Diagnostic value of trait antinuclear antibodies and multiple immunoglobulin production in autoimmune diseases

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1 Received: 18 September 2017 Accepted: 25 October 2017 DOI: /jcla RESEARCH ARTICLE Diagnostic value of trait antinuclear antibodies and multiple immunoglobulin production in autoimmune diseases Liping Wan 1,2 Hong Zhu 2 Yanan Gu 2 Hui Liu 1 1 Department of Clinical Immunology, Dalian Medical University, Dalian, Liaoning Province, China 2 Department of Clinical Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning Province, China Correspondence Hui Liu, Department of Clinical Immunology, Dalian Medical University, Dalian, China. liuhui60@sina.com Background: Our article aims to evaluate the proportion of monospecific antinuclear antibodies (ANA) and polyclonal ANAs in patients with autoimmune diseases based on the results of an ANA panel and to evaluate the efficiency of trait ANAs as a novel diagnostic tool. This study also aims to investigate immunoglobulin production in autoimmune diseases by detecting different antibodies. Methods: The serum ANA profile of 634 patients with autoimmune diseases was analyzed using the immunoblot method. A specific formula was developed in an effort to calculate the theoretical proportion of monospecific ANA (TPM) in different disease groups. Different IgM, IgG, and IgE variants for several pathologies were detected. Results: The observed proportions of monospecific ANAs (OPM) were all lower than the predicted TPM in autoimmune diseases. Polyclonal ANAs were predominant in patients with systemic lupus erythematosus (SLE). There were statistical differences in OPM and TPM in all disease groups (P <.001). Receiver operating characteristic curve (ROC curve) analysis of trait ANAs between the SLE group and the control groups indicated an area under the curve of Differences were found in IgM of Toxoplasma gondii (TOXO) and IgG of hepatitis C virus (HCV) and Treponema pallidum (TP) when comparing the various disease groups to the control group. Conclusion: The higher TPM suggests that polyclonal differentiation is the major mechanism of ANA in autoimmune diseases. Trait ANA is potentially a valuable new index for diagnosis in SLE. Further investigation is needed to understand the link between B- cell differentiation and autoimmune diseases. KEYWORDS antinuclear antibody, autoimmune disease, immunoglobulin, trait ANA 1 INTRODUCTION Autoimmune diseases may be defined as sustained pathologies in which dysfunctional immune activation results in pathological immune responses that target either cellular or organ- specific selfantigens. Recent studies on the epidemiology of systemic lupus erythematosus (SLE) report that global incidences and prevalences of SLE are increasing. 1 The etiology of the majority of autoimmune diseases remains unknown, many factors including genetics, the microbiome, and environmental toxins could influence or induce autoimmunity and eventually systemic organ disease. 2-4 All autoimmune diseases are driven by the innate and the adaptive immune response, and disease specificity is often defined by the presence of IgG autoantibodies. 5 Autoantibodies to a large number of selfantigens are usually found in sera of patients with autoimmune diseases. However, in spite of the role played by these antibodies in pathogenesis and progression of autoimmune diseases, their direct role has never been clarified. Therefore, the key to understanding autoimmune diseases with unknown etiology could be in the generation of these autoantibodies. J Clin Lab Anal. 2018;32:e wileyonlinelibrary.com/journal/jcla 2017 Wiley Periodicals, Inc. 1 of 7

2 2 of 7 These pathogenic autoantibodies are produced by plasma cells differentiated from activated autoreactive B cells. In some autoimmune diseases including SLE and rheumatoid arthritis (RA), autoantibodies are usually present several years prior to a diagnosis. 6,7 Some autoantibodies can be predictive for disease development in asymptomatic subjects. 8 In this study, we detected ANAs associated with specific autoimmune diseases as a basis for developing new diagnostic tools and deepening our understanding of the ANA s role in autoimmune disease. Indirect immunofluorescence (IIF) is still the recommended method for highly sensitive analysis of antibodies using cellular autoantigenic targets. However, in clinical laboratories, ANA detection by IIF analysis has a high false- positive rate. This is particularly true for Sjögren syndrome In our study, the ANA profile (a confirmatory test of ANA) was established by specific immunoblot assay in which test strips were coated with parallel lines of highly purified antigens. For the evaluation of incubated test strips, we used the EUROLineScan software. Signal intensity can be read after scanning. Outcomes can be used to establish grade and signal intensity for each autoantigen- detecting antibody, and these can be used to classify the ANA profile. Although the autoantibody response is broad, some ANAs are very specific and can be used as diagnostic criteria in some diseases. 12 Usually, many different autoantibodies can be found in a single sample. To understand the features and clinical significance of these autoantibodies, we investigated 634 cases of autoimmune disease and evaluated the diagnostic efficiency of ANAs in a number of autoimmune diseases including SLE, RA, primary Sjögren syndrome (pss), and vasculitis. We also evaluated the proportion of specific immunoglobulin (Ig) groups, including M, G, and E to establish whether there were more Ig types in patients with autoimmune disease. disease and 27 control group samples were collected randomly to detect different Ig. 2.2 Detection of ANA profile (IgG) Blood samples (3 ml) were collected from 643 patients and 61 healthy individuals. Serum was separated by centrifugation at 2630 g for 10 minutes. The EUROLINE Antibodies against nuclear antigens (IgG) test kit (EUROIMMUN medizinische Labordiagnostika AG, Lübeck, Germany) were used. The EUROLINE test kit provides a qualitative in vitro assay for human autoantibodies of the IgG class to 15 different nuclear antigens. The test kit contains test strips coated with parallel lines of highly purified antigens. They were first moistened and then incubated in the first reaction step with patient serum (diluted 1:101 with the sample buffer) for 30 minutes. If samples were positive, specific IgG antibodies would bind to the corresponding antigenic site. To detect the bound antibodies, a second incubation was carried out using an enzyme- labeled monoclonal anti- human IgG (enzyme conjugate) catalyzing a color reaction. In the test kit, the enzyme conjugate was alkaline phosphatase- labeled anti- human IgG (goat). Substrate solution was nitroblue tetrazolium chloride/5- bromo- 4- chloro- 3- indolylphosphate. For automated incubation with the EUROBlotMaster, select the program Euro01 AAb EL30. The whole experimental process was performed according to the procedure provided by the automatic system. The EUROLineScan program was used for digital evaluation of the strips once dry. According to signal intensity of bands the results can be classified into five grades: (0 5), ± (6 10), 1 + (11 25), 2 + (26 50), 3 + (>50). The EUROLINE test strips have been coated with 15 antigens including nrnp/sm, Sm, SSA, Ro- 52, SSB, Scl- 70, PM- Scl, Jo- 1, CENP B, PCNA, dsdna, Nucleosomes, Histones, Rib.P- protein, and AMA M2. 2 METHODS 2.1 Subjects In total, 643 serum samples were collected from hospitalized patients and outpatients at the First Affiliated Hospital of Dalian Medical University, Dalian, China, from September 2015 to November These patients were clinically evaluated by their physicians and diagnosed with specific autoimmune diseases including SLE (213, 33.13%, male 23, female 190, age ± years), RA (277, 43.08%, male 59, female 218, age ± years), and other autoimmune diseases (primary Sjögren syndrome, scleroderma, vasculitis, Behcet disease, and dermatomyositis 153, 23.79%, male 27, female 126, age ± years). All patients in the SLE group fulfilled the classification criteria for SLE. 12 All patients in RA group fulfilled the RA classification criteria. 13 In other autoimmune disease groups, all patients fulfilled corresponding diagnostic criteria for the specific disease. There were also 61 healthy individuals (10 male and 51 female, aged ± years) evaluated as a control during the same period; these individuals had to have no prior history of any autoimmune disease. Among all these samples, 187 autoimmune 2.3 Calculation of observed proportion of monospecific ANA (OPM) and theoretical proportion of monospecific ANA (TPM) Calculation of observed proportion of monospecific ANA OPM = (number of monospecific samples/total number) 100% Calculation of theoretical proportion of monospecific ANA Principle: In this test, there were 15 antigens that were random variants; for example, in one sample, if only one antigen was positive and all 14 others were negative, the result was considered monospecific. In other words, the probability of monospecificity was calculated on the basis of a joint probability: one positive variant and 14 negative variants had to be found in a single sample to qualify as monospecific. According to the principle of probability theory, joint probability is the product of individual probabilities. Theoretically, monospecific probability for an antigen (A) is equal to the product of positive probability of antigen A and negative probability of all other antigens A n n=1 (1 A n ) n=1 A, A n is negative probability of antigen A. A n = (num- n ber of negative of antigen A/total number) 100%. The sum of the

3 3 of 7 monospecific probabilities of the 15 autoantigens is theoretical proportion of monospecific ANA. 2.4 Definition of trait ANA If more than one antigen were found positive in one sample, the IgG with highest ratio was considered as the trait ANA. 14 Ratio = sample signal intensity/control signal intensity. 2.5 Total serum IgE and rheumatoid factor (RF) detection Total serum IgE and RF were detected by immunonephelometry using the BN II system (Siemens Healthcare Diagnostic Product GmbH, Marburg, Germany). 2.6 Detection of IgG antibodies to hepatitis C virus (HCV) and Treponema pallidum (Syphilis TP assay) The chemiluminescent microparticle immunoassay (ARCHITECT i2000sr, Abbott Laboratories, Abbott Park, IL, USA) was used for the detection of HCV IgG and Syphilis TP assay. The ARCHITECT isystem calculates the cutoff (CO) using the mean chemiluminescent signal (RLU). S/CO = Sample RLU/Cutoff RLU. Specimens with S/CO value 1.0 were considered reactive. 2.7 Detection of IgG antibody to human immunodeficiency virus (HIV) ELISA was conducted using diagnostic kit for antibody to HIV detection (Zhuhai Livzon Diagnostics Inc., Zhuhai, China) using an Addcare ELISA 600 immunoassay workstation (Yantai Addcare Bio- Tech Co. Ltd., Yantai, China). Specimens with S/CO value 1.0 were considered reactive. 2.8 Detection of IgM antibodies to Toxoplasma gondii (Toxoplasma), cytomegalovirus (CMV), and rubella virus (RV) IgM antibodies to Toxoplasma, CMV, and RV were analyzed using a Toxoplasma IgM test kit (F. Hoffmann- La Roche, Ltd., Basel, Switzerland), a CMV IgM test kit (F. Hoffmann- La Roche, Ltd.), and a RV IgM test kit (F. Hoffmann- La Roche, Ltd.) with an electrochemiluminescence assay system (Cobas e 601, F. Hoffmann- La Roche, Ltd.). Reference ranges (value of S/CO) for healthy Chinese population are as follows: Toxoplasma IgM ( ), CMV IgM ( ), and RV IgM ( ). 2.9 Statistical analysis All data were analyzed using SPSS (Statistical Package for Social Sciences) 20.0 software (Chicago, IL, USA). Data were considered to be statistically significant when P <.05. A chi- squared test and nonparametric independent test were used to determine the betweengroup differences in numerical data. Nonparametric binomial tests were used to evaluate OPM and TPM. ROC analysis was carried out to evaluate the diagnostic efficacy for trait ANA. 3 RESULTS 3.1 Results of ANA profile in 643 patients with autoimmune diseases and 61 healthy individuals Fifteen types of IgG antibodies were detected. No cross- reactions with other autoantibodies have been found (Figure 1). The ANA profile results are as shown: the top three antibodies were anti- Ro- 52, anti- SSA, and anti- RNP in the SLE group. While the top three antibodies were anti- Ro- 52, anti- SSA, and anti- SSB in the RA group. Finally, the top three antibodies were anti- Ro- 52, anti- SSA, and anti- SSB in the others groups (Table 1). 3.2 Numbers of ANA (IgG), TPM, and OPM in different autoimmune diseases The number of positive ANAs (IgG) in one sample was 0-8. The highest number of ANAs (IgG) was 8 in patients with SLE. The highest number of ANAs (IgG) was 5 in patients with RA (Table 2). If only one IgG ANA was positive, it was called monospecific ANA. The proportions of monospecific ANAs were different in different disease groups (Table 3). If more than one ANAs were found positive, it was called polyclonal ANA. The proportions of polyclonal ANA were different in different disease groups A n n=1 (Table 3). TPM was calculated using the formula (1 A n ) n=1 A for n all of the different disease groups. In all groups, the OPM values were lower than the TPM values. Significant differences were found between TPM and OPM in different autoimmune diseases (Table 2). 3.3 Results of IgM for Toxoplasma, CMV, and RV, IgG for TP, HCV, and HIV, and total IgE Results of IgM for Toxoplasma, CMV, and RV, IgG for TP, HCV, and HIV, and total IgE in different autoimmune diseases are in Table 4. When the data were analyzed, the P value was <.05 in anti- Toxoplasma FIGURE 1 Results of ANA profile after detection. The result of the first strip (ANA-3/528-22): negative, the result of the second strip (ANA- 3/555-64): anti-ssb antibody grade 3, anti-ro52 antibody grade 3 and anti-ssa antibody grade 3

4 4 of 7 TABLE 1 Positive number and proportion of IgG in different autoimmune diseases Diseases Antigen SLE Positive rate (%) RA Positive rate (%) Others Positive rate (%) Control Positive rate (%) Total nrnp/sm Sm SSA SSB Scl Jo CENP B dsdna Nucleosomes Histones Rib.P.protein PM- Scl PCNA Ro AMA- M SLE, systemic lupus erythematosus group; RA, rheumatoid arthritis group; Others, other autoimmune diseases group; Control, control group. TABLE 2 Numbers of IgG ANA, TPM, and OPM in different autoimmune diseases Number of positive ANA SLE RA Others Control Total TPM OPM P value <.001 <.001 <.001 SLE, systemic lupus erythematosus group; RA, rheumatoid arthritis group; Others, other autoimmune diseases group; Control, control group; TPM, theoretical proportion of monospecific ANA; OPM, observed proportions of monospecific ANA. TABLE 3 Proportions of monospecific ANA and polyclonal ANA in different autoimmune diseases (%) Disease Positive rate 3.4 Evaluation of diagnostic efficiency The receiver operating characteristic curve (ROC curve) analyses using the trait ANA as an indicator for the control and autoimmune disease groups are shown in Figure 2. The area under the curve (AUC) was found to be for SLE, while the AUC was found to be for RA and for other autoimmune diseases. 4 DISCUSSION Monospecific ANA proportion Polyclonal ANA proportion SLE RA Others Control SLE, systemic lupus erythematosus group; RA, rheumatoid arthritis group; Others, other autoimmune diseases group; Control, control group. IgM group, anti- HCV IgG group, and anti- TP group. However, in anti- Toxoplasma IgM group, differences were found, when comparing the various disease groups to the control group. In the anti- HCV IgG group and the anti- TP group, differences were only found between the SLE group and control (Table 5). The highest RF was 2500 IU/mL. In this study, the features of ANA (IgG) in autoimmune diseases were analyzed. The definition and calculation method of TPM were proposed using probability theory. In different disease groups (SLE, RA, and other autoimmune diseases), TPM and OPM values were all significantly different (P <.001). OPM values were all lower than

5 5 of 7 TABLE 4 Results of IgM for Toxoplasma, CMV, and RV, IgG for TP, HCV, and HIV, and total IgE Disease Percentiles Toxoplasma (S/ CO) CMV (S/CO) RV (S/CO) TP (S/CO) HCV (S/CO) HIV (S/CO) Total IgE (IU/mL) Control 25th th th SLE 25th th th RA 25th th th Others 25th th th SLE, systemic lupus erythematosus group; RA, rheumatoid arthritis group; Others, other autoimmune diseases group; Control, control group; Toxoplasma, Toxoplasma gondii; CMV, cytomegalovirus; RV, rubella virus; TP, Treponema pallidum, HCV, hepatitis C virus; HIV, human immunodeficiency virus. TABLE 5 Comparison of IgM, IgG, and IgE between autoimmune disease groups and control (P value) Diseases IgM antibodies P value IgG antibodies P value Toxoplasma RV CMV TP HCV HIV IgE P value RA.006 a None None.057 a.108 a None None SLE.003 a None None.009 a.046 a None None Others.000 a None None.243 a.152 a None None Total SLE, systemic lupus erythematosus group; RA, rheumatoid arthritis group; Others, other autoimmune diseases group; Toxoplasma, Toxoplasma gondii; CMV, cytomegalovirus; RV, rubella virus; TP, Treponema pallidum; HCV, hepatitis C virus; HIV, human immunodeficiency virus. a Compare with control group; none: when the total P value is over.05, comparisons of autoimmune disease group and control group were not made. FIGURE 2 Evaluations of diagnostic efficacy for trait ANA in different autoimmune diseases. A, Systemic lupus erythematosus group; B, rheumatoid arthritis group; C, other autoimmune diseases group the corresponding TPM value. If monospecific ANAs were present at random in autoimmune diseases, significant differences between the TPM and OPM values would not be found. This suggested that there were more rare patients in whom monospecific ANAs were found than predicted. On the contrary, polyclonal ANAs which means there were more than one ANA in a single patient were common.

6 6 of 7 In the SLE group, the proportion of polyclonal ANAs was 96.11% (173/180). In the RA group, there was no significance between the proportion of polyclonal ANAs and monospecific ANAs. It suggested that the mechanism for production of autoantibodies in SLE and RA is different because of their different antigenic determinants. The etiology of autoimmune disease is unclear; however, the function of B cells and plasma cells in the pathogenesis of autoimmune diseases has been established. B cells play an important role in developing into antibody- secreting plasmablasts, plasma cells, and memory B cells. One study from other laboratory found significantly elevated levels of double negative memory B cells and decreased CD27IgDIgM memory B cells in peripheral blood of patients with SLE compared to control participants. Other groups also reported similar results. 15,16 In ANA profiles, grades of IgG to 15 different antigens are detected. To observe whether other grades of antibodies are also affected, a variety of IgM (including Toxoplasma, CMV, and RV), IgG (TP, HCV, and HIV), and IgE (total IgE) antibodies were detected in patients with autoimmune diseases. Only anti- Toxoplasma IgM was significantly different between the three disease groups. It is not yet known whether there is cross- reactivity between Toxoplasma antigens and self- antigens. In addition, RF may be increased in some autoimmune diseases. RF can interfere both positively and negatively in immunoassays. 17 According to the instructions from the reagent manufacturer, when RF is <3720 IU/mL, there will be no false- positive results in anti- Toxoplasma IgM assays. In this study, RF was detected in all the samples, which were all <3720 IU/mL. This means that it was possible to exclude the false- positive results. In IgG detection, there were significant differences in anti- TP IgG and anti- HCV IgG between different groups, but this difference was only significant between the SLE and control groups. There were no significant differences in IgE detection. In autoimmune diseases, the elevation of different classes of antibodies suggests that polyclonal antibodies are not limited to IgG. Other antibodies from IgG and IgM to pathogens for infectious diseases were also affected. We have a hypothesis that more antibodies would be found to increase, if the scope for detection was extended. Among different autoimmune diseases, there are more types of Ig in the SLE group than the other diseases. It is consistent with the higher polyclonal proportion of IgGs in the ANA panel results for SLE. Above all, B- cell differentiation in SLE was more extensive and complex than other diseases. Further study is needed to enclose the nature of the disorder of B- cell function and the mechanism of autoimmunity caused by abnormal function. The ANA panel results showed the following: the top three antibodies were Ro- 52 SSA, RNP in the SLE group, Ro- 52 SSA, SSB in the RA group, and Ro- 52 SSA, SSB in the others group. Antibodies to the Ro autoantigen are one of the most frequent serological markers of autoimmunity in connective tissue diseases. The high positive rates of Ro- 52 and SSA in a variety of disease were consistent with other studies. 18,19 In our study, native SSA antigen was purified by affinity chromatography from bovine spleen and thymus. For Ro- 52, the corresponding human cdna has been expressed with the baculovirus system in insect cells. Anti- Ro- 52 is a nonspecific autoantibody in the autoimmune diseases including SLE, RA, SS/SLE overlap syndrome, neonatal lupus, and primary biliary cirrhosis. It is not important for the diagnosis and differential diagnosis of autoimmune diseases. However, it has been found that anti- Ro- 52 antibody is related to some clinical manifestations and organ lesions. 20,21 Meaning that it may play an important role in the pathogenesis of autoimmune diseases. Therefore, Ro- 52 combined with other autoantibodies could be more meaningful. In this study, the positive rate of SSA in the SLE group was 53.05%, which was slightly higher than the results reported in the literature. 22 The positive rate of SSA in RA was 13.72%, and 44.44% in other groups. Anti- Smith antibody was identified as a specific antibody in the diagnosis of SLE in our study, the positive rate was 16.43% (35/213). 12,23 The positive rate of dsdna, which is another specific antibody in SLE, was 28.64% (61/213). 12,24 Otherwise, the positive rate of the ANA profile was 84.51% in SLE. To make full use of an ANA profile and applying it in a diagnostic process, we propose the trait ANA concept, which uses the maximum ratio of signal intensity in immunoblot assays, which can also be interpreted as the highest concentration of autoantibody in samples. The application of the signal intensity is more reliable than grade. For example, in a polyclonal ANA result, there may be several autoantibodies with the same grade but different signal intensity. In order to avoid the batch to batch variations, the ratio between signal intensity and control signal intensity was used to correct. Diagnostic efficiency of trait ANA was evaluated using a ROC curve for the different diseases. For the SLE group, the area under the curve was 0.916, which suggests that this could be an effective laboratory diagnostic indicator. In the other disease groups, the area under the curve is slightly lower than that in the SLE group. The efficiency of this test in the RA group was 0.646, which suggests that trait ANA is not suitable for the diagnosis of RA; this may be related the low polyclonal proportion of ANA panels in this group. In summary, polyclonal ANAs (IgG) are common in autoimmune diseases. Trait ANA (IgG) was a useful parameter for the diagnosis of SLE. In addition to the positive ANA (IgG) profiles, some antibodies against infectious pathogens (IgM and IgG) were also significantly different in a few of the autoimmune diseases. It remains unclear what role B cells and plasma cells play in the pathogenesis of autoimmune diseases; however, ANA may prove a useful diagnostic tool and offer some good targets for future research. Further investigation about B- cell differentiation is needed to make inferences on pathogenic mechanisms. ORCID Liping Wan REFERENCES 1. Margery-Muir AA, Bundell C, Nelson D, Groth DM, Wetherall JD. Gender balance in patients with systemic lupus erythematosus. 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