The detection of pathology-specific antibody profiles is a key diagnostic criterion for the characterization of patients with connective tissue disease.
The most used serological tests are the detection of anti- nuclear antibodies (ANA), extractable nuclear antigens (ENA), and anti-dsDNA. The current nomenclature and acronyms for ANA and ENA are being re-evaluated; however, due to their widespread use, they will be retained as such in this chapter.
In recent years, the demand for these tests has increased significantly, due in part to the acquisition of new evidence on their clinical relevance but also to an inappropriate pre scription, not well quantified, for:
• Insufficient communication between clinicians and laboratories, resulting in disagreement on the diagnostic significance of available tests and test results
• Different analytical techniques available for the same marker, availability of new target antigens
• Lack of both homogeneous terminologies and diagnostic algorithms, resulting in inhomogeneous behaviour
The in-depth evaluation of the clinical significance and diagnostic rationale of the laboratory’s ANA, ENA and dsDNA tests is at the heart of the approach to connective tis sue disease, identifying screening tests and in-depth tests that may allow an appropriate request to the clinical question.
The presence of ANA in serum is considered a marker of high diagnostic significance in collagen diseases.
The positive ANA test represents, in fact, a classificatory criterion in the diagnostic definition of non-organ-specific autoimmune diseases, such as systemic lupus erythematosus (SLE), scleroderma/progressive systemic sclerosis (SSc), mixed connective tissue disease (MCTD), Sjogren’s syn drome (SS), dermatomyositis-polymyositis (DM/PM) and undetermined connective tissue disease (UCTD).
On the contrary, rheumatoid arthritis (RA) does not express this marker: This pathology frequently presents a serum negativity towards the autoantigens commonly evaluated in the diagnosis of connective tissue diseases. For this diagnosis, the combined evaluation of rheumatoid factors (RATest) and anti-cyclic citrullinated peptide antibodies (anti-CCP) is of particular interest.
Therefore, the detection of ANA in the serum of patients is an important diagnostic tool to allow the clinician a more precise classification of anamnestic and clinical pictures that are often blurred.
In recent years, commercial test offerings in this field have become numerous as the analytical techniques available for the same marker.
It is, therefore, necessary to make a reasoned choice of diagnostic tests and analytical methods in which the laboratory must be able to play a central role.
ANA are autoantibodies belonging to all classes of immunoglobulins, most often to the IgG class, directed against cellular constituents common to all cell types (non-organ-specific antibodies).
The study of the structural and functional characteristics of the target antigens and the characterization of the single antigen/antibody systems has made it possible to define close correlations between serological picture and clinical status, and in some conditions, ANA represent a real marker of dis ease, which, due to the precocity of appearance, assumes a high predictive and prognostic value.
Due to its high diagnostic sensitivity, the ANA test executed IFI method remains the gold standard and represents the first level of the diagnostic algorithm.
However, since these autoantibodies, especially at low titre, are also found in healthy subjects and/or those with non-autoimmune diseases, it is necessary to perform the ANA test and the related in-depth examinations (ENA and dsDNA) according to a correct sequence to improve the diagnostic process.
Based on all the above considerations, the application of the ANA Reflex is justified. The ANA Reflex allows the autoimmunology laboratory to evaluate the fluoroscopic pat tern, the positivity titre and the clinical information and to assess whether and which tests should be performed as in depth examinations.
In order to be effective, the ANA Reflex must be an administrative tool valid for all prescribers both in hospitals and in the territory, but with the freedom to be applied in a “reflexive” way by the laboratory, which becomes responsible for the governance of the whole subsequent process.
In the presence of a positive ANA-IFI result, a greater diagnostic and prognostic power can be achieved with the characterization of antibody specificities directed against the different nuclear antigens through molecular tests.
The determination of specific antibodies directed towards the so-called ENA-, DNA- or RNA-associated proteins is useful for the diagnosis of SLE, neonatal lupus, SS, MCTD, PM/DM and SSc, with variable sensitivity and specificity, as reported in the literature. The variety of autoantigens recognized by ANA is extremely wide.
Anti-ENA antibodies in serum can be detected by several techniques currently used in the laboratory: enzyme immunoassay (ELISA), immunoblot (IB), and immunoassay (EIA).
For the choice of the method, it is necessary to keep in mind some factors: clinical location (reference laboratory or not), laboratory budget and, consequently, cost of reagents, organization and “experience” of the laboratory and level of dialogue between laboratory and clinician.
The ideal method should meet the criteria of clinical sensitivity, precision and accuracy, ease of execution, easy avail ability and low cost; at present, there is no method that can meet all these requirements alone.
Considering the characteristics of the available methods, a correct diagnostic procedure for the detection of anti-ENA can only make use of several methodological approaches that the autoimmunology laboratory must be able to put in place.
The diagnostic and prognostic usefulness of the anti- dsDNA antibody assay has led to the development of several techniques for their identification: radiobinding techniques (Farr technique), indirect immunofluorescence (IFI) on Crithidia Luciliae and enzyme immunoassay techniques (ELISA).
Anti-dsDNA antibodies are highly specific for SLE as they are almost exclusively found in SLE and are the tenth criterion for SLE according to the American College of Rheumatology.
If their concentration is high, they are prognostic of a relapse even if SLE is quiescent; moreover, their presence, in the absence of clinical symptoms, is indicative of sub clinical SLE, being negative in drug-induced SLE and positive in less than 2% of cases with other autoimmune diseases. Numerous studies have documented that the con centration of anti-dsDNA antibodies correlates with the clinical course of SLE and lupus nephritis and that an increase in antibody titre may precede clinical flare by a few weeks.
The research of ANA in IFI is penalized by several critical issues, such as the subjectivity of the reader, the qualification and experience of the reader, the variability related to different substrates and the technology of the microscope. All this, together with the consolidation of the laboratories, has led to a workload of microscopic activity that is not always sustain able where there is a shortage of personnel dedicated to auto immune diagnostics and there is no generational turnover and no insertion and training of new professionals. These problematic aspects have seen, in recent years, the emergence of new organizational and technological strategies to cope with the increasing numbers of tests and the contraction of healthcare personnel.
Recently, automated reading systems have been introduced in the laboratories, which mainly allow a screening between negative and positive samples and among the latter a “potential” attribution of the patterns of positivity and an estimation of the titre with the possibility of archiving the images as a partial solution to the problems mentioned above. The technological offer is of platforms with different characteristics that, however, must be dropped in their own organization, taking advantage of the benefits of standardization that, however, come only after an insertion and an evaluation that requires an alignment with their own microscopic practice. Certainly, the possibility of archiving images and, more generally, the integration of these systems in the management island of autoimmunity can make the daily work more efficient.
The scientific literature has produced several comparisons and considerations on the systems on the market, analyzing their strengths and weaknesses. Also these technologies, like the others, must be consciously included and governed by a structured and robust experience in the field of autoimmunology.
Considering the objective difficulties in numbers and organization, studies of comparative evaluation of ANA in IFI and solid phase ANA in immunoassay have also increased. There are different technologies and combinations of antigens on the market. It is a question of considering the comparison and possible combination of two different screening approaches. There are studies on the combination of the two tests in the clinical diagnostic pathway, and there are studies on the use of the solid phase as initial screening, followed by immunofluorescence. In addition to sensitivity and specificity reasoning, it is necessary for each laboratory to evaluate its own population, informatics and organizational strategy and clinical algorithm. Certainly, these choices, if shared across a regional territory, increase the strength of an algorithm.
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