Autoimmunity Reviews 5 (2006) 10 – 17
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J.G.M.C. Damoiseaux, J.W. Cohen Tervaert / Autoimmunity Reviews 5 (2006) 10–17                           11


 Acknowledge...
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replaced by...
J.G.M.C. Damoiseaux, J.W. Cohen Tervaert / Autoimmunity Reviews 5 (2006) 10–17                  13


Examples include anti...
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be evaluated includ...
J.G.M.C. Damoiseaux, J.W. Cohen Tervaert / Autoimmunity Reviews 5 (2006) 10–17                              15


nized for...
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(b40%) for these d...
J.G.M.C. Damoiseaux, J.W. Cohen Tervaert / Autoimmunity Reviews 5 (2006) 10–17                                     17


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Ana To Ena

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Details on ANA and ENA as they relate to testing, diagnosis, Lupus, Autoimmune diseases, etc

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Ana To Ena

  1. 1. Autoimmunity Reviews 5 (2006) 10 – 17 www.elsevier.com/locate/autrev From ANA to ENA: How to proceed? J.G.M.C. Damoiseaux *, J.W. Cohen Tervaert Department of Clinical and Experimental Immunology, University Hospital Maastricht, P. Debeyelaan 25, 6229 HX Maastricht, The Netherlands Received 18 April 2005; accepted 14 May 2005 Available online 13 June 2005 Abstract Anti-nuclear antibodies (ANA), as detected by indirect immuno-fluorescence, are hallmarks of autoimmune connective tissue diseases. Identification of the specificity for extractable nuclear antigens (ENA) is warranted because this may further differentiate between the distinct types of autoimmune connective tissue diseases. In recent years several different ENA, as recognized by ANA, have been identified and the knowledge of the molecular structure has been expanded. Together with technical developments this has enabled the introduction of several new anti-ENA antibody detection systems. In this review we will discuss the main logistic aspects of anti-ENA antibody testing that have to be solved in order to come to a consensus in order to deal with new developments in this field. We conclude that: 1. a positive ANA test should, depending on the titre and pattern, be followed by an anti-ENA antibody assay, 2: to fully appreciate the value of the new anti-ENA antibody detection systems a large, multicenter clinical evaluation is required, and 3: proper interpretation of reported test results requires that the clinician is aware of the way anti-ENA antibodies are detected and reported. D 2005 Elsevier B.V. All rights reserved. Keywords: Anti-nuclear antibodies; Extractable nuclear antigens; Methods; Clinical evaluation Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2. Follow-up of ANA by ENA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3. New methods for anti-ENA antibody detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 4. Clinical relevance of anti-ENA antibody results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 5. Procedure and interpretation of anti-ENA antibody detection . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 * Corresponding author. Tel.: +31 43 3881433; fax: +31 43 3884164. E-mail address: jdam@limm.azm.nl (J.G.M.C. Damoiseaux). 1568-9972/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2005.05.007
  2. 2. J.G.M.C. Damoiseaux, J.W. Cohen Tervaert / Autoimmunity Reviews 5 (2006) 10–17 11 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Take-home messages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 than the immuno-diffusion assays, but the clinical 1. Introduction relevance of this information may be ill-defined. In Anti-nuclear antibodies (ANA) are directed to dis- this review we will discuss the main logistic aspects of tinct constituents of the nucleus and ANA are tradi- ENA testing. This is especially relevant since the tionally identified by the indirect immuno-fluorescent detection of these antibodies is more and more done technique (IFT) [1]. In daily practice, also antibodies by using new methodologies. to cytoplasmic antigens, like the aminoacyl-tRNA- synthetases, are referred to as ANA. Salt extraction of nuclear proteins was originally used for identifying 2. Follow-up of ANA by ENA the antigens that are recognized by ANA, hence the name extractable nuclear antigens (ENA). Although The first question to be asked is if all positive ANA several antigens, like topoisomerase-I (Topo-I), are tests should be followed by anti-ENA antibody test- not extractable in this way, in this review we will ing. ANA testing was traditionally performed on liver refer to these antigens as ENA in order to cover all tissue sections, but the substrate has now been of the most relevant anti-ENA antibodies that are tested for in a routine laboratory. Table 1 The presence of ANA is associated with autoim- Auto-antibody specificities associated with HEp-2 and HEp-2000 patterns mune connective tissue diseases. Their detection is even one of the diagnostic criteria for systemic ANA pattern Associated antigens lupus erythematosus (SLE) [2]. Further analysis of dsDNAa; histones; Topo-Ib Homogeneous Speckled the reactivity to extractable nuclear antigens (ENA) Ku; Mi-2; RNA polymerase II/III; Sm; SSA; SSB; Topo-I; U1-RNP may be of help to discriminate between the different Atypic speckledc SSA; SSB types of autoimmune connective tissue diseases. For Nucleolar Fibrillarin, NOR-90, PM/Scl instance, antibodies to the Smith (Sm) antigen are (75 and 100); RNA polymerase I; specific for SLE [2], while the presence of anti-Sjo- ¨ To/Th Centromere CENP-A; CENP-B; CENP-C gren’s Syndrome (SS)A and/or-SSB antibodies is a Nuclear dots p80-coilin hallmark for Sjogren’s Syndrome [3]. Besides having ¨ Mitotic spindle CENP-F; MSA-2; NuMA diagnostic potential, the detection of anti-ENA anti- PCNA PCNA bodies is also of prognostic importance. The presence Cytoplasmic tRNA synthetases, a.o. Jo-1, PL-7, of anti-SSA in the circulation of the mother may cause and PL-12; RibP proteins P0, P1, and P2; SRP; also cell-organelles neonatal lupus erythematosus and/or congenital heart (mitochondria, lysosomes, and block [4,5], while the presence of anti-Topo-I antibo- Golgi apparatus) dies predict a more severe course of disease in sys- a Abbreviations: dsDNA, double stranded DNA; CENP, centro- temic sclerosis (SSc) [6]. mere protein; MSA, mitotic spindle apparatus; NOR, nucleolus Originally, most of these anti-ENA antibody spe- organizer region; NuMA, nuclear mitotic apparatus; PCNA, prolif- cificities and clinical associations were determined by erating cell nuclear antigen; PM/Scl, polymyositis/scleroderma; immuno-assays based on immuno-diffusion [7,8]. RibP, ribosomal P; RNP, ribonucleoprotein; Sm, Smith; SSA, Sjo-¨ gren’s Syndrome A; SSB, Sjogren’s Syndrome B; SRP, signal ¨ However, further knowledge on the structure of the recognition particle; Topo-I, topoisomerase-I. autoantigens, the identification of other specificities, b Antigens that are commonly used for anti-ENA antibody testing and recombinant technology has enabled the launch of in routine laboratories are presented in bold. many different anti-ENA antibody detection systems c The atypic speckled pattern is typical for the HEp-2000 cells [9–11]. These systems give a lot more information which are transfected with the gene encoding SSA-60.
  3. 3. 12 J.G.M.C. Damoiseaux, J.W. Cohen Tervaert / Autoimmunity Reviews 5 (2006) 10–17 replaced by HEp-2 cells. These cells have large nuclei include the homogeneous, speckled, nucleolar, and and contain a considerable number of dividing cells, centromere pattern. In case of the HEp-2000 cells an displaying mitotic figures, which enables the detection atypic speckled or SSA-pattern may be recognized as of antigens that are differentially expressed during the well. With the exception of the centromere pattern, the cell cycle. The HEp-2 cells may be transfected with clinical relevance of ANA patterns is considered to be additional antigens, such as the human 60 kDa SSA limited and further testing is warranted to definitely antigen (HEp-2000 cells) in order to increase the establish the antigen specificity of the ANA (Table 1). spectrum of relevant antigens [12]. Improvements in However, both the titre and the pattern give informa- the ANA substrate have strongly reduced the number tion that may be helpful to identify the antigen spec- of samples that are ANA negative, but positive for ificity by testing for antibodies to the most anti-ENA antibodies. In our experience with HEp- conventional ENA, including U1-ribonucleoproteins 2000 cells this is less than 2% (n = 700). Results of (U1-RNP), Sm, SSA, SSB, centromere protein ANA testing should be reported as positive or nega- (CENP)-B, Topo-I, and Jo-1 (Histidyl-tRNA synthe- tive, and if positive include a titre. The titre is impor- tase). Indeed, low titre ANA and ANA with a nucle- tant because low titre ANA may be present in healthy olar or miscellaneous pattern, like mitotic spindle, individuals upon aging. Up to 30 different ANA have a low probability to reveal a positive result in staining patterns have been described today including a conventional anti-ENA antibody test. Nevertheless both nuclear as well as cytoplasmic staining patterns. these ANA patterns may harbour antibodies to ENA Obviously, some of these patterns are very rare and with clinical significance, but due to their low occur- most technicians in routine laboratories are not trained rence rates in most routine laboratories they are not to distinguish them all. The most common patterns incorporated in the standard anti-ENA antibody test. Homogeneous Speckled Atypic Speckled Nucleolar Centromere Cytoplasmic 100 100 100 100 100 100 75 75 75 75 75 75 ND 50 50 50 50 50 50 25 25 25 25 25 25 0 0 0 0 0 0 L M H L M H L M H L M H L M H L M H • Anti-ENA test • Anti-ENA test • Anti-ENA test • Anti-ENA test • Anti-ENA test • Anti-ENA test if high titre if medium or in all cases is not required in all cases for for anti-Jo-1 • Most likely • Consider • Consider other ANA positive high titre ANA confirmation • Also test for positive anti-SSA with other SSc of anti-CENP-B specificities: anti-dsDNA or without associated see Table 1 antibodies anti-SSB anti-ENA Fig. 1. Further anti-ENA antibody testing in ANA positive samples. Upper panels show distinct immuno-fluorescence anti-nuclear antibodies (ANA) patterns as indicated. The middle panels reveal the relation between ANA pattern and titre and subsequent antibody positivity (%) for the conventional extractable nuclear antigens (ENA; U1-RNP, Sm, SSA, SSB, CENP-B, Topo-I, and Jo-1). The L(ow), M(edium), and H(igh) titre refers to dilutions of 1:80 (L), N1 : 80 and b1 : 1280 (M), and z1 : 1280 (H), respectively. The lower panels contain the recommendations for further anti-ENA antibody testing depending on ANA pattern and titre. Abbreviations: dsDNA, double stranded DNA; ND, not determined.
  4. 4. J.G.M.C. Damoiseaux, J.W. Cohen Tervaert / Autoimmunity Reviews 5 (2006) 10–17 13 Examples include antibodies to RNA polymerase I flow cytometry [11,15,16]. These multiplex assays and III, To/Th, fibrillarin, and polymyositis/scleroder- enable the detection of multiple autoantibodies as ma (PM/Scl) which reveal a nucleolar staining pattern separate entities at the same time. The line-blots will and may designate important subpopulations of reveal qualitative antibody reactivity to antigens that patients with SSc. High titre ANA, and especially are applied as distinct lines on a membrane, while ANA with a atypic speckled or centromere pattern, flow cytometry will give quantitative results based on will nearly always enable the identification of the reactivity with a mixture of bead subsets that are each antigen specificity of the ANA. This is supported by labelled with a unique combination of internal fluo- a study performed in our routine laboratory where rescent signal and antigen. Nevertheless, the new anti-ENA antibody testing was performed in all sam- techniques have been widely introduced in routine ples of patients that tested ANA positive for the first immunology laboratories because they are less labo- time during a period of 2 years (Fig. 1). Based on rious and more easy to read. There are some clear these data a scheme can be devised for further anti- differences between the traditional anti-ENA antibody ENA antibody testing in ANA positive samples assays using cell extracts and the new techniques depending on the titre and pattern (Fig. 1). This using purified or recombinant antigens. While approach will speed up the diagnostic process because immuno-diffusion and immuno-blotting give informa- the clinician will have the results after a single labo- tion about the identity of the recognized antigen, i.e. ratory request without additional costs of useless anti- confluence of precipitation lines and molecular ENA antibody testing. weight, respectively, the new techniques do not enable to discriminate between reactivity to the antigen of choice or to impurities of the antigen preparation. Furthermore, the exposition of antigen epitopes will 3. New methods for anti-ENA antibody detection be different depending on the type of antigen compo- Anti-ENA antibody testing was originally per- sition: recombinant antigens may be poorly glycosy- formed by immuno-diffusion based techniques using lated, mixing of antigen subunits may not result in thymic and/or spleen extracts [7,8]. Also immuno- proper refolding of the antigen, and the use of sub- blotting of cell extracts from for instance HeLa-cells units as separate entities (split antigens) will also (nuclear and cytoplasmic) was often used for the result in a different three dimensional structure. The detection of antibodies to ENA [13]. Although these difference between recombinant and native antigens is techniques are quite laborious and results are difficult most apparent in case of anti-SSA60 sera from to read, the use of cell extracts enabled the identifica- patients with SLE and Sjogren’s Syndrome (A. ¨ tion of new antigenic entities. In addition, these stud- Wiik, personal communication). ies revealed that some ENA consist out of several Obviously, the distinct anti-ENA antibody assays subunits. Moreover, it appeared that distinct ENA that are available nowadays will reveal different are attached to the same RNA molecule [14]. For results that have to be carefully evaluated in terms instance, U1-RNP consists of 3 subunits (68 kDa, of clinical relevance. In particular, the introduction of A, and C) and Sm is composed of 6 subunits (B, new antigenic entities and split antigens in the multi- BV, D, E, F, and G), while both are linked to U1- plex assays requires special attention. RNA molecules. Similarly, SSA-60 is coupled togeth- er with SSB to Y-RNA. The Ro-52 component, also referred to as SSA-52, seems to be separated from this 4. Clinical relevance of anti-ENA antibody results complex, at least in the resting cell. All this pioneering work has been, and still is, of utmost importance for The second question about anti-ENA antibody the development of new techniques that are based on testing is: what is the proper choice of anti-ENA purified native or even recombinant antigens. Since antibody assay to be used? For making a good choice the early eighties these antigens were first employed between the distinct anti-ENA antibody assays that in enzyme-linked immunosorbent assay (ELISA), and are available, it is important to have information on more recently in multiplex-assays, like line-blots and the clinical relevance of the obtained results. Items to
  5. 5. 14 J.G.M.C. Damoiseaux, J.W. Cohen Tervaert / Autoimmunity Reviews 5 (2006) 10–17 be evaluated include test characteristics for diagnosis anti-ENA antibody reactivity in the control popula- and prognosis of a certain disease. For instance, while tion may precede eventual development of autoim- anti-Sm antibodies are part of the diagnostic criteria mune connective tissue disease [19]. for SLE, antibodies to the split antigen SmD are Altogether, it will be evident that the mere com- considered far more specific for SLE than antibodies parison of anti-ENA antibody assays can be expressed to SmB. Antibodies against the BV/B proteins are often in terms of concordance rates and discrepancies, but found in non-Sm sera, mostly via cross reaction [17]. without proper clinical evaluation the value of the data However, in a recent evaluation of an anti-ENA anti- obtained is rather limited. In this respect it is worth- body line-blot, antibodies to SmB appeared to be while to mention that several initiatives are currently more specific and more sensitive for SLE than anti- being undertaken to establish a large scale internation- bodies to SmD [16]. al serum collection with well defined clinical data in Different approaches are being used for evalua- order to enable the clinical evaluation of new devel- tion of anti-ENA antibody assays. First, consecutive opments in auto-antibody testing. samples of routine anti-ENA antibody screening may be included for comparison of different meth- ods and/or (commercial) assays. Obviously this ap- 5. Procedure and interpretation of anti-ENA proach will reveal discrepancies in results for the antibody detection reactivity to separate anti-ENA antibodies. It appeared, for instance, that ELISA’s are more sen- There is a consensus statement that a combination sitive than immuno-diffusion assays [18]. The clin- of two or more techniques should be used to detect ical relevance of this increased sensitivity is all specificities with an adequate efficiency [17], the difficult to interpret. For instance, what is the clin- final question is how to report the data obtained to ical relevance of anti-SSB antibodies in a patient the clinician. Although many countries do not allow presenting with fatigue and increased erythrocyte multiple test systems for economical reasons, the sedimentation rate, or of anti-CENP-B antibodies need for additional tests is based on the fact that in a young woman with Raynaud’s phenomenon? some techniques as well as some commercial assays For correct interpretation it is important to know may be sub-optimal for detecting a certain (conven- the eventual diagnosis of the patients. Since anti- tional) anti-ENA antibody specificity. Again based bodies to ENA may be present several years before on a proper clinical evaluation an algorithm has to be clinical symptoms become apparent or a definite devised how to deal with the data obtained by diagnosis can be made [19], it may take several multiple assays. For instance, if there is good con- years before the data obtained can be evaluated in cordance for anti-SSB antibodies between both test the right way. The second approach for evaluation systems, one may decide that SSB is reported pos- of anti-ENA antibody assays includes samples of itive only if both tests are positive. On the other well defined patient populations. Due to prevalence hand, if one of the two tests is known to be superior of disease this concerns most often patients with in testing for anti-Topo-I antibodies, than this test SLE or Sjogren’s Syndrome. These diseases enable ¨ should be decisive for reporting the results. The the evaluation of antibodies to RNP, Sm, SSA, and clinician should not be bothered with separate reports SSB. Evaluation of antibodies to Topo-1 and Jo-1 of both test systems. However, the clinician should is hampered by the fact that these antibodies are be acquainted with the test characteristics as well as present in the minority of patients with SSc (20– the algorithm that is used for reporting the results. 30%) and myositis (25–35%), respectively, and be- Interpretation and reporting of results is further ham- cause these diseases are relatively rare. Parameters pered by the fact that two anti-ENA antibody assays that are evaluated include sensitivity and specificity. may measure distinct antigenic entities. For instance, For the latter aspect samples are often included antigens like U1-RNP, Sm, and SSA may be ana- from healthy controls and/or specimens selected lysed as whole antigen, or as split antigen. For U1- for their potential biologic interference, instead of RNP there is some consensus that at least 2 out of proper disease controls [20]. However, here again, the 3 subunits (68 kDa, A, and C) should be recog-
  6. 6. J.G.M.C. Damoiseaux, J.W. Cohen Tervaert / Autoimmunity Reviews 5 (2006) 10–17 15 nized for being referred to as positive for anti-U1- pattern in conjunction with the clinical information. RNP antibodies. As already mentioned there is dis- An example is given in Table 2. However, one cussion on the clinical relevance of anti-SmD versus should be aware that low titre ANA may be ob- anti-SmB antibodies [16,17]. Also the clinical rele- scured by the presence of high titre ANA with an vance of antibodies to Ro-52 (SSA52) has been overlapping pattern in the same sample. Also, the disputed. Although antibodies to SSA are not spe- clinical information of a patient at first presentation cific for any of the autoimmune connective tissue may not be specific for the autoimmune connective diseases, these antibodies may be prognostic for the tissue disease that is diagnosed during long-term development of neonatal lupus and/or congenital follow-up. An exception here is neonatal lupus heart block [4,5]. Interestingly, it was recently and/or congenital heart block which is typically as- reported that maternal auto-antibodies directed to sociated with anti-Ro-52 antibodies. For logistic rea- the Ro-52 protein correlate with prolongation of sons it may be easier to analyse the whole array of fetal atrioventricular time and heart block [21]. conventional anti-ENA antibodies in samples that are This indicates that detection of antibodies specific positive for ANA in relevant titres and patterns (Fig. for Ro-52 is clinically relevant in pregnant women 1), but only reactivities that are relevant in the and positive test results should be reported to the context of the clinical information should be reported clinician. to the clinician (Table 2). In case of a strong clinical Finally, the question arises whether a selection suspicion for SSc or myositis, testing for additional can be made for testing on antibody reactivity to anti-ENA antibody specificities may be indicated the conventional ENA (U1-RNP, Sm, SSA, SSB, since marker antibodies, i.e. antibodies to Topo-I CENP-B, Topo-I, and Jo-1) based on the ANA and Jo-1, respectively, have a low sensitivity Table 2 Algorithm for directed anti-ENA antibody testing based on ANA detection and clinical information ANA-pattern Z Homogeneous Speckled Atypic Nucleolar Centromere Cytoplasmic speckleda Clinical suspicion x SLEb dsDNA, Sm, SSA, Sm, SSA, SSB SSA, SSB – – RibP proteins Histones Neonatal lupus SSA SSA, SSB SSA, SSB – – – Sjogren’s syndrome ¨ – SSA, SSB SSA, SSB – – – Systemic sclerosis Topo-I Topo-I, U1-RNP, – Fibrillarin, To/Th, CENP-B Ku, RNA-Polymerase I, RNA-Polymerase PM/Scl III Myositis – Ku, Mi-2 – PM/Scl – Aminoacyl-tRNA transferases (Jo-1, PL-7, PL12), SRP Overlap syndromesc dsDNA, Sm, SSA, U1-RNP, Topo-I, SSA, SSB PM/Scl CENP-B Jo-1 Topo-I Ku, Sm, SSA, SSB Congenital SSA SSA SSA – – – heartblock Miscellaneousd dsDNA, Sm, SSA, Sm, SSA, SSB, SSA, SSB – CENP-B Jo-1 Topo-I Topo-I, U1-RNP a The atypic speckled pattern is typical for the HEp-2000 cells which are transfected with the gene encoding SSA-60. b Abbreviations: ANA, anti-nuclear antibodies; CENP, centromere protein, dsDNA, double stranded DNA; ENA, extractable nuclear antigens; PM-Scl, polymyositis/scleroderma; RibP, ribosomal P; RNP, ribonucleoprotein; SLE, systemic lupus erythematosus; Sm, Smith; SSA, Sjogren’s ¨ Syndrome A; SSB, Sjogren’s Syndrome B; SRP, signal recognition particle; Topo-I, topoisomerase-I. ¨ c Selection of anti-ENA antibody testing should be based on the clinical overlap that is being considered. d Miscellaneous implies that no clinical information is given together with the laboratory request.
  7. 7. 16 J.G.M.C. Damoiseaux, J.W. Cohen Tervaert / Autoimmunity Reviews 5 (2006) 10–17 (b40%) for these diseases. Additional antigenic en- Take-home messages tities of interest for myositis include Ku, Mi-2, PL-7 (threonyl-tRNA synthetase), PL-12 (alanyl-tRNA ! ANA titre and pattern give information on the ne- synthetase), PM-Scl, and signal recognition particles cessity to proceed with anti-ENA antibody testing. (SRP) [22], while in SSc antibodies to RNA poly- ! High through-put anti-ENA antibody assays en- merase I and III, To/Th, fibrillarin, Ku, and PM-Scl abling detection of multiple antigenic specificities, may have added value [23]. Obviously, also detec- including novel antigens and split antigens, are tion systems for these additional specificities require available nowadays, but evaluation of its clinical further clinical evaluation. relevance is at present lacking. ! There is a high demand for a large scale interna- tional serum collection with well defined clinical data, including long term follow-up data, in order 6. Conclusions to enable the clinical evaluation of new develop- In the discussion on how to proceed from a pos- ments in auto-antibody testing. itive ANA test to anti-ENA antibody testing more ! Optimal algorithms for anti-ENA antibody test- questions than answers are generated. With respect to ing and reporting of results relies on a close the first question if a positive ANA should always be collaboration between clinicians and the labora- followed by anti-ENA antibody testing our own data tory staff. reveal that both the titre and pattern give a clue to which samples could be excluded from further testing for antibodies to the conventional ENA. For the References proper appreciation of the clinical value of the new anti-ENA antibody detection systems evaluations [1] Holborrow E, Weir D, Johnson G. A serum factor in lupus should not be restricted to the mere comparisons of erythematosus with affinity for tissue nuclei. Br Med J different assay systems, but should include evaluation 1957;2:732 – 4. based on clinical data. The set-up of a large scale [2] Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, international serum collection with well defined clin- Cooper NS, et al. The american rheumatism association 1987 ical data, including follow-up data, to enable the revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315 – 24. clinical evaluation of new developments in auto-an- [3] Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Al- tibody testing, would be of great help to identify the exander EL, Carsons SE, et al. Classification criteria for pros and cons of all anti-ENA antibody detection Sjogren’s syndrome: a revised version of the European criteria assays. Given the consensus that two distinct meth- proposed by the American–European Consensus Group. Ann ods for anti-ENA antibody detection should be used Rheum Dis 2002;61:554 – 8. [4] Watson RM, Lane AT, Barnett NK, Bias WB, Arnett FC, in parallel, a clear algorithm for reporting the results Provost TT. Neonatal lupus erythematosus. A clinical, sero- to the clinician should be available. Obviously, the logical and immunogenetic study with review of the literature. clinician should be acquainted with the test character- Medicine (Baltimore) 1984;63:362 – 78. istics as well as the algorithm that is used for report- [5] Singsen BH, Akhter JE, Weinstein MM, Sharp GC. Congenital ing the results. Finally, the clinician can be of help to complete heart block and SSA antibodies: obstetric implica- tions. Am J Obstet Gynecol 1985;152:655 – 8. the laboratory by giving relevant clinical information [6] Hu PQ, Fertig N, Medsger Jr TA, Wright TM. Correlation of enabling more relevant testing, for instance in case of serum anti-DNA topoisomerase I antibody levels with disease SSc or myositis. severity and activity in systemic sclerosis. Arthritis Rheum 2003;48:1363 – 73. [7] Tan EM, Kunkel HG. Characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic Acknowledgements lupus erythematosus. J Immunol 1966;96:464 – 71. [8] Clark G, Reichlin M, Tomasi Jr TB. Characterization of a We thank Professor Allan Wiik (Statens Serum soluble cytoplasmic antigen reactive with sera from patients Institut, Copenhagen, Denmark) for helpful comments with systemic lupus erythmatosus. J Immunol 1969;102: during the preparation of the manuscript. 117 – 22.
  8. 8. J.G.M.C. Damoiseaux, J.W. Cohen Tervaert / Autoimmunity Reviews 5 (2006) 10–17 17 [9] Bizzaro N, Tozzoli R, Tonutti E, Piazza A, Manoni F, Ghir- the detection of antibodies to extractable nuclear antigens. Ann ardello A, et al. Variability between methods to determine N Y Acad Sci in press. ANA, anti-dsDNA and anti-ENA autoantibodies: a collabora- [17] van Venrooij WJ, Charles P, Maini RN. The consensus work- tive study with the biomedical industry. J Immunol Methods shops for the detection of autoantibodies to intracellular anti- 1998;219:99 – 107. gens in rheumatic diseases. J Immunol Methods 1991; [10] Pottel H, Wiik A, Locht H, Gordon T, Roberts-Thomson P, 140:181 – 9. Abraham D, et al. Clinical optimization and multicenter val- [18] Orton SM, Peace-Brewer A, Schmitz JL, Freeman K, Miller idation of antigen-specific cut-off values on the INNO-LIA WC, Folds JD. Practical evaluation of methods for detection ANA update for the detection of autoantibodies in connective and specificity of autoantibodies to extractable nuclear anti- tissue disorders. Clin Exp Rheumatol 2004;22:579 – 88. gens. Clin Diagn Lab Immunol 2004;11:297 – 301. [11] Rouquette AM, Desgruelles C, Laroche P. Evaluation of the [19] Arbuckle MR, McClain MT, Rubertone MV, Scofield RH, new multiplexed immunoassay, FIDIS, for simultaneous quan- Dennis GJ, James JA, et al. Development of autoantibodies titative determination of antinuclear antibodies and compari- before the clinical onset of systemic lupus erythematosus. N son with conventional methods. Am J Clin Pathol 2003; Engl J Med 2003;349:1526 – 33. 120:676 – 81. [20] Wiik AS, Gordon TP, Kavanaugh AF, Lahita RG, Reeves W, [12] Fritzler MJ, Miller BJ. Detection of autoantibodies to SS-A/Ro van Venrooij WJ, et al. Cutting edge diagnostics in rheuma- by indirect immunofluorescence using a transfected and over- tology: the role of patients, clinicians, and laboratory scientists expressed human 60 kD Ro autoantigen in HEp-2 cells. J Clin in optimizing the use of autoimmune serology. Arthritis Lab Anal 1995;9:218 – 24. Rheum 2004;51:291 – 8. [13] de Rooij DJ, van de Putte LB, Habets WJ, Verbeek AL, van [21] Salomonsson S, Sonesson SE, Ottosson L, Muhallab S, Olsson Venrooij WJ. The use of immunoblotting to detect antibodies T, Sunnerhagen M, et al. Ro/SSA autoantibodies directly bind to nuclear and cytoplasmic antigens. Clinical and serological cardiomyocytes, disturb calcium homeostasis, and mediate associations in rheumatic diseases. Scand J Rheumatol congenital heart block. J Exp Med 2005;201:11 – 7. 1988;17:353 – 64. [22] Brouwer R, Hengstman GJ, Vree Egberts W, Ehrfeld H, Bozic [14] Lerner MR, Boyle JA, Hardin JA, Steitz JA. Two novel classes B, Ghirardello A, et al. Autoantibody profiles in the sera of of small ribonucleoproteins detected by antibodies associated European patients with myositis. Ann Rheum Dis 2001; with lupus erythematosus. Science 1981;211:400 – 2. 60:116 – 23. [15] Hoffman IE, Peene I, Meheus L, Huizinga TW, Cebecauer L, [23] Harvey GR, Butts S, Rands AL, Patel Y, McHugh NJ. Clinical Isenberg D, et al. Specific antinuclear antibodies are associated and serological associations with anti-RNA polymerase anti- with clinical features in systemic lupus erythematosus. Ann bodies in systemic sclerosis. Clin Exp Immunol 1999; Rheum Dis 2004;63:1155 – 8. 117:395 – 402. [16] Damoiseaux J, Boesten K, Giesen J, Austen J, Cohen Tervaert JW. Tervaert. Evaluation of a novel line-blot immunoassay for Anti-oxLDL antibodies: marker for arterial thrombosis in antiphospholipid syndrome? Antibodies against oxidized low-density lipoproteins (anti-oxLDL antibodies) are involved in the development of atherosclerosis in animal models, but their role in human is not clear. In this study Bacarevic M. et. al. (Clin Lab 2005;51:279-83) investigated the relationship between the presence of ant-oxLDL antibodies and the presence of anti-beta2glycoprotein I (beta2GPI) antibodies, anticardiolipin antibodies and lupus anticoagulant. The authors also analyzed the relationship between the appearance of anti-oxLDL antibodies and clinical signs of antiphospholipid syndrome (APLS). The study included three groups of patients: 27 patients with primary APLS, 20 with secondary APLS associated with systemic lupus erythematosus (SLE) and 13 patients with SLE. They found that the presence of anti-oxLDL antibodies was associated with a history of arterial thrombosis in patients with secondary APLS (chi2 = 8.89, p b 0.01) and in patients with primary APLS (chi2 = 4.64, p b 0.05). Also, the appearance of anti-oxLDL antibodies was associated with the presence of anti-beta2GPI antibodies (chi2 = 4.25, p b 0.05), which was not dependent on diagnosis. These preliminary observations have to be confirmed in a larger study.

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