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 40.4

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 ).

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

Venn diagram representation of major clinico-serologic correlations in the primary AI-CTDs that impact the skin. Some targets of autoantibodies are specific (boxed with clinical associations) while others are seen in the context of overlapping clinical features. High levels of U1RNP autoantibody with overlapping features of all four of these AI-CTDs defines the concept of mixed connective tissue disease. CADM, clinically amyopathic dermatomyositis; IFIH1, interferon induced with helicase C domain-containing protein 1; MDA5, melanoma differentiation-associated protein 5; p155, a 155 kDa polypeptide; RF, rheumatoid factor; RNP, ribonucleoprotein, TIF1-γ, transcriptional intermediary factor 1 gamma.

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

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  historical and technical aspects of ANA assays

  
  
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  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.

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.

Detection of antinuclear antibodies (ANA) by indirect immunofluorescence.

Utilizing Hep-2 tumor cells as the substrate, patterns of nuclear immunofluorescence include: homogeneous ( A ); peripheral ( B ); speckled ( C ); nucleolar ( D ); and centromeric ( E ). E is illustrated on a chromosome preparation in metaphase arrest.

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 .