Autoantibodies Encountered in Patients with Autoimmune Connective Tissue Diseases


Key features

  • Autoantibodies can be of significant value in the diagnosis, management, and prognosis of autoimmune connective tissue diseases (AI-CTDs), but their interpretation depends on the type of autoantibody and the specific AI-CTD

  • The classic ANA assay remains the entrée into the world of AI-CTD serology, and understanding its limitations is critical to clinical decision making

  • The evolution of clinical immunology laboratory technology has altered the significance of some of the original clinical–serologic correlations reported at the time of discovery of the autoantibodies. Basic understanding of key technical issues related to current laboratory methodologies is therefore important

  • There has been a trend toward adopting solid-phase immunoassay techniques (e.g. ELISA) to detect many autoantibodies. For several autoantibodies, including anti-SSA/Ro and anti-double-stranded (ds) DNA antibodies, this has resulted in some decrease in disease specificity

  • Certain autoantibodies have significant disease specificity and thus can be of great diagnostic value: anti-dsDNA and anti-Smith (Sm) for systemic lupus erythematosus (SLE); anti-Mi-2 for classic dermatomyositis; anti-Jo-1 for antisynthetase syndrome; anti-topoisomerase-1 (Scl-70), anti-RNA polymerase III, and anti-centromere for different clinical forms of systemic sclerosis; and cytoplasmic antineutrophil cytoplasmic antibodies (c-ANCA) for granulomatosis with polyangiitis. However, most autoantibodies fall into the disease-nonspecific category

  • The absolute blood levels of a few autoantibodies can correlate positively with underlying autoimmune disease activity (e.g. anti-dsDNA in SLE, c-ANCA in granulomatosis with polyangiitis), but in most cases the titer does not correlate with disease activity

Introduction

Autoimmune connective tissue diseases (AI-CTDs) represent polygenic clinical disorders that have heterogeneous and overlapping clinical features. Sometimes they are referred to as “autoimmune rheumatic diseases”. These disorders are characterized by an immune dysregulation that includes the production of antibodies to self-antigens, termed autoantibodies (aAb). The aAb in patients with AI-CTD often target structures vital to cellular metabolism and division.

When the diagnosis of an AI-CTD is being considered, the most commonly requested laboratory test is measurement of antinuclear antibodies (ANA). ANA are commonly defined as aAb that target primarily nuclear components, including DNA or small nuclear ribonucleoproteins (snRNP), and are detected by the fluorescent antinuclear antibody (FANA) test. Elevated titers of ANA are seen in multiple AI-CTD, from systemic lupus erythematosus (SLE) to systemic sclerosis (SSc) and dermatomyositis (DM). These aAb reflect basic inflammatory events in tissue, but rarely carry pathogenic potential (the exception being ANCA). ANCA target cytoplasmic structures and assist in the diagnosis of systemic vasculitides (see Ch. 24 ).

If used appropriately, clinical laboratory testing for ANA can be useful in the diagnosis and management of AI-CTD patients . In order to maximize the clinical utility of aAb testing in this setting, familiarity with the serologic tests currently used to identify these aAb, as well as the disease associations of the various aAb, is helpful. This chapter focuses on these latter two aspects, while Chapter 41, Chapter 42, Chapter 43, Chapter 44, Chapter 45 contain an in-depth discussion of the various AI-CTDs. Therefore, the molecular identity of the various autoantigens and their clinical associations are summarized in table form (see Tables 40.2–40.4 ). An effort will also be made to illustrate the often complex relationships that can exist between the presence and relative amounts of these aAb, disease diagnosis, and prognosis. In addition, the basic principles of clinical utility as related to the various aAb laboratory tests (e.g. sensitivity, specificity) will be briefly addressed. The related subjects of circulating immune complexes and cryoglobulins are discussed in Chapter 4, Chapter 23, Chapter 24 .

Table 40.2
Autoantibodies associated with lupus erythematosus.
aAb, autoantibody; AI-CTD s , autoimmune connective tissue diseases; ANA, antinuclear antibodies; DLE, discoid lupus erythematosus; DM/PM, dermatomyositis/polymyositis; IF, immunofluorescence; LE, lupus erythematosus; MCTD, mixed connective tissue disease; PCNA, proliferating cell nuclear antigen; RA, rheumatoid arthritis; RNP, ribonucleoprotein; SCLE, subacute cutaneous lupus erythematosus; SjS, Sjögren syndrome; SLE, systemic lupus erythematosus; SSc, systemic sclerosis.
Adapted from .
AUTOANTIBODIES ASSOCIATED WITH LUPUS ERYTHEMATOSUS
Target Median prevalence * Molecular specificity Clinical associations
High specificity for SLE
dsDNA 60% Double-stranded (native) DNA LE nephritis and monitoring activity of nephritis
Sm 10–30% of
Caucasians;
30–40% of
Asians and African-Americans
Splicesome RNP (ribonucleoprotein particles involved in splicing pre-mRNA)
rRNP 7–15%; 40% of Asians Ribosomal P proteins (proteins involved in ribosome function) Neuropsychiatric LE
Low specificity for SLE
ANA (most common IF patterns: homogeneous, peripheral) 99%
ssDNA 70% Denatured DNA Possible risk for SLE in DLE patients; also seen in RA, DM/PM, MCTD, SSc, SjS, morphea
C1q 60% C1q component of complement Severe SLE, hypocomplementemic urticarial vasculitis syndrome
PCNA 50% A component of multiprotein complexes involved in cellular proliferation
U1RNP 50% Splicesome RNP Overlapping features with other AI-CTDs; MCTD (100%)
SSA/Ro 50% hYRNP (quality control function for misfolded RNA molecules) SCLE (75–90%), neonatal LE/congenital heart block (99%), SCLE–SjS overlap, primary SjS (70%); associated with vasculitis
SSB/La 20% hYRNP SCLE (30–40%), SCLE–SjS overlap, primary SjS (40%); occurs in conjunction with SSA/Ro
Cardiolipin 50% Cardiolipin, a negatively charged phospholipid Recurrent spontaneous abortions, thrombocytopenia, and hypercoagulable state in SLE (cutaneous manifestations include livedo reticularis, leg ulcers, acral infarction/ulceration, hemorrhagic cutaneous necrosis); similar associations in primary antiphospholipid antibody syndrome; clinical manifestations have strongest association with IgG class of anti-cardiolipin
β2 glycoprotein I 25% An important cofactor for cardiolipin in cardiolipin aAb assays Relatively high risk of thrombosis in SLE and primary antiphospholipid antibody syndrome
Histones 40% Histones Drug-induced SLE; also RA, SLE, and SSc with pulmonary fibrosis (in conjunction with other aAb)
Rheumatoid factor 25% Fc portion of IgG Nonspecific
Ku 10% DNA end-binding repair protein complex Overlap with other AI-CTDs such as DM/PM, SSc
Alpha-fodrin 10% An actin-binding protein found at the periphery of chromaffin cells that may be involved in secretion SjS

* Based on most common assay techniques currently employed in clinical immunology laboratories. Note that these figures represent the authors' best estimates based on most recently published data.

Listed in decreasing order of prevalence within categories.

Table 40.3
Autoantibodies encountered in the idiopathic inflammatory dermatomyopathies (DM/PM).
ANA, antinuclear antibodies; CADM, clinically amyopathic DM; CHB, congenital heart block; DM/PM, dermatomyositis/polymyositis; IF, immunofluorescence; IFN, interferon; LE, lupus erythematosus; RNP, ribonucleoprotein; SCLE, subacute cutaneous lupus erythematosus; SjS, Sjögren syndrome; SLE, systemic lupus erythematosus; SSc, systemic sclerosis.
Adapted from .
AUTOANTIBODIES ENCOUNTERED IN THE IDIOPATHIC INFLAMMATORY DERMATOMYOPATHIES
Target Median prevalence * Molecular specificity Clinical association
High specificity for DM/PM
p155 80% (clinically amyopathic); 20–30% (classic) Transcriptional intermediary factor 1 gamma (TIF1-γ) Clinically amyopathic DM; in adult-onset classic DM, increased risk of malignancy; extensive cutaneous involvement; mucocutaneous findings include palatal erythema (“ovoid patches”), psoriasiform lesions, and hypopigmented and telangiectatic (“red on white”) patches
Mi-2 15% Helicase nuclear proteins Gottron papules/sign, shawl sign, cuticular telangiectasias, cuticular overgrowth/dystrophy
Jo-1 20% Histidyl tRNA synthetase Antisynthetase syndrome
PL-7 5% Threonyl tRNA synthetase Antisynthetase syndrome
PL-12 3% Alanyl tRNA synthetase Antisynthetase syndrome
OJ Rare Isoleucyl tRNA synthetase Antisynthetase syndrome
EJ Rare Glycyl tRNA synthetase Antisynthetase syndrome, possibly increased frequency of skin changes
SRP 5% Signal recognition particle (intracytoplasmic protein translocation) Fulminant DM/PM, cardiac involvement
Fer Rare Elongation factor 1-α
Mas Rare Small RNA
MDA5/CADM-140 10–15% of Caucasians; 10–45% of adult Asians; 5 (in the UK) –35 (in Japan)% of juveniles Melanoma differentiation-associated protein 5 (MDA5)/IFN induced with helicase C domain protein 1 (IFIH1) Clinically amyopathic DM, rapidly progressive interstitial lung disease, cutaneous ulcerations with associated vasculopathy, tender palmar papules, oral pain and ulceration
NXP-2 1–15% of adults; 20–25% of juveniles Nuclear matrix protein
(MORC family CW-type zinc finger 3 [MORC3])
In adults, associated with malignancy, subcutaneous edema, and calcinosis; in juveniles, associated with more severe muscle disease and calcinosis
Low specificity for DM/PM
ANA (most common IF patterns: speckled, nucleolar) 40% Clinically amyopathic DM (65%)
ssDNA 40% Single-stranded DNA SLE, SSc, morphea
PM-Scl (PM-1) 10% Ribosomal RNA processing enzyme Overlap with SSc
SSA/Ro (especially 52 kDa Ro) 15% hYRNP Overlap with SjS, SCLE, neonatal LE/CHB, SLE
U1RNP 10% Splicesome RNP Overlap with other AI-CTDs
Ku 3% DNA end-binding repair protein complex Overlap with SSc
U2RNP 1% Splicesome RNP Overlap with SSc

* Using current assay techniques.

Table 40.4
Autoantibodies associated with systemic sclerosis (SSc) and morphea (localized scleroderma).
The hallmark aAb of SSc are shaded in orange. Patients with SSc can also develop additional aAb including: anti-endothelial cell aAb which may induce apoptosis of endothelial cells, but are nonspecific; anti-NAG-2 aAb which are expressed on vascular endothelial cells and fibroblasts and may also induce endothelial cell apoptosis; anti-U11/U12 RNP antibodies (in ≤3% of SSc patients), which have been associated with pulmonary fibrosis; and anti-topoisomerase IIα aAb which are more commonly seen in patients with morphea (~75% of patients with morphea in one series). ANA, antinuclear antibodies; ECM, extracellular matrix; HMG, high mobility group; IF, immunofluorescence; NAG-2, tetraspan novel antigen-2; PDGFR, platelet-derived growth factor receptor; RNP, ribonucleoprotein.
Adapted from .

Chapter Organization

Although the various AI-CTDs are distinct clinical entities, they can have features in common. Consequently, each AI-CTD has aAb specific to that particular disorder as well as other aAb that are shared with the other diseases. In addition, some patients can display overlapping clinical features of several different AI-CTDs and, as a result, an overlap in the pattern of aAb production ( Fig. 40.1 ).

Fig. 40.1, Correlation between clinical and serologic manifestations of autoimmune connective tissue diseases (AI-CTDs).

This chapter is organized based upon two major considerations in the use of aAb testing:

  • historical and technical aspects of ANA assays

  • aAb associated with AI-CTDs that have cutaneous manifestations.

Historical Perspective

One can better appreciate the evolution of thought that has occurred in this area by considering the change in methodology used to identify and measure these aAb over the past 60 years . In 1948, the bone marrow from a patient with SLE was noted to contain polymorphonuclear neutrophils that had phagocytosed nuclear material from degenerated cells. This became known as the LE cell phenomenon and occurred in the presence of antibody to DNA. The presence of these cells became an indicator of SLE. Approximately 10 years later, it was shown that indirect immunofluorescence could be used to detect ANA (i.e. the FANA test), providing a more sensitive assay for SLE.

Another decade later, Tan and co-workers employed the Ouchterlony double immunodiffusion technique to identify and define precipitating serum aAb that reacted with saline extractable nuclear antigens (ENA) such as nuclear ribonucleoprotein (nRNP), Sm, SSA/Ro, and SSB/La . Unfortunately, the Ouchterlony double immunodiffusion technique is time-consuming and expensive. However, a more efficient adaptation of the double immunodiffusion technique, counterimmunoelectrophoresis, can be used as an alternative to the less expensive solid-phase immunoassays (e.g. e nzyme- l inked i mmuno s orbent a ssay [ELISA]) that can be less specific (see below) .

In 1979, the molecularly and genetically defined ANA era began as the result of work by Lerner and Steitz, who employed immunodiffusion and Western blotting to define the molecular identities of the nRNP and Ro:La families of ribonucleoprotein autoantigens. These observations generated the notion that the various AI-CTD aAb might be more efficiently and cost-effectively assayed by using purified and/or recombinant forms of the various autoantigens in solid-phase immunoassays such as the ELISA. While less expensive and more convenient, there are a number of drawbacks to employing the ELISA and related immunoassay techniques to detect ANA such as anti-SSA/Ro and anti-dsDNA aAb, in particular increased sensitivity and decreased specificity. Newer techniques (e.g. solid-phase assays, proteome microarrays, Luminex xMAP® technology) are now being used more frequently, allowing for the simultaneous profiling of all relevant aAb in a given patient. Understanding the actual immunochemical techniques used to detect and quantify the various types of ANA can be helpful to the clinician, but is beyond the scope of this chapter. The reader is referred to a review by Griesmacher and Peichl .

FANA: the Classic ANA Assay

Although there is debate about its role in the diagnosis and management of AI-CTD , the classic ANA indirect immunofluorescence assay (i.e. FANA) is still the most clinically efficient screening test for systemic autoimmune disorders such as SLE . A number of potential pitfalls must be considered when interpreting the results of the ANA assay ( Table 40.1 ). The following discussion will focus on the key aspects of interpreting ANA test results.

Table 40.1
Major issues related to the interpretation of results of antinuclear antibody (ANA) assays.
AI-CTD, autoimmune connective tissue disease.
MAJOR ISSUES RELATED TO THE INTERPRETATION OF RESULTS OF ANTINUCLEAR ANTIBODY (ANA) ASSAYS
  • Methods of detecting and reporting ANA (e.g. use of a human tumor cell line substrate leads to increased sensitivity but decreased specificity)

  • Subjective nature of ANA assay endpoint

  • “Normal” versus “abnormal” ANA levels

  • Patient age

  • Drug-induced ANA

  • ANA can be seen in disorders other than AI-CTDs

  • Sontheimer's corollary to Greenwald's Law of Lupus

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 (or cytoplasm) of mammalian cells. Both the pattern and the titer are reported , with the former consisting of morphological descriptors that reflect the localization of the autoantigen. The reported titer represents the last dilution at which the ANA pattern is detectable, and a titer of 1 : 40 or greater is considered “positive” (see below). 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 fluorochrome-conjugated antiserum that is specific for human immunoglobulin (the aAb) that is bound to nuclei in the cell substrate ( Fig. 40.2 ). Earlier versions of the ANA assay utilized rodent cell lines and the latter lacked some of the autoantigens present in human cell nuclei (e.g. SSA/Ro). Thus, sera from some SLE patients could be negative when assayed on rodent cells, especially if the predominant aAb were anti-SSA/Ro (i.e. “ANA-negative SLE”). This occurred in up to 15% of certain SLE patient populations, particularly those enriched in anti-SSA/Ro aAb-associated disorders such as subacute cutaneous LE (SCLE) and Sjögren syndrome (SjS). Nowadays, because of the use of human Hep-2 cells, only ~1–2% of SLE patients are ANA-negative. Thus, “ANA-negative SLE” is predominantly an historical phenomenon.

Fig. 40.2, Detection of antinuclear antibodies (ANA) by indirect immunofluorescence.

An important caveat with regard to the FANA test is that determination of the titer (which reflects the serial serum dilutions necessary for fluorescence to disappear) depends upon subjective interpretation by a laboratory technician. Consequently, a single serum specimen can have an ANA titer result that varies within a two-tube dilution range. In other words, a serum sample reported as having an ANA titer of 1 : 320 might be read as 1 : 160 or 1 : 640 upon repeat testing in the same laboratory within the same timeframe. 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 1 : 160), many clinical laboratories in the US continue to report ANA results using a titer system. More recently, automated systems have been introduced in an attempt to decrease variability and increase cost-effectiveness; however, no pattern is reported and sometimes the result is simply positive or negative with no titer provided .

“Normal” Versus “Abnormal” ANA Values

The ANA titer that is considered to be abnormal can vary significantly, depending upon how the assay is performed and interpreted. The commercial ANA kits that are used most commonly in laboratories today usually indicate that an ANA titer of 1 : 40 is considered abnormal, presumably because accepting such relatively low levels of ANA as being abnormal retains a high degree of sensitivity of the ANA assay in detecting systemic AI-CTD. However, using such low ANA titer cut-offs creates a lot of positive, but clinically insignificant, ANA test results, i.e. it reduces specificity. A number of studies that have compared ANA results in SLE populations with those in normal control populations have indicated that a titer of <1 : 160, using a human tumor cell line substrate, has little clinical utility. For example, in one report based upon 15 international laboratories, the ANA positivity rate in a population of healthy individuals (ages 20–60 years) was 13.3% at 1 : 80, 5.0% at 1 : 160, and 3.3% at 1 : 320 . Of note, the elderly, relatives of those with SLE, and patients with other autoimmune disorders (e.g. autoimmune thyroid disease), as well as healthy individuals, may have abnormally elevated ANA titers.

Clinical Significance of ANA

You're Reading a Preview

Become a Clinical Tree membership for Full access and enjoy Unlimited articles

Become membership

If you are a member. Log in here