© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12275
APMIS
Evaluation of antinuclear antibodies by indirect immunofluorescence and line immunoassay methods0: four years0 data from Turkey _ ASLI GAMZE SENER, ILHAN AFSAR and MUSTAFA DEMIRCI Ataturk Training and Research Hospital Medical Microbiology Laboratory, Izmir Katip Celebi University, Yesilyurt, Izmir, Turkey
_ Demirci M. Evaluation of antinuclear antibodies by indirect immunofluorescence and line Sener AG, Afsar I, immunoassay methods0 : four years0 data from turkey. APMIS 2014. The presence of antinuclear antibodies (ANAs), directed against intracellular antigens, is a hallmark of systemic autoimmune rheumatic diseases. The indirect immunofluorescence (IIF) assay is among the most commonly used routine methods for ANA detection as the screening test. The objective of the study was to evaluate ANA patterns in a 4-year period retrospectively. All 19 996 serum samples that were sent to the Laboratory of Medical Microbiology of the tertiary Hospital by any hospital department between 1 January 2009 and 1 January 2013 with a request to test for ANA, anti-ENA or both were included in the study. Of these samples, 4375 (21.9%) were ANA-IIF-positive and 15621 (78.1%) were ANA-IIF-negative. The presented ANA-positive samples consisted of 2392 (54.67%) homogenous, 818 (18.70%) speckled, 396 (9.05%) centromere, 242 (5.53%) nucleolar, 213 (4.87%) nuclear dots, 178 (4.07%) cytoplasmic (except for actin and golgi), 24 (0.55%) actin, 9 (0.21%) golgi, 53 (1.21%) nuclear membrane and 50 (1.14%) mixed pattern. Totally 7800 samples were examined by LIA. Of these samples, 3440 were positive and 4307 were negative with IIF and LIA. In addition, 22 samples were detected as IIF-positive but LIA-negative, whereas the rest 31 samples were IIF-negative but LIA-positive. ANA patterns in 22 IIF-positive samples were homogenous (9), speckled (5), golgi (4), cytoplasmic (3) and nucleolar (1). SSA/Ro-52, SSB/La and Scl-70 positivity were detected in 31 IIF-negative/LIA-positive samples by LIA. The present study comes forward with its overall scope, which covers 4-year data obtained in tertiary hospital located in the western part of Turkey. Key words: Antinuclear antibody; immunofluorescence assay; line assay. Asli Gamze Sener, Izmir Katip Celebi University, Ataturk Training and Research Hospital Medical Microbiology Laboratory, Yesilyurt, Izmir, Turkey. e-mail: [email protected]
The presence of antinuclear antibodies (ANAs), directed against intracellular antigens, is a hallmark of systemic autoimmune rheumatic diseases. The indirect immunofluorescence (IIF) assay is among the most commonly used routine methods for ANA detection as the screening test (1). This standard method has been used for over 40 years as a first-step screening test for autoimmune diseases and is still the standard method. Although the ANA test has a nearly 100% sensitivity for the diagnosis of SLE, it is not specific for this diagnosis and is frequently positive in other systemic autoimmune rheumatic diseases (SRD) such as scleroderma (SSc), polymyositis/ dermatomyositis (PM/DM) and Sj€ ogren’s syndrome (SjS) as well (2). In addition, ANA-positive samples Received 4 December 2013. Accepted 25 February 2014
should be subjected to more specific assays for the diagnosis (3). The reliable detection of an increased number of disease-specific autoantibodies would be beneficial for the accurate and early diagnosis of SRD. Line blot assay represents a reliable and sensitive alternative to more time-consuming procedures (3, 4). The objective of the study was to evaluate ANA patterns in a 4-year period retrospectively. The present study comes forward with its overall scope which covers the 4-year data obtained in tertiary hospital located in the western part of Turkey.
PATIENTS AND METHODS All antinuclear antibody and extractable nuclear antibody (ENA) performed in the laboratory over a 4-year period
1
SENER et al.
(from January 2009 to January 2013) were reviewed. A total of 19 996 serum samples were screened for ANA by IIF. The serum samples which were positive for ANA by IIF method or those which were negative by IIF method but requested by the rheumatologists were further processed for identification of the specific antibodies by line immunoassay (LIA). The specimens were from 12 148 new or follow-up patients with various SRD: SLE; n = 6971, Sjogren’s syndrome (Sjogren; n = 1814), systemic sclerosis (SS; n = 203), mixed connective tissue disease (MCTD; n = 591), overlap syndrome (n = 36), polymyositis/dermatomyositis (PM/DM; n = 294), rheumatoid arthritis (RA; n = 2150), vasculitis (n = 89) from rheumatology department and 7848 patients with non-SRD from gastroenterology, nephrology, neurology, oncology and general internal medicine according to the medical record review. Samples were evaluated by fluorescence microscope (Eurostar II, L€ ubeck, Germany) by the first author. The number of specimens which are re-evaluated due to technical failure does not exceed one or two per year. Serum samples were processed in dilution of 1:100 using HEp-20-10/liver biochip (Monkey) (EUROIMMUN AG, L€ ubeck, Germany) and conjugated with specific antihuman IgG (EUROIMMUN AG). The fluorescence intensity was evaluated at 9400, semi-quantitatively from 1+ to 4+ relative to the intensity of the positive (4+) and negative control. The test result was discarded if the positive control sample failed to show the precise results. ENAs were identified with LIA (ANAProfile 3, EUROIMMUN AG). This test identifies 15 different autoantibodies against nRNP, Sm, SS-A, Ro52, SS-B, Scl-70, PM/Scl, Jo-1, CENP B, PCNA, dsDNA, nucleosome, histone, Rib-P and M2. All assays were performed and interpreted according to the manufacturers’ instructions. Our laboratory participates in the external quality assurance program of the Euroimmune AG (L€ ubeck, Germany). In the course of this program, three specimens which are sent to our laboratory twice a year are evaluated with IIF and LIA for auto-antibodies and the results are sent online. Until now, the rate of correct results has been 99–100% (589 laboratories included).
RESULTS Among these 19 996 samples, 4375 (21.9%) were ANA-IIF-positive and 15 621 (78.1) were ANAIIF-negative. The presented ANA-positive samples consisted of 2392 (54.67%) homogenous, 818 (18.70%) speckled, 396 (9.05%) centromere, 242 (5.53%) nucleolar, 213 (4.87%) nuclear dots, 178 (4.07%) cytoplasmic (except for actin and golgi), 24 (0.55%) actin, 9 (0.21%) golgi, 53 (1.21%) nuclear membrane and 50 (1.14%) mixed pattern (Table 1). Totally 7800 samples were examined by LIA. Of these samples, 3440 were positive and 4307 were negative with IIF and LIA. In addition, 22 samples were detected as IIF-positive but LIA-negative, whereas the rest 31 samples were IIF-negative but LIA-positive (Table 2). ANA patterns in 22 IIF-positive samples were homogenous (9), speckled 2
Table 1. Patterns and number of ANA-IIF-positive samples Pattern n % Homogenous 2392 54.67 Speckled 818 18.70 Centromere 396 9.05 Nucleolar 242 5.53 Nuclear dots 213 4.87 Cytoplasmic1 178 4.07 Nuclear membrane 53 1.21 Mixed 50 1.14 Actin 24 0.55 Golgi 9 0.21 1 Except for actin and golgi.
Table 2. The results of IIF and LIA IIF-positive/LIA-positive IIF-negative/LIA-negative IIF-positive/LIA-negative IIF-negative/LIA-positive
3440 4307 22 31
(5), golgi (4), cytoplasmic (3) and nucleolar (1). SSA/Ro-52, SSB/La and Scl-70 positivity were detected in 31 IIF-negative/LIA-positive samples by LIA. Among the 31 samples, four showed monoreactivity on the LIA (Table 3).
DISCUSSION The introduction of human cancer cell lines (human laryngeal cancer cell line HEp-2 is the standard) as a substrate for the ANA test significantly increased the sensitivity in the detection of ANA in patients with SRD. However, an increase in positive results in healthy individuals decreased the specificity. One multicenter study reported that 31.7% of normal individuals were ANA-positive at 1:40 dilution, which was decreased to 13.3% at 1:80, and 5.0% at 1:160 dilution. As ~95% of SLE were still positive at 1:160 dilution, raising the negative cut-off titer
Table 3. Laboratory and clinical details for 31 samples testing positive by LIA and negative by IIF LIA Diagnosis SS-A(+)Ro52(+) Myalgia Polymyositis/dermatomyositis Rheumatoid arthritis Systemic sclerosis Non-SRD Systemic lupus erythematosus SS-A(+)Ro52(+) SS-B(+)Anti ds-DNA(+) SS-B(+) Systemic lupus erythematosus Vasculitis Scl(+) Systemic sclerosis
n 3 8 5 3 6 2 1 1 2
© 2014 APMIS. Published by John Wiley & Sons Ltd
EVALUATION OF ANTINUCLEAR ANTIBODIES
from 1:40 to 1:160 may improve the distinction between a clinically significant ANA result and a positive ANA result occurring in a normal individual (2, 5). Although ‘ANA-negative’ SLE is reported (6, 7), it is not clear whether this is the result of a technical artifact or whether a subgroup of SLE exists. Most ANA-negative patients are positive in DNA or extractable nuclear antigens (ENAs) assays (3). Therefore, ANA IIF and LIA have been used for detection of autoantibodies in our laboratory. The interpretation of most nuclear staining patterns is relatively straightforward, and they have been reported as being nuclear, centromere or nucleolar. A positive nuclear staining result is reported with a more detailed staining pattern, such as speckled, homogeneous, or peripheral in our laboratory. In addition, unusual staining patterns in nuclei, such as those for the nuclear mitotic apparatus, have not been informed to the clinicians (2). The homogenous pattern was the most common ANA pattern in the present study. In another study, the most common immunofluorescent patterns being homogeneous (39%), speckled (20%), mixed (17%), nucleolar (8%), Ro (7%) and centromere (4%) were reported (8). Sebastian et al. (9) informed that the most common ANA pattern was homogenous (45.4%) and second was speckled (35.6%). They reported that 21 (17.2%) of the ANA-IIF-positive samples showed negativity with line immunoassay. ANA-IIF-negative with line immunoassay-positive samples was discussed by researchers. In our results, of the 31 IIF-negative/ LIA-positive samples, SSA/Ro-52, SSB/La and Scl70 positivity were detected in 25, 4, and 2, respectively, by LIA. Vos et al. (10) informed that sera missed by screening with ANA-IIF the reactivity in the lineblot test was often against Ro/SSA and La/ SSB antigens. Their data imply that the laboratory should perform anti-ENA tests only in those patients with at least one criterion for systemic autoimmune disease, as has been reported previously for the ANA-IIF test. The finding of antiSSA/Ro reactivities in IIF-negative samples is not unexpected. Not only are anti-SSA/Ro reactivities seen in samples with low fluorescence intensities on IIF (11), but it has also been suggested that antiSSA/Ro antibodies should also be analyzed in samples with negative IIF on HEp-2 (12). Hoffman et al. argued that when clinical suspicion for rheumatic connective tissue disease is present, testing for ENAs should be performed; it should also be performed when IIF is negative. They emphasized that there is a risk of missing anti-SSA/Ro, anti-SSB and anti-Jo1 antibodies, which have an established clinical relevance (13). Peene et al. demonstrated in a
© 2014 APMIS. Published by John Wiley & Sons Ltd
large and consecutive patient population that serum samples with borderline ANA fluorescence might contain anti-SSA or anti-SSB antibodies (11). In the present study, 22 IIF-positive/LIA-negative samples were detected. Vos et al. (10) reported that a positive result with ANA-IIF together with negative results in LIA may indicate the presence of anti-dsDNA antibodies. In their results, a positive test for anti-dsDNA antibodies (Farr assay) was found in three of 23 ANA-positive samples with a negative result in LIA. However, antidsDNA was one of the 15 antigenic lines on LIA in our study. IIF- positive/LIA-negative samples were 25.7% in the other study (14). Multiplex technologies such as LIA have the advantage of simultaneous ANA testing for multiple reactivities (15). The study of Lee et al. had the benefit of examining a large population with various types of SRD and gathering data on a wide group of autoantibodies including less common diseasespecific ones. In the study, sera from non-SRD were associated with IIF()/LIA(+) and monoreactivity and anti-SS-A()/Ro52(+) on the LIA. Among the IIF()/LIA(+) patients, two cases, which were SS-A(+),Ro52(+),SS-B(+) and anti ds-DNA(+), were monitored in rheumatology polyclinic. Consequent to laboratory results and clinical findings, the two cases were evaluated as SLE. There are some articles about the prevalence of SS-A/SS-B antibodies in different patient groups. Metskula K et al. (16) showed that the prevalence of SS-A/SS-B antibodies in a general random population might be higher than thought previously as it was detected in up to 16.5% of persons including a significant number of those with HLA-DR3 or/ and DR11 alleles and with antibodies against enteroviruses. Abid et al. (17) collected data related to the clinical manifestations and laboratory investigations of systemic lupus erythematosus (SLE) patients in the eastern part of Saudi Arabia. In the retrospective study, they informed that antinuclear antibodies were positive in 44 (95.7%), antidsDNA in 38 (42.6%), anti-Ro SSA and La SSB in 38 (82.6%). In another study, it was aimed to characterize the manifestations of clinical symptoms and signs, primary rheumatic diseases, and other autoantibodies in paediatric patients with positive anti-SSA and/or anti-SSB antibodies (18). They informed that among twenty enrolled patients, seventeen of them had systemic lupus erythematosus; four of them were diagnosed as SS with positive Schirmer test. Among the studies reviewed, the capacity and structure of the hospital and the patient profile are important for the comparison of results. While some of the studies (9, 10, 14) resemble our study,
3
SENER et al.
given the specimens from different clinics were reviewed, some (8, 13) differed by examining only the patients with SRD. This has to be taken into consideration while discussing the results. The clinical impact of rare immunofluorescent patterns should be discussed. Nuclear dots, nuclear membrane, anti actin and anti golgi patterns were detected in 299 patients. Vermeersch P et al. (19) defined rare patterns as patterns occurring in
APMIS
Evaluation of antinuclear antibodies by indirect immunofluorescence and line immunoassay methods0: four years0 data from Turkey _ ASLI GAMZE SENER, ILHAN AFSAR and MUSTAFA DEMIRCI Ataturk Training and Research Hospital Medical Microbiology Laboratory, Izmir Katip Celebi University, Yesilyurt, Izmir, Turkey
_ Demirci M. Evaluation of antinuclear antibodies by indirect immunofluorescence and line Sener AG, Afsar I, immunoassay methods0 : four years0 data from turkey. APMIS 2014. The presence of antinuclear antibodies (ANAs), directed against intracellular antigens, is a hallmark of systemic autoimmune rheumatic diseases. The indirect immunofluorescence (IIF) assay is among the most commonly used routine methods for ANA detection as the screening test. The objective of the study was to evaluate ANA patterns in a 4-year period retrospectively. All 19 996 serum samples that were sent to the Laboratory of Medical Microbiology of the tertiary Hospital by any hospital department between 1 January 2009 and 1 January 2013 with a request to test for ANA, anti-ENA or both were included in the study. Of these samples, 4375 (21.9%) were ANA-IIF-positive and 15621 (78.1%) were ANA-IIF-negative. The presented ANA-positive samples consisted of 2392 (54.67%) homogenous, 818 (18.70%) speckled, 396 (9.05%) centromere, 242 (5.53%) nucleolar, 213 (4.87%) nuclear dots, 178 (4.07%) cytoplasmic (except for actin and golgi), 24 (0.55%) actin, 9 (0.21%) golgi, 53 (1.21%) nuclear membrane and 50 (1.14%) mixed pattern. Totally 7800 samples were examined by LIA. Of these samples, 3440 were positive and 4307 were negative with IIF and LIA. In addition, 22 samples were detected as IIF-positive but LIA-negative, whereas the rest 31 samples were IIF-negative but LIA-positive. ANA patterns in 22 IIF-positive samples were homogenous (9), speckled (5), golgi (4), cytoplasmic (3) and nucleolar (1). SSA/Ro-52, SSB/La and Scl-70 positivity were detected in 31 IIF-negative/LIA-positive samples by LIA. The present study comes forward with its overall scope, which covers 4-year data obtained in tertiary hospital located in the western part of Turkey. Key words: Antinuclear antibody; immunofluorescence assay; line assay. Asli Gamze Sener, Izmir Katip Celebi University, Ataturk Training and Research Hospital Medical Microbiology Laboratory, Yesilyurt, Izmir, Turkey. e-mail: [email protected]
The presence of antinuclear antibodies (ANAs), directed against intracellular antigens, is a hallmark of systemic autoimmune rheumatic diseases. The indirect immunofluorescence (IIF) assay is among the most commonly used routine methods for ANA detection as the screening test (1). This standard method has been used for over 40 years as a first-step screening test for autoimmune diseases and is still the standard method. Although the ANA test has a nearly 100% sensitivity for the diagnosis of SLE, it is not specific for this diagnosis and is frequently positive in other systemic autoimmune rheumatic diseases (SRD) such as scleroderma (SSc), polymyositis/ dermatomyositis (PM/DM) and Sj€ ogren’s syndrome (SjS) as well (2). In addition, ANA-positive samples Received 4 December 2013. Accepted 25 February 2014
should be subjected to more specific assays for the diagnosis (3). The reliable detection of an increased number of disease-specific autoantibodies would be beneficial for the accurate and early diagnosis of SRD. Line blot assay represents a reliable and sensitive alternative to more time-consuming procedures (3, 4). The objective of the study was to evaluate ANA patterns in a 4-year period retrospectively. The present study comes forward with its overall scope which covers the 4-year data obtained in tertiary hospital located in the western part of Turkey.
PATIENTS AND METHODS All antinuclear antibody and extractable nuclear antibody (ENA) performed in the laboratory over a 4-year period
1
SENER et al.
(from January 2009 to January 2013) were reviewed. A total of 19 996 serum samples were screened for ANA by IIF. The serum samples which were positive for ANA by IIF method or those which were negative by IIF method but requested by the rheumatologists were further processed for identification of the specific antibodies by line immunoassay (LIA). The specimens were from 12 148 new or follow-up patients with various SRD: SLE; n = 6971, Sjogren’s syndrome (Sjogren; n = 1814), systemic sclerosis (SS; n = 203), mixed connective tissue disease (MCTD; n = 591), overlap syndrome (n = 36), polymyositis/dermatomyositis (PM/DM; n = 294), rheumatoid arthritis (RA; n = 2150), vasculitis (n = 89) from rheumatology department and 7848 patients with non-SRD from gastroenterology, nephrology, neurology, oncology and general internal medicine according to the medical record review. Samples were evaluated by fluorescence microscope (Eurostar II, L€ ubeck, Germany) by the first author. The number of specimens which are re-evaluated due to technical failure does not exceed one or two per year. Serum samples were processed in dilution of 1:100 using HEp-20-10/liver biochip (Monkey) (EUROIMMUN AG, L€ ubeck, Germany) and conjugated with specific antihuman IgG (EUROIMMUN AG). The fluorescence intensity was evaluated at 9400, semi-quantitatively from 1+ to 4+ relative to the intensity of the positive (4+) and negative control. The test result was discarded if the positive control sample failed to show the precise results. ENAs were identified with LIA (ANAProfile 3, EUROIMMUN AG). This test identifies 15 different autoantibodies against nRNP, Sm, SS-A, Ro52, SS-B, Scl-70, PM/Scl, Jo-1, CENP B, PCNA, dsDNA, nucleosome, histone, Rib-P and M2. All assays were performed and interpreted according to the manufacturers’ instructions. Our laboratory participates in the external quality assurance program of the Euroimmune AG (L€ ubeck, Germany). In the course of this program, three specimens which are sent to our laboratory twice a year are evaluated with IIF and LIA for auto-antibodies and the results are sent online. Until now, the rate of correct results has been 99–100% (589 laboratories included).
RESULTS Among these 19 996 samples, 4375 (21.9%) were ANA-IIF-positive and 15 621 (78.1) were ANAIIF-negative. The presented ANA-positive samples consisted of 2392 (54.67%) homogenous, 818 (18.70%) speckled, 396 (9.05%) centromere, 242 (5.53%) nucleolar, 213 (4.87%) nuclear dots, 178 (4.07%) cytoplasmic (except for actin and golgi), 24 (0.55%) actin, 9 (0.21%) golgi, 53 (1.21%) nuclear membrane and 50 (1.14%) mixed pattern (Table 1). Totally 7800 samples were examined by LIA. Of these samples, 3440 were positive and 4307 were negative with IIF and LIA. In addition, 22 samples were detected as IIF-positive but LIA-negative, whereas the rest 31 samples were IIF-negative but LIA-positive (Table 2). ANA patterns in 22 IIF-positive samples were homogenous (9), speckled 2
Table 1. Patterns and number of ANA-IIF-positive samples Pattern n % Homogenous 2392 54.67 Speckled 818 18.70 Centromere 396 9.05 Nucleolar 242 5.53 Nuclear dots 213 4.87 Cytoplasmic1 178 4.07 Nuclear membrane 53 1.21 Mixed 50 1.14 Actin 24 0.55 Golgi 9 0.21 1 Except for actin and golgi.
Table 2. The results of IIF and LIA IIF-positive/LIA-positive IIF-negative/LIA-negative IIF-positive/LIA-negative IIF-negative/LIA-positive
3440 4307 22 31
(5), golgi (4), cytoplasmic (3) and nucleolar (1). SSA/Ro-52, SSB/La and Scl-70 positivity were detected in 31 IIF-negative/LIA-positive samples by LIA. Among the 31 samples, four showed monoreactivity on the LIA (Table 3).
DISCUSSION The introduction of human cancer cell lines (human laryngeal cancer cell line HEp-2 is the standard) as a substrate for the ANA test significantly increased the sensitivity in the detection of ANA in patients with SRD. However, an increase in positive results in healthy individuals decreased the specificity. One multicenter study reported that 31.7% of normal individuals were ANA-positive at 1:40 dilution, which was decreased to 13.3% at 1:80, and 5.0% at 1:160 dilution. As ~95% of SLE were still positive at 1:160 dilution, raising the negative cut-off titer
Table 3. Laboratory and clinical details for 31 samples testing positive by LIA and negative by IIF LIA Diagnosis SS-A(+)Ro52(+) Myalgia Polymyositis/dermatomyositis Rheumatoid arthritis Systemic sclerosis Non-SRD Systemic lupus erythematosus SS-A(+)Ro52(+) SS-B(+)Anti ds-DNA(+) SS-B(+) Systemic lupus erythematosus Vasculitis Scl(+) Systemic sclerosis
n 3 8 5 3 6 2 1 1 2
© 2014 APMIS. Published by John Wiley & Sons Ltd
EVALUATION OF ANTINUCLEAR ANTIBODIES
from 1:40 to 1:160 may improve the distinction between a clinically significant ANA result and a positive ANA result occurring in a normal individual (2, 5). Although ‘ANA-negative’ SLE is reported (6, 7), it is not clear whether this is the result of a technical artifact or whether a subgroup of SLE exists. Most ANA-negative patients are positive in DNA or extractable nuclear antigens (ENAs) assays (3). Therefore, ANA IIF and LIA have been used for detection of autoantibodies in our laboratory. The interpretation of most nuclear staining patterns is relatively straightforward, and they have been reported as being nuclear, centromere or nucleolar. A positive nuclear staining result is reported with a more detailed staining pattern, such as speckled, homogeneous, or peripheral in our laboratory. In addition, unusual staining patterns in nuclei, such as those for the nuclear mitotic apparatus, have not been informed to the clinicians (2). The homogenous pattern was the most common ANA pattern in the present study. In another study, the most common immunofluorescent patterns being homogeneous (39%), speckled (20%), mixed (17%), nucleolar (8%), Ro (7%) and centromere (4%) were reported (8). Sebastian et al. (9) informed that the most common ANA pattern was homogenous (45.4%) and second was speckled (35.6%). They reported that 21 (17.2%) of the ANA-IIF-positive samples showed negativity with line immunoassay. ANA-IIF-negative with line immunoassay-positive samples was discussed by researchers. In our results, of the 31 IIF-negative/ LIA-positive samples, SSA/Ro-52, SSB/La and Scl70 positivity were detected in 25, 4, and 2, respectively, by LIA. Vos et al. (10) informed that sera missed by screening with ANA-IIF the reactivity in the lineblot test was often against Ro/SSA and La/ SSB antigens. Their data imply that the laboratory should perform anti-ENA tests only in those patients with at least one criterion for systemic autoimmune disease, as has been reported previously for the ANA-IIF test. The finding of antiSSA/Ro reactivities in IIF-negative samples is not unexpected. Not only are anti-SSA/Ro reactivities seen in samples with low fluorescence intensities on IIF (11), but it has also been suggested that antiSSA/Ro antibodies should also be analyzed in samples with negative IIF on HEp-2 (12). Hoffman et al. argued that when clinical suspicion for rheumatic connective tissue disease is present, testing for ENAs should be performed; it should also be performed when IIF is negative. They emphasized that there is a risk of missing anti-SSA/Ro, anti-SSB and anti-Jo1 antibodies, which have an established clinical relevance (13). Peene et al. demonstrated in a
© 2014 APMIS. Published by John Wiley & Sons Ltd
large and consecutive patient population that serum samples with borderline ANA fluorescence might contain anti-SSA or anti-SSB antibodies (11). In the present study, 22 IIF-positive/LIA-negative samples were detected. Vos et al. (10) reported that a positive result with ANA-IIF together with negative results in LIA may indicate the presence of anti-dsDNA antibodies. In their results, a positive test for anti-dsDNA antibodies (Farr assay) was found in three of 23 ANA-positive samples with a negative result in LIA. However, antidsDNA was one of the 15 antigenic lines on LIA in our study. IIF- positive/LIA-negative samples were 25.7% in the other study (14). Multiplex technologies such as LIA have the advantage of simultaneous ANA testing for multiple reactivities (15). The study of Lee et al. had the benefit of examining a large population with various types of SRD and gathering data on a wide group of autoantibodies including less common diseasespecific ones. In the study, sera from non-SRD were associated with IIF()/LIA(+) and monoreactivity and anti-SS-A()/Ro52(+) on the LIA. Among the IIF()/LIA(+) patients, two cases, which were SS-A(+),Ro52(+),SS-B(+) and anti ds-DNA(+), were monitored in rheumatology polyclinic. Consequent to laboratory results and clinical findings, the two cases were evaluated as SLE. There are some articles about the prevalence of SS-A/SS-B antibodies in different patient groups. Metskula K et al. (16) showed that the prevalence of SS-A/SS-B antibodies in a general random population might be higher than thought previously as it was detected in up to 16.5% of persons including a significant number of those with HLA-DR3 or/ and DR11 alleles and with antibodies against enteroviruses. Abid et al. (17) collected data related to the clinical manifestations and laboratory investigations of systemic lupus erythematosus (SLE) patients in the eastern part of Saudi Arabia. In the retrospective study, they informed that antinuclear antibodies were positive in 44 (95.7%), antidsDNA in 38 (42.6%), anti-Ro SSA and La SSB in 38 (82.6%). In another study, it was aimed to characterize the manifestations of clinical symptoms and signs, primary rheumatic diseases, and other autoantibodies in paediatric patients with positive anti-SSA and/or anti-SSB antibodies (18). They informed that among twenty enrolled patients, seventeen of them had systemic lupus erythematosus; four of them were diagnosed as SS with positive Schirmer test. Among the studies reviewed, the capacity and structure of the hospital and the patient profile are important for the comparison of results. While some of the studies (9, 10, 14) resemble our study,
3
SENER et al.
given the specimens from different clinics were reviewed, some (8, 13) differed by examining only the patients with SRD. This has to be taken into consideration while discussing the results. The clinical impact of rare immunofluorescent patterns should be discussed. Nuclear dots, nuclear membrane, anti actin and anti golgi patterns were detected in 299 patients. Vermeersch P et al. (19) defined rare patterns as patterns occurring in
