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A Multicenter Analysis of Subjectivity of Indirect Immunofluorescence Test in Antinuclear Antibody Screening

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A Multicenter Analysis of Subjectivity of Indirect Immunofluorescence Test in Antinuclear Antibody Screening Arch Rheumatol 2019;34(3):326-333 doi: 10.5606/ArchRheumatol.2019.7310 ORIGINAL ARTICLE A Multicenter Analysis of Subjectivity of Indirect Immunofluorescence Test in Antinuclear Antibody Screening Vildan TURAN FARAŞAT1, Talat ECEMİŞ1, Yavuz DOĞAN2, Aslı Gamze ŞENER3, Gülfem TEREK ECE4, Pınar ERBAY DÜNDAR5, Tamer ŞANLIDAĞ6 1Department of Medical Microbiology, Manisa Celal Bayar University, Faculty of Medicine, Manisa, Turkey 2Department of Medical Microbiology, Dokuz Eylül University, Faculty of Medicine, Izmir, Turkey 3Department of Medical Microbiology, Katip Çelebi University, Faculty of Medicine, Izmir, Turkey 4Department of Medical Microbiology, Izmir Medicalpark Hospital, Izmir, Turkey 5Department of Public Health, Manisa Celal Bayar University, Faculty of Medicine, Manisa, Turkey 6Department of Medical Microbiology, Manisa Celal Bayar University, Faculty of Medicine, Manisa, Turkey ABSTRACT Objectives: This study aims to evaluate the interpretation of the antinuclear antibody (ANA)-indirect immunofluorescence (IIF) test results based on the interpreter-related subjectivity and to examine the inter-center agreement rates with the performance of each laboratory. Patients and methods: The ANA-IIF testing was carried out in a total of 600 sera and evaluated by four laboratories. The inter-center agreement rates were detected. The same results given by the four centers were accepted as gold standard and the predictive values of each center were calculated. Results: The inter-center agreement was reported for ANA-IIF test results from 392 of 600 (65.3%) sera, while 154 of 392 results were positive. Four study centers reported 213 (35.5%), 222 (37.0%), 266 (44.3%), and 361 (60.2%) positive test results, respectively. In terms of the patterns, the highest and lowest positive predictive values were 72.3% and 42.7%, respectively, while the highest and lowest negative predictive values were 99.6% and 61.5%, respectively. The agreement for semi-quantitative evaluation at three levels of fluorescence intensity stated by four centers was detected in 100 sera at 87% 3(+), while the other two levels were 6% and 7%. The highest predictive value for the highest fluorescence intensity of 3(+) was found to be 71.9%. Conclusion: Significant differences may be observed among laboratories in terms of qualitative results, patterns, and semi-quantitative determination of the fluorescence intensity in the ANA-IIF testing, particularly at low fluorescence intensity levels and in those with speckled patterns. In case of any discrepancy between ANA-IIF test and clinical prediagnosis, the test should be repeated in another laboratory, if necessary. Keywords: Antinuclear antibodies; autoimmune rheumatic diseases; indirect immunofluorescence; subjectivity. Systemic autoimmune rheumatic diseases The term ANA is based on historical are a wide, heterogeneous group of diseases development in the understanding of this group characterized by the occurrence of antinuclear of diseases; however, currently, this term indicates antibodies (ANAs) directed against several antibodies against both nuclear and cytoplasmic intracellular targets. Therefore, detection of ANAs antigens. Indirect immunofluorescence (IIF) test is of utmost importance in the diagnosis of these has been used for detecting these antibodies diseases.1,2 for more than five decades and considered the Received: November 21, 2018 Accepted: January 10, 2019 Published online: April 22, 2019 Correspondence: Talat Ecemiş, MD. Celal Bayar Üniversitesi Tıp Fakültesi Tıbbi Mikrobiyoloji Anabilim Dalı, 45030 Manisa, Turkey. Tel: +90 236 - 231 19 20 e-mail: [email protected] Citation: Turan Faraşat V, Ecemiş T, Doğan Y, Şener AG, Terek Ece G, Erbay Dündar P, et al. A multicenter analysis of subjectivity of indirect immunofluorescence test in antinuclear antibody screening. Arch Rheumatol 2019;34(3):326-333. ©2019 Turkish League Against Rheumatism. All rights reserved. Subjectivity of ANA-IIF Test 327 gold standard screening method owing to its The inclusion criteria for the laboratories high sensitivity.3 By binding to cells from human were as follows: having staff microbiologists laryngeal epithelial carcinoma-2 (HEp-2) cell line, and technicians with a minimum of five-year which are specifically used for this test, ANAs create experience in reading and interpreting ANA-IIF images (patterns) which not only demonstrate the tests; and the inter-center distances not exceeding presence of ANAs but that may also predict the 50 km to protect test slides in transport. prognosis for a specific ANA type, which makes A code unrelated to the above order was IIF test more than a screening test and superior to randomly assigned to each center as Center 1, 4 other screening modalities. The ANA titers may Center 2, Center 3, and Center 4. The Serology be determined with IIF tests, and fluorescence Laboratory of Manisa Celal Bayar University, intensity may be expressed semi-quantitatively as Faculty of Medicine performed and interpreted a 5 currently practiced in many laboratories. Based total of 100 ANA-IIF tests every day. Test slides on the currently established ANA disease testing were delivered to other centers during the same algorithm, the second-line reflex tests including day in containers with light and heat insulation. enzyme-linked immunosorbent assay (ELISA) Tests were assessed by interpreters within the using extractable nuclear antigens (ENAs), flow same day. cytometry or immunoblotting techniques, which can detect multiple anti-ENA antibodies, are used Test sera were selected independently from to detect specific antibodies, when the initial the clinical diagnosis. A total of 600 sera with screening tests produce ANA positivity.6-8 positive and negative results were obtained from the centers and included in the study. All sera Although the ANA-IIF test has been were collected at Manisa Celal Bayar University, considered the gold standard screening test, this Faculty of Medicine. The sera were stored at technique involves a number of time-consuming -70°C until analysis. The IIFT Mosaic Basic manual procedures with standardization issues.5,9 Profile 3A® (Euroimmun®, Luebeck, Germany) The major challenge and limitation of the test containing both human HEp-2 cells and ANA-IIF testing is that it requires competent monkey liver cells was used in the ANA-IIF and experienced interpreters and subjectivity. testing. Tests were conducted in sera diluted Although subjectivity is an established fact 1/100 in accordance with the manufacturer’s in routine laboratory practice, the degree of instructions. subjectivity has been investigated in a limited number of studies in the literature to date. For the assessment of ANA-IIF tests, the Therefore, in this study, we aimed to evaluate sera were randomly distributed into slides. All ANA-IIF tests were assessed in a double-blind the interpretation of the ANA-IIF test results fashion. The Guidelines for the Laboratory based on the interpreter-related subjectivity and Diagnosis of Autoantibodies of the Society for to examine the inter-center agreement rates with Clinical Microbiologists of Turkey was used in the the performance of each laboratory. assessment of ANA patterns, and fluorescence microscopes of the same brand (EUROStar II®, Euroimmun®, Luebeck, Germany) were used in PATIENTS AND METHODS all study centers.10 The fluorescence intensity This multicenter study was conducted was semi-quantitatively rated at three levels and between January 2016 and September 2017 expressed as 1(+), 2(+), 3(+). in medical microbiology laboratories of four Considering multiple patterns in sera with tertiary hospitals. The ANA-IIF analyses were ANA positivity in the ANA-IIF test, the test assessed by specialists who were competent results were divided into two categories based on in reading and interpreting ANA-IIF testing. the pattern diagnosed in the analysis: complete The interpreter-assessed positive/negative rates, agreement and partial agreement. Agreement patterns detected in the analyses and semi- with at least one pattern was considered partial quantitative analysis of fluorescence intensities agreement, while agreement for all patterns were compared to each other. The results were was considered complete agreement, if multiple interpreted and statistically analyzed. patterns were detected in the same sera sample. Arch Rheumatol 328 Table 1. Patterns and their numbers used by the centers in the assessment of ANA-IIF tests Center 1 Center 2 Center 3 Center 4 SP MP Total SP MP Total SP MP Total SP MP Total n n n % n n n % n n n % n n n % NS 61 32 93 29 66 11 77 32.6 54 6 60 24.2 110 69 179 39.2 NDFS 39 9 48 15 15 0 15 6.4 14 1 15 6 23 2 25 5.5 NH 35 13 48 15 51 8 59 25 42 8 50 20.2 21 19 40 8.8 CR 24 11 35 10.9 13 9 22 9.3 13 9 22 8.9 24 17 41 9 C 14 3 17 5.3 15 2 17 7.2 14 4 18 7.3 13 4 17 3.7 N 14 14 28 8.7 12 4 16 6.8 26 3 29 11.6 25 16 41 9 CS 14 24 38 11.8 5 1 6 2.5 16 7 23 9.3 42 55 97 21.2 SLP 2 1 3 0.9 3 0 3 1.3 - - - - - - - - CLF 2 1 3 0.9 3 0 3 1.3 2 1 3 1.2 1 4 5 1.1 CE - - - - - - - - 5 1 6 2.4 - - - - NDF - - 1 0.3 0 7 7 3.0 4 6 10 4 - - 4 0.9 CDFS - - - - 2 0 2 0.8 1 1 2 0.8 - - - - NM 1 3 4 1.2 2 1.1 3 1.3 2 4 6 2.4 2 2 4 0.9 IB - - - - 1 0 1 0.4 2 0 2 0.8 0 1 1 0.2 CFF 0 1 1 0.3 - - - - 0 1 1 0.4 1 1 2 0.4 SF 1 1 2 0.6 - - 4 1.7 - - - - 0 1 1 0.2 CPS - - - - 1 0 1 0.4 1 0 1 0.5 - - - - Total 207 114 321 100 189 47 236 100 196 52 248 100 262 195 457 100 SP: Single pattern; MP: Multiple patterns; NS: Nuclear speckled; NDFS: Nuclear dense fine speckled; NH: Nuclear homogeneous; CR: Cytoplasmic reticular; C: Centromere; N: Nucleolar; CS: Cytoplasmic speckled; SLP: Scl-70 like pattern; CLF: Cytoplasmic fibrillar linear; CE: Centrosome; NDF: Nuclear dots few; CDFS: Cytoplasmic dense fine speckled; NM: Nuclear membrane; IB: Intracellular bridge; CFF: Cytoplasmic fibrillar filamentous; SF: Spindle fibers; CPS: Cytoplasmic polar speckled.
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