simplified overview for dermatologists about autoantibodies in connective tissue diseases

1. Auto antibodies in Autoimmune
Connective Tissue Diseases
BY
Mostafa A. Sanad

2. INTRODUCTION
• The autoimmune connective tissue diseases (AI-CTDs) are a group
of polygenic clinical disorders often having heterogeneous and
overlapping clinical features; the term ‘rheumatic diseases’ can be
used synonymously for AI-CTD. It should be noted that the older
designation,‘collagen vascular diseases’, is an obsolete terminology
that should be avoided.
• A hallmark of these disorders is the production of circulating
autoantibodies(aAb) that have been identified by various
immunochemical techniques. For convenience, these aAb have
been referred to as a group as ‘antinuclear antibodies’ (ANA).
However, the term ANA is also used to describe the results of a
specific immunofluorescence microscopy assay that detects
circulating aAb that are reactive with the nuclei of human cells.

3. Our discussion will necessarily focus on the clinical significance of the various aAb
associated with AI-CTDs that have expression in the skin (i.e. lupus erythematosus
[LE], dermatomyositis[DM], systemic sclerosis [SSc]).

4. ANA
• THE CLASSICAL ANA ASSAY
• Importance of Technical Aspects of the ANA
Assay
• Age-Related ANA Results
• Drug-Induced ANA/SLE
• Immunofluorescence Patterns
• Clinical Value of aAb in Diagnosis Versus Disease
Activity Monitoring
• How Does One Evaluate a Positive ANA Result in
a Patient

5. THE CLASSICAL ANA ASSAY
• Although there continues to be debate about its role in
the diagnosis and management of AI-CTD4,5, the
classical ANA indirect immunofluorescence assay is still
considered to be the most clinically efficient screening
test for systemic autoimmune disorders such as SLE.
• A number of the potential pitfalls and questions must
be considered when interpreting the results of the ANA
assay. The following discussion will be framed around
these issues, and a more detailed description of these
issues can be found elsewhere.

6. Importance of Technical Aspects of
the ANA Assay
• As the name implies, the ANA assay identifies antibodies present in serum that
bind to autoantigens present in the nuclei of mammalian cells. The current
version of the ANA assay used in almost all clinical laboratories employs a
human tumor cell line such as Hep-2 for the nucleated cell substrate. aAb are
detected with a fluorochromeconjugated antiserum that is specific for human
immunoglobulin (the aAb) that is bound to nuclei in the cell substrate
• In earlier versions of the ANA assay, rodent cells rather than human cells were
used as the substrate.
• Thus, in the past, some sera from SLE patients (especially those containing
predominately anti-Ro aAb) were negative in the rodent cellbased ANA assay
(i.e. ‘ANA-negative SLE’).
• This occurred in up to 15% of certain SLE patient populations (especially those
enriched in anti-Ro aAb-associated disorders such as subacute cutaneous LE
(SCLE) and Sjِ gren’s syndrome (SjS).
• However, only ~1–2% of SLE patients today are ANA-negative using a human
tumor cell line-based substrate such as Hep-2. Thus, ‘ANA-negative SLE’ is
predominantly a historical phenomenon and of little current clinical
significance.

7. Importance of Technical Aspects of
the ANA Assay
• It is also important to remember that the determination of
ANA assay results is via the subjective interpretation by a
laboratory technician of the disappearance of nuclear
fluorescence. Thus, one might expect a certain degree of
inherent variability in ANA results.
• Such variability is more often encountered when different
laboratory technicians read and report ANA results
• Although an attempt has been made by the World Health
Organization to standardize reporting of ANA results with
an international unit system (e.g. 1 IU rather than a titer of
160), virtually all clinical laboratories in the US continue to
report ANA results using a titer system.

8. ‘Normal’Versus ‘Abnormal’ ANA
Values
• The commercial ANA kits that are used most commonly in
laboratories today usually indicate that an ANA titer of 40
or 80 is considered abnormal.
• However, using such low ANA titer cut-offs creates a lot of
positive but clinically insignificant ANA test results.
• A number of studies that have compared ANA results in SLE
populations with those in normal control populations have
indicated that a titer of <160 using a human tumor cell line
substrate has little clinical utility.
• It also must be realized that some otherwise healthy
individuals can produce ‘abnormal’ levels of ANA. For
example, approximately 5% of otherwise healthy young
individuals will have an ANA titer of 160 or higher.

9. Age-Related ANA Results
• One aspect of normal human aging is that
older individuals more often produce elevated
levels of ANA in the absence of associated
systemic autoimmune disease.
• Up to 15% of otherwise healthy individuals 55
years of age and older can be found to have a
significantly elevated ANA titer of no apparent
clinical consequence.

10. Drug-Induced ANA/SLE
• A number of drugs are known to be capable of triggering a positive
ANA and/or a drug-induced SLE syndrome (e.g. procainamide,
hydralazine, isoniazid, chlorpromazine, phenytoin, methyldopa). An
important dermatologic drug, minocycline, has recently been added
to this list. The predominant clinical manifestations of drug-induced
SLE are musculoskeletal symptoms (arthritis, arthralgia, myalgia)
and serositis (pleuritis, pericarditis). Skin involvement is seen much
less commonly in the drug-induced form of SLE compared to
idiopathic SLE.
• Antihistone aAb represent a serologic marker of drug-induced ANA
and the classical form of drug-induced SLE. A different set of drugs
(e.g. hydrochlorothiazide,diltiazem, griseofulvin, terbinafine) has
been reported as capable of triggering anti-Ro aAb production as
well as the cutaneous lesions of SCLE. Antihistone aAb do not
appear to be produced in this setting.

11. Induction of ANA and Anti-dsDNA Antibodies by
Biologic Inhibitors/Blockers of Tumor Necrosis Factor-
a• TNF-a blockade by recombinant proteins has become increasingly
common in the treatment of an expanding number of disorders.
• To date, there have been at least 40 reported cases of lupus or a
lupus-like syndrome associated with etanercept but none
developed nephritis (although one case of etanercept-associated
nephritis has been described).
• Infliximab, a chimeric monoclonal anti-TNF-a antibody, is approved
for psoriatic arthritis and severe psoriasis. Compared to
etanercept,infliximab induces a higher rate of aAb formation.
• TNF-a inhibitor-induced lupus differs from typical drug induced
lupus in several ways. Firstly, skin manifestations, including malar
rash, SCLE, DLE, photosensitivity and purpura, are
prominent.Secondly, ANA and anti-dsDNA antibodies are
predominant, not antihistone antibodies.

12. Immunofluorescence Patterns

13. Immunofluorescence Patterns
• Various patterns of nuclear immunofluorescence
can be observed in the ANA assay (i.e.
homogeneous , peripheral, speckled, nucleolar
and centromeric.
• With one exception, these patterns have little
disease specificity and are of little value in the
management of patients. The exception is the
centromeric which is seen predominantly in
patients having the limited cutaneous form of SSc
(CREST syndrome).

14. Clinical Value of aAb in Diagnosis Versus Disease
Activity Monitoring
• In everyday practice, these laboratory tests can be used to
establish the diagnosis of an AI-CTD and to monitor the
level of systemic activity of an AI-CTD over time.
• Most aAb serve one of these two functions. As an example,
the Sm aAb is highly specific for SLE and thus has
considerable diagnostic value. However, the absolute
amounts of anti- Sm aAb present in the circulation of a
patient at different points in time do not correlate strongly
enough with SLE disease activity to have practical clinical
value in patient management.
• Likewise, there is not much value in serial determinations
of titers of the ANA assay to monitor disease activity in SLE.
However, some aAb have both types of clinical value (e.g.
anti-dsDNA; antineutrophil cytoplasmic antibodies [ANCA]).

15. How Does One Evaluate a Positive ANA Result
in
a Patient Suspected of Having SLE?
• This question would likely be answered differently by
different specialists. Rheumatologists might initially
order a broader array of laboratory tests in order to
expedite diagnosis and prognostic assessment in
patients having active SLE that is capable of assuming
many confusing forms of clinical expression.
• However, dermatologists usually see a highly selected
population of LE patients by virtue of their having
some form of cutaneous involvement that at times can
be the only significant clinical manifestation of the
autoimmune disease process (e.g. discoid LE, SCLE).

16. How Does One Evaluate a Positive ANA Result in
a Patient Suspected of Having SLE?

17. AUTOANTIBODIES ENCOUNTERED IN LUPUS
ERYTHEMATOSUS
• The various aAb encountered in SLE are subdivided into those that
areand are not specific for SLE.
• aAb to Sm and dsDNA are specific and prevalent enough to
represent one of the 11 ACR classification criteria for SLE . aAb to
single-stranded (ss) DNA are also seen in LE but are not at all
specific for this disorder. Rising levels of anti-dsDNA aAb correlate
positively with SLE disease activity (especially when associated with
falling complement values). High levels can reflect an increased risk
for LE nephritis.
• The popular ELISA assays preferentially detect low avidity anti-
dsDNA aAb that are not as specific for SLE as are high-avidity anti-
dsDNA aAb. It is recommended that all positive antidsDNA aAb
ELISA results be confirmed by a more specific assays that are
adaptable to the clinical immunology laboratory, such as
counterimmunoelectrophoresis.

18. AUTOANTIBODIES ENCOUNTERED IN LUPUS
ERYTHEMATOSUS
• Anti-Ro (SS-A) and -La (SS-B) aAb were originally thought to react
exclusively with cytoplasmic RNP and were thus designated as cytoplasmic
aAb. However, subsequent work has documented that these aAb react
with proteins present in human cytoplasmic RNP (hYRNP) that are found
in both the cytoplasm and nucleus.
• Anti-Ro aAb can occur alone; however, it is extremely rare for anti-La aAb
to occur without anti-Ro aAb also being present.
• As with anti-dsDNA aAb assays, it is important to understand certain
technical considerations relating to anti-RNP aAb assays, such as those
used to measure anti-Ro, -La, -U1RNP and -Sm aAb. A number of these
aAb were originally identified by Ouchterlony double immunodiffusion,
which is a less sensitive but highly specific technique for identifying such
aAb. However, this assay has been replaced by solidphase immunoassays
such as ELISA . The latter tend to be more sensitive but less specific for
detecting anti-RNP aAb
• Ten percent of normal individuals had Ro antibody by ELISA, with less than
0.3% having such antibodies by immunodiffusion.

20. AUTOANTIBODIES ENCOUNTERED IN
DERMATOMYOPATHIES
• Patients with adult-onset clinically amyopathic DM appear to have a
somewhat higher prevalence of positive ANAs (63%) than do
patients with classical DM or PM.
• Anti-Jo-1 and anti-Mi-2 are better-established myositis-specific
aAb;however, their prevalence is so low that they do not have much
utility in a routine clinical setting.
• Anti-Jo-1 is one of several myositis-specific aAbs . The presence of
one or more of these ‘anti-synthetase’ aAb correlates positively
with a subset of mostly PM and some DM patients that have an
increased risk of arthritis, Raynaud’s phenomenon, and interstitial
lung disease (this clinicoserologic constellation has been referred to
as the ‘anti-synthetase syndrome’). The mechanic’s hand skin lesion
was originally described to occur in this setting.
• Anti-Mi-2 aAb are seen in highest frequency in patients having the
hallmark skin lesions of DM (Gottron’s papules, Gottron’s sign,
shawl sign).

22. AUTOANTIBODIES ENCOUNTERED IN SYSTEMIC
SCLEROSIS AND LOCALIZED SCLERODERMA
(MORPHEA)
• The two aAb having the greatest clinical utility within the
scleroderma spectrum of illnesses are those that react with a
centromere protein (CENP-B) and a DNAunwinding enzyme,
topoisomerase I (originally named Scl-70). Anticentromere aAb
correlate most strongly with the limited cutaneous form of SSc
(CREST syndrome) while the DNA topoisomerase I specificity (anti-
Scl-70) is seen more commonly in the more severe form of SSc
marked by the presence of diffuse cutaneous scleroderma
• While there tends to be very little clinical overlap between SSc and
localized scleroderma (morphea), some degree of serologic overlap
has been described. aAb to fibrillin-1, histones and ssDNA have
been found in 30–50% of patients with localized scleroderma. As
with classical ANA, these antibodies have been observed more
often in patients who have the more widespread forms of localized
scleroderma, such as linear scleroderma and generalized morphea.

25. Biomarkers
• By the US Food and Drug Administration (FDA),biological marker or
biomarker is defined as a characteristic that is objectively measured and
evaluated as an indicator of normal biologic processes, pathogenic
processes,or biological responses to a therapeutic intervention
• So, it’s not just aAs, There are several classes of biomarkers that are widely
used in drug development.
• Diagnostic biomarkers are used to identify specific diseases or abnormal
conditions
• Prognostic biomarkers are used to categorize patients by degree of risk for
disease occurrence or progression.
• Predictive biomarkers are baseline characteristics and are used to group
patients by their likelihood for response to a particular therapeutic
intervention. The ability to predict favorable or unfavorable responses to a
particular therapy may help to select the appropriate target population(s)
with increased likelihood of response to that treatment.

26. Biomarkers
• Pharmacodynamic (PD) biomarkers are markers of a certain
pharmacological response and are generally used to guide the dose
selection or dosing regimens for clinical trials. The ideal PD markers
are typically downstream of the drug targets, such as the use of
type I interferon (IFN) signatures for anti-type I IFN therapy in SLE.
• Surrogate biomarkers or surrogate endpoints are biomarkers used
to substitute for a clinical efficacy endpoint. Clinical endpoint is a
characteristic or variable that reflects how a patient feels, functions
or survives.It is the most credible assessment of the benefits and
risks of an investigational drug in clinical trials.Surrogate endpoint is
expected to reasonably likely predict clinical benefit (or harm, or
lack of benefit or harm), based on epidemiologic, therapeutic,
pathophysiologic,or other evidence. However, a single biomarker is
generally not able to account for all the effects that may result from
a treatment.