proteomic analysis of serum autoantibodies, cytokines and
chemokines enables stratification of patients with RA into
molecular subgroups [41] (Table 1).

Unauthenticated
Download Date | 11/18/14 9:05 PM

1732Foti etal.: Laboratory approach to RA patients

Table 1Factors involved in rheumatoid arthritis.
Molecules and receptors
PTPN22
TCR/BCR signaling
TNFAIP
Ubiquitin editing enzyme; TNFR signaling and
NFkB pathway inhibitor
TRAF1
TNFR signaling and NF-kB pathway regulator
NKp46
Activation of NK
Transcription factors
REL
NF-kB pathway component
STAT4
IFN- pathway regulator
Cytokines and receptors
IL-2/IL-21
T cells regulation
CTL4
T cells costimulation inhibitor
CD40
B/T cells costimulator; production of IgM, TNF-,
IL-2
TNF-
Proinflammatory; Pro-osteoclastogenesis
(RANKL)
IL-6
Proinflammatory; Pro-osteoclastogenesis
(RANKL)
IL-1
Moderate pro-inflammatory
IL-17
Blocking anti-osteoclastogenetic factors (IL-4,
IFN-; IL-12)
RANKL
Osteoclast differentiation
M-CSF
Essential for osteoclast differentiation
IL-15
Pro-osteoclastogenesis inducing RANKL
IL-8
Chemotactic factor
IL-22
NK activation
IFN-
Proinflammatory response, NK autocrine function
Enzymes
PAD4
Enzymatic peptide citrullination
Polymorphisms
VEGF-A
Early onset
MMP-1; MMP-3 Matrix degradation; hypoxic effect

Biomarkers
The most widely accepted diagnostic criteria for RA
suggest that laboratory tests can make an essential contribution. Despite the fact that several markers can be measured by widely validated analytical procedures, not all of
them allow early diagnosis as they only become positive
in the well-established active phase of the disease. On

the basis of international guidelines, their assay is still

used as corollary procedure for determining a first diagnosis [42]. Evidence-based laboratory medicine (EBLM)
predicts that a laboratory biomarker should have several
properties: it should be present in a biosample which is
representative of the disease localization; recorded from
a patient, and thus specifically linked to his/her disease;
and it has to be measured by a standardized and reproducible analytical methodology. The ideal biomarker
should clearly indicate: health/disease condition; high/
low exposure to risk factors; effectiveness/no response to
therapy. Thus, each different laboratory biomarker can be
used properly in a selected phase of clinical decision (predictivity, diagnosis, prognosis, monitoring therapeutic
efficacy or adverse effects), and in all cases, their choice
must be related to a real improvement of the patient management [43] (Figure 1).
Common and uncommon laboratory biomarkers of
RA are summarized in Table 2.
RF is the prototypic marker of RA for diagnostic and
prognostic purposes, since it is associated with joint
destruction, even if is not clearly indicated as a mediator
of these effects. Moreover, in seropositive RA patients, the
levels of RF are not related to bone damage and status of
the disease [44]. For several years, evaluation of RF Ig isotypes has been used to enhance the serological diagnosis
of RA [45]. More recently, it has been proposed as a useful
tool to predict patient response to biological therapy
[46]. Several proteolytical enzymes can be involved in
the pathogenesis of RA. Between those, metalloproteinases represent a family of important factors which cause
the destruction of articular tissue. The role that proteolytic enzymes play in RA can be summarized as follows:
matrix metalloproteinases (MMP 1,2,3,9); cystein proteinases (cathepsin B,H,L); serin proteinase (elastase, PA,
cathepsin G); and aspartic acid proteinase (cathepsin D).
MMP-3 can be a very useful marker for the prediction of
joint destruction [47]. MMP-3 (stromelisyn-1) is expressed
by synovial and articular cells, fibroblasts, chondroblasts,

Figure 1Evolutive steps in RA.

Unauthenticated
Download Date | 11/18/14 9:05 PM

Foti etal.: Laboratory approach to RA patients1733

Table 2Laboratory markers in RA.

Biohumoral
Rheumatoid factor (RF) IgM, IgA, IgG
hsCRP
ESR
Metalloproteinases: MMP-3
Autoantibodies
ANA (cytoplasmic pattern endosomes-lysosomes)
ACPA (anti-citrullinated peptides) (vimentin, type II collagen,
-enolase, fibrinogen)
Antibodies antithrombospondin-1
Anti-hnRNP A2/B1 (RA33)
Cytokines and molecular markers
IL-1, IL-6, TNF-, IL-10, IL-17, VEGF, MCP-1
Cellular and genetic markers
Treg lymphocytes
NK, NKT
HLA DRB1, PTPN22 Arg620Trp, STAT4, TRAF1/C5

and osteoclasts. It acts upon extracellular substrates of

cartilages, such as fibronectin, collagen IV and V, elastin,
proteoglycans, but also together with other MMPs in the
disruption of cartilages [48]. It is present in the synovial
fluid during the active phase of disease and its levels correlate with serum concentrations, independently from
patients age and severity of the disease. It is produced
very early during the course of RA and elevated serum
levels can be predictive of articular disruption, within
612 months from the event. Together with RF, it can be
a laboratory marker of active phlogosis and predictive of
invalidant evolution [49]. Polymorphisms in the vascular
endothelial factor A (VEGF-A) gene and MMP-1,3 intergenic
locus may influence the age of onset of RA [50]. Currently,
a more specific immunological profile has been proposed
to evaluate each patient at the onset of disease, or their
relatives in order to predict the risk of disease. All antiCCP and RF isotypes analyzed occurred more commonly
in unaffected first-degree relatives from multicase families
than in the controls, but had different isotype distribution
from the patients with RA [51].
Anti-CCP-2 antibodies of both the IgG and IgA isotypes pre-dated the onset of RA by years; also, both IgG
and IgA anti-CCP-2 antibodies predicted the development
of RA, with the highest predictive value for IgG anti-CCP-2
antibodies; individuals who subsequently developed RA
had increased concentrations of several cytokines and
chemokines years before the onset of symptoms of joint
disease [52].
The immunological profile consists in the definition
of haplotype, evaluation of ACPA, and ANA autoantibodies (cytoplasmic pattern), measurement of plasma levels
of Th1 and Th2 cytokine networks. A seminal paper has

settled down the importance to identify a complete profile

of RA-associated antibodies (AAB) to improve early diagnosis of the disease and provide prognostic and theragnostic indications. Anti-CCP antibodies demonstrate the
best diagnostic performances for profiling, thus they
must be used as first-line screening and identification of
subgroups of patients. The use of multiplex assays may
facilitate a wider implementation of profiling [53]. Moreover, a positive result of the immunoenzymatic evaluation of anti-citrullinated peptides (ACPA) against several
structural proteins has to be considered the highest
specific biomarker for RA. Even if in some patients IgM
anti-second generation CCP-2 autoantibodies appear at
the initial phase of the disease and seem to persist more
frequently, both the IgG and IgA isotypes of anti-CCP-2
antibodies predate the onset of RA by years; the highest
positive predictive value is associated with IgG anti-CCP-2
autoantibodies. However, ACPA is not an appropriate
marker to monitor the disease [54, 55]. Autoantibodies
against citrullinated proteins can be an important factor
of osteoclastogenesis. Plasma cells produce ACPA with
specificity for citrullinated vimentin, which binds to osteoclast precursor cells and stimulates the release of TNF,
in turn enhancing the differentiation of these cells into
mature osteoclasts. During the osteoclast differentiation
process, the production of PAD2 enzyme is induced by
calcium. The activity of PAD2 leads to the citrullination of
vimentin, which is abundantly expressed on the surface
of osteoclast-lineage cells [56]. Discrimination between
RA and non-RA patients could be performed using CCP
antibodies evaluation, especially in RF-negative patients
[57]. Evidence to date suggests that the positivity of antiCCP antibodies may be a predictor of RA onset. This analytical data can have a higher positive predictive value in
a population at risk, rather than in a general one, thus
the evaluation of haplotype profile can improve the early
diagnostic outcome. A multi-biomarker test for the assessment of disease activity in RA patients has been performed
using CAMERA study. This test may be complementary
to the available disease activity laboratory evaluation to
improve patient care and outcomes [58]. Four protein biomarkers, whose mass/charge (m/z) values have been well
established, were identified as novel biomarkers related to
RA, adding great value for laboratory diagnosis of RA [59].
In a recent study, a Multiple Biomarker Disease Activity
(MBDA) score algorithm has been tested. It uses the same
equation of the Disease Activity Score DAS28-CRP, which
takes into account the C-reactive protein (CRP), and biomarkers useful to predict the Swollen Joint Count (SJC28),
Tender Joint Count (TJC28), and general health (VAS-GH)
as components of the equation (PTJC=predicted TJC;

Unauthenticated
Download Date | 11/18/14 9:05 PM

1734Foti etal.: Laboratory approach to RA patients

PSJC=predicted SJC; PVAS-GH=predicted VAS-GH). By
using a Venn diagram to predict potential markers, Curtis
etal. evaluated and validated this multiple biomarker test
to assess RA disease activity with a score, expressed from
1 to 100 [60]. More recently, using a multi-stage approach,
the same group tested 130 candidate biomarkers, and
ultimately set up an algorithm with 12 protein biomarkers to obtain an MBDA score for RA patients, with no
effects from common comorbidities [61]. A study from a
Japanese research group has demonstrated that MMP-3
ELISA method works well in the assessment of MMP-3
serum levels in routinely evaluated RA patients and the
test may be important to better characterize the disease
outcome in the follow-up [62]. The results of this study
suggest that serum MMP-3 can be considered as a predictor of joint destruction in RA, treated with a non-biological disease modifying anti-rheumatic drug. Variations
of the MMP-1 and MMP-3 genes affect the serum levels of
MMP-1 and -3 and disease activity. MMP-3 is significantly
associated with disease activity, inflammatory mediators
and cartilage breakdown, making it a potential biomarker
of disease severity, but seemingly less useful than CRP
and SAA as a biomarker of disease activity in early RA. A
conclusive remark is that several closely linked polymorphisms in the MMP-1-MMP-3 loci have an important role in
determining the circulating levels of these MMPs in RA,
and that MMP-3 polymorphism is associated with the level
of disease activity over time [63].
Recently, using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) tools which is a randomeffects method, Zhang et al. were able to summarize
sensitivities, specificities, positive and negative likelihood
ratio (LR+ and LR, respectively), and diagnostic odds
ratio from 17 studies. They concluded that based upon
their high specificity and moderate sensitivity, anti-CCP-2
and anti-CCP-3 played an important role in confirming the
diagnosis of RA. Anti-CCP-3 did not have better diagnostic
performance than anti-CCP-2, but anti-CCP-2 had evident
heterogeneity compared to anti-CCP-3, especially in American patients [64].
From pathogenic and clinical evidence, we may
suggest a new approach of laboratory medicine to evaluate patients in all different evolutive phases of RA. First,
the haplotype of the proband should be characterized,
and in case of positivity, the relatives must be invited to
take the test. This can be useful to predict the risk level
of developing the disease. As mentioned before, internationally adopted clinical criteria imply that the laboratory
data are important to make the diagnosis. Together with
basic laboratory analytical evaluation, in our opinion it
is appropriate to measure RF and ACPA. If both tests are

positive, in order to completely and correctly evaluate

the patient, a cytokine profile and MMP-3 measurements
should be undertaken. The first can be obtained by multiparametric microarray and allows the patient immunological response, the prevalence of Th1 or Th2, cytokine
network and the production of Th17 to be evaluated. The
latter can be considered the biomarker of disease aggressiveness and progression. This complete laboratory evaluation can produce a correct and personalized therapeutic
treatment and prognostic evaluation [65]. In the future, a
significant improvement can be obtained by the evaluation of genomics and proteomics arrays, in order to characterize the individual patient profile at diagnosis and its
response to therapeutic treatment.

Conclusions
The evaluation of multiple biomarker profiles is fundamental in order to enrol the patient in a specific target
therapy treatment. This is most likely to be effective and
safe based on similarities between patients disease characteristics and the cohort that responds to therapy. In this
prospective, analysis of multiple biomarkers, and especially of post-translational biomarkers in RA, can help to
stratify the patients population in different subsets that
exhibit different outcomes and respond to specific therapeutic treatment.
In summary:
1. RA is a complex disease with different clinically
aspects and evolutionary forms.
2. HLA-DRB1 shared epitope and PTPN22 (gain-offunction of Lyp which inhibits T-lymphocytes
activation) are two genes strongly associated with the
risk of RA.
3. IL-6 and TNF- play a central role in RA. IL-17
can be measured either in synovial fluid either in
compromised tissues.
4. Treg CD4+CD25hi lymphocytes are predominant in
synovial fluid, rather than in blood. Their levels can
be normalized as effect of anti-TNF therapy.
5. NKp46 cytofluorimetric assay can account for NK
activation.
6. Anti-CCP antibodies are specifically present during
RA and support distinction between clinically and
genetically different forms of RA.
7. All environmental factors which can alter antigenic
tolerance against anti-CCP antibodies are not yet
defined.
8. Anti-CCP-antibodies are present in serum several
years before the early phase of disease (pre-clinical

Unauthenticated
Download Date | 11/18/14 9:05 PM

Foti etal.: Laboratory approach to RA patients1735

window); anti-CCP antibodies and high levels of

MMP-3 are associated with bone erosion and with a
worse clinical evolution of the disease, if present in
the synovial fluid.
9. The time-course measurement of the cytokine network
can be effective for setting prognosis and therapeutic
efficacy.
10. Multiparametric laboratory evaluation can be the
correct approach in order to define different subgroups
of patients and tailor a personalized therapy.
Conflict of interest statement
Authors conflict of interest disclosure: The authors
stated that there are no conflicts of interest regarding the
publication of this article.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared

References
1. Gabriel SE, Michaud K. Epidemiological studies in incidence,
prevalence, mortality, and comorbidity of the rheumatic diseases. Arthritis Res Ther 2009;11:229.
2. Dannecker GE, Quartier P. Juvenile idiopathic arthritis: classification, clinical presentation and current treatments. Horm Res
2009;72:412.
3. Cimmino MA, Salvarani C, Macchioni P, Montecucco C, Fossaluzza V, Mascia MT, etal. Extra articular manifestations in
587 Italian patients with rheumatoid arthritis. Rheumatol Int
2000;19:2137.
4. Cavagna L, Boffini N, Cagnotto G, Inverardi F, Grosso V, Caporali
R. Atherosclerosis and rheumatoid arthritis: more than a simple
association. Mediat Inflamm 2012;2012:147354.
5. Gauhar UA, Gaffo AL, Alaron GS. Pulmonary manifestations of
rheumatoid arthritis. Sem Respir Crit Care Med 2007;28:
43040.
6. Lauretis A, Veeraraghavan S, Renzoni A. Connective tissue
disease-associated interstitial lung disease: how does it differ
from IPF? How should the clinical approach differ? Chronic Resp
Dis 2011;8:5382.
7. Turesson C. Extra-articular rheumatoid arthritis. Curr Opin Rheumatol 2013;25:3606.
8. Anderson JJ, Wells G, Verhoeven AC, Felson DT. Factors predicting response to treatment in rheumatoid arthritis: the importance of disease duration. Arthritis Rheum 2000;43:229.
9. Pincus T, Kavanaugh A, Sokka T. Benefit/risk of therapies for
rheumatoid arthritis: underestimation of the side effects or
risks of RA leads to underestimation of the benefit/risk of therapies. Clin Exp Rheumatol 2004;22(Suppl 35):211.
10. Oliver JE, Silman AJ. Risk factors for the development of rheumatoid arthritis. Scand J Rheumatol 2006;35:16974.

11. Deane KD, El-Gabalawy H. Pathogenesis and prevention of

rheumatoid disease: focus on preclinical RA and SLE. Nat Rev
Rheumatol 2014;10:21228.
12. Nakken B, Munthe LA, Konttinen YT, Sandberg AK, Szekanecz Z,
Alex P, etal. B-cells and their targeting in RA, current concepts
and future perspectives. Autoimmun Rev 2011;11:2834.
13. Burmester GR, Feist E, Drner T. Emerging cell and cytokine
targets in rheumatoid arthritis. Nat Rev Rheumatol 2014;10:
7788.
14. Imboden BJ. The immunopathogenesis of rheumatoid arthritis.
Annu Rev Pathol Mech Dis 2009;4:41734.
15. Smolen JS, Alehata D. Monitoring rheumatoid arthritis. Curr
Opin Rheumatol 2011;23:2528.
16. Smolen JS, Alehata D, Redlich K. The pathogenesis of rheumatoid arthritis: new insights from old clinical data? Nat Rev
Rheumatol 2012;8:23543.
17. MacKay K, Eyre S, Myerscough A, Milicic A, Barton A, Laval S,
etal. Whole-genome linkage analysis of rheumatoid arthritis
susceptibility loci in 252 affected sibling pairs in the United
Kingdom. Arthritis Rheum 2002;46:6329.
18. Raychaudhuri S, Sandor C, Stahl EA, Freudenberg J, Lee HS, Jia
X, etal. Five amino acids in three HLA proteins explain most
of the association between MHC and seropositive rheumatoid
arthritis. Nat Genet 2012;44:2916.
19. Hammer J, Gallazzi F, Bono E, Karr RW, Guenot J, Valsasnini
P, etal. Peptide binding specificity of HLA-DR4 molecules:
correlation with rheumatoid arthritis association. J Exp Med
1995;181:184755.
20. Tzenas du Montcel S, Michou L, Petit-Teixeira E, Osorio J,
Lemaire I, Lasbleiz S, etal. New classification of HLA-DRB1
alleles supports the shared epitopes hypothesis of rheumatoid
arthritis susceptibility. Arthritis Rheum 2005;52:10638.
21. Marchesan JT, Gerow EA, Schaff R, Taut AD, Shin SY, Sugai J,
etal. Porphyromonas gingivalis oral infection exacerbates the
development and severity of collagen-induced arthritis. Arthritis
Res Ther 2013;15:R186.
22. Yoo SA, You S, Yoon HJ, Kim DH, Kim HS, Lee K, etal. A novel
pathogenic role of the ER chaperone GRP78/BiP in rheumatoid
arthritis. J Exp Med 2012;209:87186.
23. McInnes I, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Immunol 2007;7:42942.
24. Frey S, Derer A, Messebacher ME, Baeten DL, Bugatti S, Montecucco C, etal. The novel cytokine interleukine-36a is expressed
in psoriatic and rheumatoid arthritis synovium. Ann Rheum Dis
2013;72:156974.
25. Larsen H, Muz B, Khong TL, Feldmann M, Paleolog EM. Differential effects of Th1 versus Th2 cytokines in combination
with hypoxia on HIFs and angiogenesis in RA. Arthritis Res Ther
2012;14:R180.
26. Moran EM, Connolly M, Gao W, McCormick J, Fearon U, Veale DJ.
IL-17A induction of angiogenesis, cell migration, and cytoskeletal rearrangement. Arthritis Rheum 2011;63:326373.
27. Pavkova Goldbergova M, Lipkova J, Pavek N, Gatterova J, Vasku
A, Soucek M, etal. RANTES, MCP-1 chemokines and factors
describing rheumatoid arthritis. Mol Immmunol 2012;52:2738.
28. Schett G, Gravellese E. Bone erosion in rheumatoid arthritis:
mechanisms, diagnosis and treatment. Nat Rev Rheumatol
2012;8:65664.
29. Sarkar S, Fox DA. Regulatory T cells in rheumatoid arthritis. Curr
Rheumatol Rep 2008;10:40512.

Unauthenticated
Download Date | 11/18/14 9:05 PM

1736Foti etal.: Laboratory approach to RA patients

30. Fogel LA, Yokoyama WM, French AR. Natural killer cells in human
autoimmune disorders. Arthritis Res Ther 2013;15:21625.
31. Antonelli A, Ferrari SM, Giuggioli D, Ferrannini E, Ferri C, Fallahi
P. Chemokine (C-X-C motif) ligand (CXCL)10 in autoimmune
diseases. Autoimmun Rev 2014;13:27280.
32. Huss RS, Huddleston JI, Goodman SB, Butcher EC, Zabel
BA. Synovial tissue-infiltrating natural killer cells in osteoarthritis and periprosthetic inflammation. Arthritis Rheum
2010;62:3799805.
33. Yang X, Zheng SG. Interleukin-22: a likely target for treatment of
autoimmune diseases. Autoimmun Rev 2014;13:61520.
34. Conigliaro P, Scrivo R, Valesini G, Perricone R. Emerging role
for NK cells in the pathogenesis of inflammatory arthropathies.
Autoimmun Rev 2011;10:57781.
35. Hueber W, Tomooka BH, Zhao X, Kidd BA, Drijfhout JW, Fries JF,
etal. Proteomic analysis of secreted proteins in early rheumatoid arthritis: anti-citrulline autoreactivity is associated with
up regulation of proinflammatory cytokines. Ann Rheum Dis
2007;66:7129.
36. Sokolove J, Bromberg R, Deane KD, Lahey LJ, Derber LA, Chandra
PE, etal. Autoantibody epitope spreading in the pre-clinical
phase predicts progression to rheumatoid arthritis. PLoS One
2012;7:e35296.
37. Hughes-Austin JM, Deane KD, Derber LA, Kolfenbach JR,
Zerbe GO, Sokolove J. Multiple cytokines and chemokines are
associated with rheumatoid arthritis-related autoimmunity in
first-degree relatives without rheumatoid arthritis: studies of
the aetiology of rheumatoid arthritis (SERA). Ann Rheum Dis
2013;72:9017.
38. Proost P, Struyf S, Loos T, Gouwy M, Schutyser E, Conings R,
etal. Coexpression and interaction of CXCL10 and CD26 in
mesenchymal cells by synergising inflammatory cytokines:
CXCL8 and CXCL10 are discriminative markers for autoimmune
arthropathies. Arthritis Res Ther 2006;8:R107.
39. Szekanecz Z, Szucs G, Szanto S, Koch AE. Chemokines in rheumatic diseases. Curr Drug Targets 2006;7:91102.
40. Noack M, Miossec P. Th17 and regulatory T cell balance in
autoimmune and inflammatory diseases. Autoimmun Rev
2014;13:66877.
41. Li XJ, Xu M, Zhao XQ, Zhao JN, Chen FF, Yu W, etal. Proteomic
analysis of synovial fibroblast-like synoviocytes from rheumatoid arthritis. Clin Exp Rheumatol 2013;31:5528.
42. Neogi T, Aletaha D. Silman AJ, Naden RL, Felson DT, Aggarwal
R, etal. American College of Rheumatology; European League
against Rheumatism. The 2010 American College of Rheumatology/European League Against Rheumatism classification
criteria for rheumatoid arthritis: phase 2 methodological report.
Arthritis Rheum 2010;62:258291.
43. Lindstrom TM, Robinson WH. Biomarkers for rheumatoid arthritis: making it personal. Scand J Clin Lab Invest 2010;70(Suppl
242):7984.
44. Aletaha D, Alasti F, Smolen JS. Rheumatoid factor determines
structural progression of rheumatoid arthritis dependent and
independent of disease activity. Ann Rheum Dis 2013;72:
87580.
45. Kleveland G, Egeland T, Lea T. Quantitation of rheumatoid factors (RF) of IgM, IgA and IgG isotypes by a simple and sensitive
ELISA. Discrimination between false and true IgG-RF. Scand J
Rheumatol Suppl 1988;75:1524.

46. Can M, Najip A, Ylmaz N, Inanc N, Yavuz S. Immunoglobulin

subtypes predict therapy response to the biologics in patients
with rheumatoid arthritis. Rheumatol Int 2013;33:145560.
47. Burrage PS, Mix KS, Brinckerhoff CE. Matrix metalloproteinases:
role in arthritis. Front Biosci 2006;11:52943.
48. Mamehara A, Sugimoto T, Sugiyama D, Morinobu S, Tsuji G,
Kawano S, etal. Serum MMP-3 as predictor of joint destruction
in RA, treated with non-biological diseases modifying anti-rheumatic drugs. Kobe J Med Sci 2010;56:E98107.
49. Houseman M, Potter C, Marshall N, Lakey R, Cawston T, Griffiths
I, etal. Baseline serum MMP-3 levels in patients with rheumatoid arthritis are still independently predictive of radiographic
progression in a longitudinal observational cohort at 8years
follow up. Arthritis Res Ther 2012;14:R30.
50. Chen Y, Mattey DL. Age at onset of rheumatoid arthritis: association with polymorfisms in the VEGFA gene and an intergenic locus between MMP 1 and 3 genes. Clin Exp Rheumatol
2012;30:8948.
51. rlestig L, Mullazehi M, Kokkonen H, Rocklv J, Rnnelid J,
Dahlqvist SR. Antibodies against cyclic citrullinated peptides
of IgG, IgA and IgM isotype and rheumatoid factor of IgM and
IgA isotype are increased in unaffected members of multicase
rheumatoid arthritis families from northern Sweden. Ann Rheum
Dis 2012;71:8259.
52. Kokkonen H, Mullazehi M, Berglin E, Hallmans G, Wadell G,
Rnnelid J, etal. Antibodies of IgG, IgA and IgM isotypes against
cyclic citrullinated peptide precede the development of rheumatoid arthritis. Arthritis Res Ther 2011;13:R13.
53. Conrad K, Roggenbuck D, Reinhold D, Drner T. Profiling of
rheumatoid arthritis associated autoantibodies. Autoimmun Rev
2010;9:4315.
54. Jaskowski TD, Hill HR, Russo KL, Lakos G, Szekanecz Z, Teodorescu M. Relationship between rheumatoid factor isotypes and
IgG anti-cyclic citrullinated peptide antibodies. J Rheumatol
2010;37:15828.
55. Szodoray P, Szabo Z, Kapitany A, Gyetvai A, Lakos G, Szanto S,
etal. Anti-citrullinated protein/peptide autobodies in association with genetic and environmental factors as indicators
of disease outcome in rheumatoid arthritis. Autoimmun Rev
2010;9:1403.
56. Wegner N, Lundberg K, Kinloch A, Fisher B, Malmstrom V, Feldmann M, etal. Autoimmunity to specific citrullinated proteins
gives the first clues to the etiology of rheumatoid arthritis.
Immunol Rev 2010;233:3454.
57. Bizzaro N, Tonutti E, Tozzoli R, Villalta D. Analytical and diagnostic characteristics of 11 2nd- and 3rd-generation immunoenzymatic methods for the detection of antibodies to citrullinated
proteins. Clin Chem 2007;53:152733.
58. Bakker MF, Cavet G, Jacobs JW, Bijlsma JW, Haney DJ, Shen
Y, etal. Performance of a multi-biomarker score measuring
rheumatoid arthritis disease activity in the CAMERA tight control
study. Ann Rheum Dis 2012;71:16927.
59. Niu Q, Huang Z, Shi Y, Wang L, Pan X, Hu C. Specific serum protein biomarkers of RA detected by MALDI-TOF-MS combined with
magnetic beads. Int Immunol 2010;22:6118.
60. Curtis JR, van der Helm-van Mil AH, Knevel R, Huizinga TW,
Haney DJ, Shen Y, etal. Validation of a novel multi-biomarker
test to assess rheumatoid arthritis disease activity. Arthritis
Care Res (Hoboken) 2012;64:1794803.

Unauthenticated
Download Date | 11/18/14 9:05 PM

Foti etal.: Laboratory approach to RA patients1737

61. Centola M, Cavet G, Shen Y, Ramanujan S, Knowlton N, Swan KA,
etal. Development of a multi-biomarker disease activity test for
rheumatoid arthritis. PLoS One 2013;8:e60635.
62. Shinozaki M, Inoue E, Nakajima A, Hara M, Tomatsu T, Kamatani N, etal. Elevation of serum matrix metalloproteinase-3 as
a predictive marker for the long-term disability of rheumatoid
arthritis patients in a prospective observational cohort IORRA.
Mod Rheumatol 2007;17:4038.
63. Ally MM, Hodkinson B, Meyer PW, Musenge E, Tikly M, Anderson
R. Serum matrix metalloproteinase-3 in comparison with acute

phase proteins as a marker of disease activity and radiographic damage in early rheumatoid arthritis. Med Inflamm
2013;2013:183653.
64. Zhang WC, Wu H, Chen WX. Meta-analysis: diagnostic accuracy
of anti-cyclic citrullinated peptide 2 antibody and anti-cyclic citrullinated peptide 3 antibody in rheumatoid arthritis. Clin Chem
Lab Med 2014;16:112.
65. Deane KD, Norris JM, Holers VM. Preclinical rheumatoid arthritis: identification, evaluation and future directions for investigation. Rheum Dis Clin North Am 2010;36:21341.

Daniela P. Foti, MD, PhD, Associate professor of Clinical P

athology
at University Magna Graecia, Catanzaro, Medical School. Her
principal scientific interest is the pathophysiology of Diabetes mellitus and the best biomedical laboratory performances to evaluate
patients affected by chronic degenerative diseases.

Eleonora Palella, MD, Post-graduate resident in Clinical Pathology at

University Magna Graecia, Catanzaro, Medical School. Her principal
scientific interest is dealing with biomedical laboratory evaluation
of patients affected by chronic degenerative diseases.

Marta Greco, MS Sci., Specialist in Clinical Pathology, Assistant

professor of Clinical Pathology at University Magna Graecia, Catanzaro, Medical School. Her principal scientific interest is dealing with
biomedical laboratory evaluation of patients affected by vascular
and chronic degenerative diseases.

Elio Gulletta, MD, PhD, Full professor and Chair of Clinical Pathology
at University Magna Graecia, Catanzaro, Medical School. His principal professional interest is dealing with pathophysiology of chronic
inflammatory diseases, and biomedical laboratory performances to
increase patient outcomes.

Unauthenticated
Download Date | 11/18/14 9:05 PM