Auto-Antibody Detection: Prevailing Practices at a Tertiary Care Hospital in Riyadh

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1 Clin. Lab. 2014;60: Copyright ORIGINAL ARTICLE Auto-Antibody Detection: Prevailing Practices at a Tertiary Care Hospital in Riyadh ADEL ALMOGREN, ZAHID SHAKOOR, RANA M. W. HASANATO, ABDULRAHMAN ALGASSIM, NAWAF A. AL-FURAIH, FAISAL A. AL-MUDAIHEEM, OMAR A. AL-ABDULKARIM Department of Pathology, College of Medicine and University Hospitals, King Saud University, Riyadh 11461, Kingdom of Saudi Arabia SUMMARY Background: Anti-nuclear antibody (ANA) test as the first level investigation for detection of auto-immune rheumatic disease has been recommended in a number of international guidelines. This study was performed to evaluate the local practice and trends of auto-antibody laboratory requests. Methods: Data were collected from 249 initial laboratory requests for first level auto-antibody detection between April 2012 and April 2013 in the Immunology Unit at King Khalid University Hospital, Riyadh. This group of patients included 151 females and 98 males (mean age 40.1 ± 21; range 4-85 years). Results: Of the total requests, ANA as a single first level investigation was requested by only 32 (13%) clinicians whereas the rest of the investigations included simultaneous testing of ANA and second level extractable nuclear antigen (ENA) auto-antibody tests. Anti-double stranded DNA (anti-dsdna) antibody was simultaneously tested with ANA in 158 patients as first level test where both the tests were positive in 44 (27.8%) patients and in 24 (15.1%) patients a negative ANA test was associated with a positive anti-dsdna antibody test. Rheumatoid factor (RF) tested positive in 04/53 (7.5%), anti-neutrophil cytoplasmic antibody (ANCA) in 01/48 (2%) and SS-A and SS-B in 03/37 (8.1%) requests as first level tests with ANA. Conclusions: Using second level auto-antibody tests in conjunction with ANA as the first line investigation does not appear to be a cost effective approach, highlighting the importance of adherence to the guidelines. ANA negative and anti-dsdna positive group of patients requires further assessment in a large scale study. (Clin. Lab. 2014;60: DOI: /Clin.Lab ) KEY WORDS auto-antibodies, ANA, anti-dsdna, autoimmune, SLE INTRODUCTION Systemic autoimmune diseases comprise a heterogeneous group of disorders characterized by the presence of disease specific antibodies against intracellular self antigens. Among several known auto-antibodies the most frequently sought for antibodies include antinuclear antibodies (ANA), antiextractable nuclear antigens (anti-ena), and anti-double stranded DNA (antidsdna) antibodies. Presence of ANA is now being considered as an integral part of the diagnostic criteria for autoimmune rheumatic disorders [1,2]. Laboratory testing for autoimmune disorders has undergone several technological advancements in the recent past and has had a significant impact on the diagnostic approaches [3,4]. In addition, because of the variable symptoms and the associated pathological lesions in systemic autoimmune disorders, the role of laboratory investigations is often critical in interpreting a wide range of clinical presentations that frequently do not support a clear cut diagnosis [5]. Lately there has been a remarkable increase in the number of requests for the detection of auto-antibodies not only because of the clinical importance but also due to the inappropriate utilization of the laboratory services [6]. Reduction of clinically inappropriate requests is vital for decreasing extra burden on available financial re- Manuscript accepted July 8, 2013 Clin. Lab. 4/

2 A. ALMOGREN et al. sources for health services [7]. In order to achieve this objective, particularly for autoimmune serology, a close collaboration between the laboratory and clinical scientists or physicians is crucial. By defining simple algorithms for autoimmune workup in daily practice a significant reduction in suffering and far higher lifetime costs for chronic disorders can be achieved with a relative ease [8]. In order to guide or select the most appropriate test for specific auto-antibodies the Italian Society of Laboratory Medicine Study Group on the Diagnosis of Autoimmune Diseases in year 2002 proposed a series of guidelines for the diagnosis and monitoring of systemic autoimmune rheumatic disorders [9]. Similarly, in the year 2000 a panel of renowned experts comprising members from the College of American Pathologists, the American College of Rheumatology and the Clinical Immunology Society, Clinical Center of the National Institutes of Health and other experts have proposed various guidelines for diagnosis of autoimmune rheumatic disorders [10]. These guidelines were prepared after scrutinizing more than 800 best relevant published articles. Among the number of proposed recommendations both guidelines, however, agreed upon ANA as the initial first level screening test to be followed by more specific autoantibody second level tests for the diagnosis of autoimmune rheumatic disorders including systemic lupus erythematosus (SLE). This study analyzes the initial auto-antibody test requests received in the Immunology laboratory at King Khalid University Hospital, Riyadh to evaluate the pattern of autoantibody test requests by clinicians from various clinics and adherence to the internationally recommended guidelines. MATERIALS AND METHODS The study was approved by the Institutional Review Board of the College of Medicine at King Saud University, Kingdom of Saudi Arabia. A total of 249 first time requests between April 2012 and April 2013 for autoantibody testing received in the Immunology Unit at King Khalid University Hospital, Riyadh were included in the study. This group of patients, comprised 151 females and 98 males with the mean age of 40.1 ± 21 years (range 4-85 years) including 9 individuals of less than 14 years of age. The most frequently sought for condition was SLE in 161 patients followed by rheumatoid arthritis in 53, recurrent fetal loss in 11, chronic liver disease in 7, idiopathic pulmonary fibrosis in 7, connective tissue disease in 6 and end stage renal disease in 4 patients. Information regarding auto-antibody test requests sent to the immunology laboratory was extracted from patient test requests. Apart from the demographic details the pattern of simultaneous testing of ANA and anti-ena antibody tests requested for the first time as the first level investigation was also noted. The ANA test was performed by using indirect immunofluorescence assay on Hep-2 cells. Results of the ANA test were expressed as positive for titers either equal to or more than 1:40 along with the description of the fluorescence pattern observed. The anti-dsdna test and other auto-antibody tests including SS-A, SS-B, Sm, RNP, Scl70, and Jo1 tests were performed by immunoenzymatic assays. RESULTS Figure 1 describes the frequency of ANA test requests by various physicians either as a single initial investigation or in combination with other auto-antibodies as an initial test request in patients with clinical suspicion of autoimmune rheumatic disease. Of the total number, only 32 (13%) of the initial requests were for only ANA test and the remaining 217 (87%) were ANA accompanied by varying numbers of other auto-antibodies in different combinations. The most common among the other auto-antibody requests with ANA was the antidsdna Ab. Among all the records screened there were 158 requests of ANA simultaneously tested with antidsdna Abs. Figure 2 shows data of association of dsdna Ab reactivity when requested together with ANA. Out of the total 158 combined requests, 44 (27.8%) patients had both the antibodies in their sera and interestingly 24 (15.1%) patients had a non-reactive ANA whereas they tested positively for the presence of anti-dsdna Abs. Table 1 describes the data for other auto-antibodies requested simultaneously with the ANA test at the outset for investigation of autoimmune rheumatic disorders. Among these, rheumatoid factor (RF) was requested simultaneously with the ANA test in 53 patients where only 4 (7.5%) tested positive for RF. This was followed by 48 requests for ANCA, 37 requests for SS-A and SS-B antibodies, 19 requests for anti-smooth muscle antibodies (ASMA), 11 requests for anti-cardiolipin antibodies (ACA) and 9 requests for anti-mitochondrial antibodies (AMA) with the reactivity of 6.6%, 2%, 8.1%, 10.5%, 0.9%, and 1.1%, respectively. Table 1. Frequency of antibodies simultaneously tested with ANA as first level investigations for investigation of autoimmune rheumatic disorders. S# Auto-antibodies No. of requests Both positive No. (%) 1. ANA + SS-A and SS-B 37 3 (8.1) 2. ANA + RF 53 4 (7.5) 3. ANA + AMA 09 1 (1.1) 4. ANA + ASMA 19 2 (10.5) 5. ANA + ANCA 48 1 (2) 6. ANA + ACA 11 1 (0.9) ANA = anti-nuclear antibody, RF = Rheumatoid factor, AMA = anti-mitochondrial antibody, ASMA = anti-smooth muscle antibody, ANCA = anti-neutrophil cytoplasmic antibody, ACA = anticardiolipin antibody. 672 Clin. Lab. 4/2014

3 AUTO-ANTIBODY DETECTION Figure 1. Frequency of anti-nuclear antibody tests requested as a single first level investigation for diagnostic workup of autoimmune rheumatic disorder. Figure 2. Simultaneous testing of anti-nuclear antibody and anti-double stranded DNA antibodies and their reactivity. Clin. Lab. 4/

4 A. ALMOGREN et al. DISCUSSION The findings of this study revealed that 13% of clinicians requested ANA as a single initial test for auto-antibody screening at KKUH. This observation indicates that a smaller proportion of clinicians adhered to the internationally recommended guidelines [10]. These guidelines recommend that in the presence of clinical suspicion of autoimmune rheumatic disorder ANA as a single test should be requested as a first level investigation and in the event of a negative result no further investigations should be performed. The findings of the present study also indicate that the majority of clinicians were tempted to investigate autoimmune rheumatic disorders by requesting additional second level auto-antibody screening tests such as antibodies against ENAs at the outset. A number of factors have been implicated in the surging numbers of requests for such tests. These include: inappropriate prescriptions partly due to inadequate knowledge, lack of compliance to the recommended guidelines, laboratory aspects such as preset profiles on laboratory request forms, delayed reporting time, and the lack of communication [6,11]. The practice of over prescription was reviewed in an Italian study following implementation of guidelines recommending ANA as the first level investigation. The study reported a significant reduction in the number of second level auto-antibody testing without jeopardizing the diagnostic efficacy and also proved to be a very cost effective measure [12]. The observations made in the present study, therefore, highlight the importance of the introduction of shared guidelines between the clinical and laboratory specialists not only to improve the diagnostic yield but also for the better and cost effective utilization of the laboratory services. Another notable finding in the present study was a higher frequency of simultaneous requests for anti-dsdna Ab and ANA and a lower percentage of other anti-ena antibodies as the first level investigations. An anti-dsdna Ab test is usually requested when a higher index of clinical presentation favors SLE [9] which is reflected by the higher frequency of test requests for SLE. A similar practice of simultaneous requests for ANA, dsdna, and other ENAs as the first level tests for detection of autoimmune rheumatic disorders has been recently reported from the USA. Following this observation, an algorithm was developed which included exclusion of anti-dsdna and other ENAs from the initial patient screening, performance of second level tests only on samples testing positive for ANA, and cutting down repeated requests for ANA. Retrospective application of this algorithm as a simulation on one year of inpatient laboratory data demonstrated a substantial (30%) cost saving without any affect on the diagnostic efficacy [13]. These observations re-emphasize the need for implementation of algorithms for auto-antibody detection in consultation with the physicians. Whereas the simultaneous assessment of ANA and antidsdna Abs revealed 27.84% reactivity of both the antibodies in the present study, it was, however, interesting to detect 15.1% of patients testing positively for antidsdna Abs with a negative ANA test. The term ANA negative SLE was initially coined in 1976 [14] and after 10 years in 1986 it was reported that 8.9% of patients with lupus are ANA negative [15]. Because of the existence of higher numbers of ANA negative lupus it was later proposed that the SLE was being under-diagnosed and actual prevalence of SLE could be higher than the reported prevalence rates [16]. In recent years, however, scientists have questioned the very existence of ANA negative SLE on technical grounds. This has been attributed to the fact that in the past ANA detection was routinely performed using rat tissues as a substrate, the introduction of Hep2 cells as a substrate for detection of ANA antibodies has proven to be more reliable and a sensitive test that has virtually eliminated false negative results [9,10,17,18]. Hep2 cell substrate was used for detection of ANA in the present study and the observed ANA negativity appears to be real. Though rarely, this phenomenon of negative ANA with positive antidsdna test has been reported previously in a group of patients where a considerably lower proportion (0.4%) of such occurrence was observed which was associated with 1.4% incidence of autoimmune disorders other than SLE [19]. The higher proportion of ANA negative and anti-dsdna positive patients detected in the present study could possibly be due to a higher number of simultaneous detection of both antibodies as first level investigations. Although this practice is not in conformity with the international recommendations, it prompts further investigations to evaluate this group of patients for the prevalence of autoimmune disorders that may remain undetected by strict adherence to the proposed algorithms for auto-antibody detection. Simultaneous testing of rheumatoid factor (RF) and ANA was requested in 53 patients as first level investigations where only 4 (7.5%) patients tested positively for RF. This pattern of investigation may help in defining a group of rheumatoid arthritis patients who are less than 40 years of age [20]. Furthermore, treatment of refractory rheumatoid arthritis with infliximab has been shown to be associated with induction of ANA and antidsdna antibodies [21]. Auto-antibody induction has also been reported in rheumatoid arthritis following treatment with anti-tumor necrosis factor antibodies which inversely correlated with the levels of specific auto-antibodies, such as RF and anti-cyclic citrullinated peptide [22]. None of the patients investigated in the present study were being treated with any disease modifying agent at the time of investigation warranting auto-antibody testing. Since the diagnosis of rheumatoid arthritis is generally made on clinical presentation and is not based on RF reactivity the frequency of simultaneous ANA and RF testing observed in the present study clearly was not cost effective. 674 Clin. Lab. 4/2014

5 AUTO-ANTIBODY DETECTION CONCLUSION ANA as a single first level test for investigation of autoimmune rheumatic disorders was requested by a smaller proportion of clinicians indicating a poor adherence to recommended algorithm. Similarly, a vast majority of second line investigations for auto-antibody detection were simultaneously requested with ANA as first level tests. This practice, though contrary to the recommended guidelines, not only contributed to avoidable workload on the laboratory resources but was also not a cost effective approach. These observations highlight the importance of implementation of recommended guidelines and call for a better collaboration between clinicians and laboratory staff. Declaration of Interest: Each author declares no conflict of interest. Funding: This study was not funded. References: 1. Tan EM, Cohen AS, Fries JF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25: Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997;40: Villalta D, Tozzoli R, Tonutti E, Bizzaro N. The laboratory approach to the diagnosis of autoimmune diseases: is it time to change? Autoimmun Rev 2007;6: Plebani M, Pittoni M, Celadin M, Bernardi D, Mion MM. Recent advances in diagnostic technologies for autoimmune diseases. Autoimmun 2009;Rev 8: Stinton LM, Fritzler MJ. A clinical approach to autoantibody testing in systemic autoimmune rheumatic disorders. Autoimmun Rev 2007;7: Walraven C, Naylor CD. Do we know what inappropriate laboratory utilization is? JAMA 1998;280: Plebani M. The clinical importance of laboratory reasoning. Clin. Chim. Acta 1999;280: Wiik AS. Diagnostic strategies for autoimmune rheumatic diseases. Z. Rheumatol 2007;66: Tozzoli R, Bizzaro N, Tonutti E, et al. Italian Society of Laboratory Medicine Study Group on the Diagnosis of Autoimmune Diseases. Guidelines for the laboratory use of autoantibody tests in the diagnosis and monitoring of autoimmune rheumatic diseases. Am J Clin Pathol 2002;117: Kavanaugh A, Tomar R, Reveille J, Solomon DH, Homburger HA. Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantibodies to nuclear antigens. American College of Pathologists. Arch Pathol Lab Med 2000;124: Hindmarsh JT, Lyon AW. Strategies to promote rational clinical chemistry test utilization. Clin Biochem 1996;29: Tampoia M, Brescia V, Fontana A, Zucano A, Morrone LF, Pansini N. Application of a combined protocol for rational request and utilization of antibody assays improves clinical diagnostic efficacy in autoimmune rheumatic disease. Arch Pathol Lab Med 2007;131: Man A, Shojania K, Phoon C, et al. An evaluation of autoimmune antibody testing patterns in a Canadian health region and an evaluation of a laboratory algorithm aimed at reducing unnecessary testing. Clin Rheumatol 2013;32: Koller SR, Johnston CL Jr, Moncure CW. Lupus erythematosus cell preparation antinuclear factor incongruity. A review of diagnostic tests for systemic lupus erythematosus. Am J Clin Pathol 1976;66: McHardy KC, Horne CH, Rennie J. Antinuclear antibody-negative systemic lupus erythematosus-how common? J Clin Pathol 1982;35: Ferreiro JE, Reiter WM, Saldana MJ. Systemic lupus erythematosus presenting as chronic serositis with no demonstrable antinuclear antibodies. Am J Med 1984;76: Wallace DJ, Linker-Israeli M. It's not the same old lupus or Sjögren's any more: one hundred new insights, approaches, and options since Curr Opin Rheumatol 1999;11: Cross LS, Aslam A, Misbah SA. Antinuclear antibody-negative lupus as a distinct diagnostic entity--does it no longer exist? QJM. 2004;97: Craig WY, Ledue TB. The antinuclear antibody assay: developing criteria for reflexive anti-dsdna antibody testing in a laboratory setting. Clin Chem Lab Med 2011;49: Jacobsen S. Young age of onset is associated with increased prevalence of circulating IgM rheumatoid factor and antinuclear antibodies at presentation in women with rheumatoid arthritis. Clin Rheumatol 2004;23: Allanore Y, Sellam J, Batteux F, Job Deslandre C, Weill B, Kahan A. Induction of autoantibodies in refractory rheumatoid arthritis treated by infliximab. Clin Exp Rheumatol 2004;22: Atzeni F, Turiel M, Capsoni F, Doria A, Meroni P, Sarzi-Puttini P. Autoimmunity and anti-tnf-alpha agents. Ann N Y Acad Sci 2005;1051: Correspondence: Adel Almogren Department of Pathology College of Medicine and University Hospitals King Saud University PO Box: 2925 Riyadh Kingdom of Saudi Arabia Tel.: Fax: almogren@ksu.edu.sa Clin. Lab. 4/