Anti-DFS70 Antibodies Among Patient and Healthy Population Cohorts in China: Results From a Multicenter Training Program Showing Spontaneous Abortion and Pediatric Systemic Autoimmune Rheumatic Diseases Are Common in Anti-DFS70 Positive Patients

Objective Anti-DFS70 antibodies correlating with the nuclear dense fine speckled (DFS) pattern in the HEp-2 indirect immunofluorescence assay (IFA) are less common in patients with systemic autoimmune rheumatic disease (SARD) than in healthy subjects and their clinical associations remain elusive. We hosted a multi-center HEp-2 IFA training program to improve the ability of clinical laboratories to recognize the DFS pattern and to investigate the prevalence and relevance of anti-DFS70 antibodies. Methods DFS pattern sera identified by HEp-2 IFA in 29 centers in China were redirected to a central laboratory for anti-DFS70 testing by line immunoblot assay (LIA), enzyme-linked immunosorbent assay (ELISA), and IFA with HEp-2 ELITE/DFS70-KO substrate. Anti-extractable nuclear antigen antibodies were measured by LIA and the clinical relevance was examined in adult and pediatric patients. Results HEp-2 IFA positive rate and DFS pattern in positive sera were 36.2% (34,417/95,131) and 1.7% (582/34,417) in the patient cohort, and 10.0% (423/4,234) and 7.8% (33/423) in a healthy population, respectively. Anti-DFS70 prevalence among sera presenting the DFS pattern was 96.0, 93.7, and 49.6% by ELISA, LIA, and HEp-2 ELITE, respectively. 15.5% (52/336) of adult and 50.0% (20/40) of pediatric anti-DFS70 positive patients were diagnosed with SARD. Diseases most common in anti-DFS70 positive patients were spontaneous abortion (28.0%) in adults and juvenile idiopathic arthritis (22.5%) in pediatric patients. Conclusion Accurate DFS pattern identification increased the detection rate of anti-DFS70 antibodies by ELISA and LIA. Anti-DFS70 antibodies are remarkably high in cases of spontaneous abortion and in pediatric SARD patients, but not prevalent in adult SARD patients.


INTRODUCTION
Antinuclear antibodies (ANA) are commonly regarded as serological hallmarks of systemic autoimmune rheumatic disease (SARD) (1). However, antibodies against the 70 kDa dense fine speckled protein (DFS70), also known as transcription coactivator p75 (2) and lens epithelium-derived growth factor (3), are purported to be an interesting immunological paradox as they are commonly detected in apparently healthy individuals (4)(5)(6)(7)(8)(9) but are rare in patients with SARD (5,10). In the last decade, many studies have focused on the clinical relevance of anti-DFS70 antibodies [reviewed in (5)] and their prevalence has been reported in some chronic inflammatory diseases (2,11,12) and cancers [e.g. prostate cancer (13,14)], but still no clear disease association has been found. In fact, the presence of isolated anti-DFS70 antibodies has been proposed to serve as a diagnostic biomarker to help rule out SARD (4,15,16), which highlights the importance of correctly identifying these antibodies in clinical laboratories.
The HEp-2 cell indirect immunofluorescence assay (HEp-2 IFA) is considered the "gold standard" method for ANA screening by the American College of Rheumatology (17). Typical images of the DFS pattern show dense fine speckled staining of interphase nuclei and strong coarse speckled staining of the metaphase plate. DFS pattern is defined as the anti-cell-2 (AC-2) pattern by the International Consensus on ANA Patterns (ICAP) (18). Due to its unique features, only trained and experienced technicians may recognize DFS pattern. Correlations between the anti-DFS70 antibodies detected by specific assays and the DFS pattern have been reported higher than 90% (19). Considering HEp-2 IFA pattern interpretation is largely dependent on the experience of the technologist, reading of the DFS pattern continues to challenge researchers and clinicians alike. Bentow et al. reported that the reading accuracy of DFS unmixed and mixed patterns were~50% and <10%, respectively, based on an international internet-based survey (20). Recently, the Autoantibody Standardization Committee has made available a reference material for anti-DFS70 antibodies Abbreviations: ANA, antinuclear antibodies; AC-2, anti-cell-2; DFS70, dense fine speckled 70 kDa antigen; CLIA, chemiluminescence immunoassay; CTD, connective tissue disease; ELISA, enzyme-linked immunosorbent assay; ENA, extractable nuclear antigen; HEp-2 IFA, HEp-2 cell indirect immunofluorescence assay; ICAP, International Consensus on Antinuclear Antibody Patterns; LIA, line immunoblot assay; NI, nuclear intensities; O.D., optical density; Rib-P, ribosomal P proteins; RA, rheumatoid arthritis; SARD, systemic autoimmune rheumatic disease; SD, standard deviations; UCTD, undifferentiated connective tissue disease.  (21), which may assist clinical laboratories in the recognition of DFS pattern to some extent. However, further efforts still need to be placed on training to recognize the DFS pattern. Our research focused on exploring cost-effective training models to improve the consistency of DFS pattern recognition in laboratories from various regions with unevenly distributed medical resources. Thus, we organized a multi-center DFS pattern identification mutual aid program. The prevalence and clinical associations of anti-DFS70 in both Han Chinese and minority populations were investigated in routine HEp-2 IFA screening cohorts from 30 centers and in healthy individuals from a physical examination cohort from one center in China.

Study Design
From July to September 2019, a total of 645 serum specimens were sent from 29 research centers across China to the organizing laboratory at the Renji Hospital, which is affiliated with Jiao Tong University (Shanghai, China). Figure 1 and Table  1 detail the distribution and specific information (e.g. type of hospital, location, etc.) of the 30 participating laboratories.
At the initiation of the study, corresponding author BZ at the Renji Hospital organizing center directed an online training course to all participating laboratories detailing how to identify the DFS pattern based on the ICAP classification criteria (22). In brief, interphase nuclei show characteristic speckles with heterogeneity in brightness and distribution, while the metaphase plate depicts a strong speckled pattern with some coarse speckles (22). At the end of each month, participating laboratories shipped sera they interpreted as DFS pattern positive during routine HEp-2 IFA tests to Renji Hospital. Upon arrival, two experienced research technicians re-tested the samples by HEp-2 IFA and reported patterns according to ICAP classification (22). Inconsistent interpretations were discussed between two independent evaluators. If they could not reach a consensus, a third evaluator adjudicated. A video conference was offered monthly to review all inconsistent ANA pattern interpretations as part of the ongoing training. Figure 2A shows a flowchart of the study design.
Additionally, the prevalence of ANA and DFS patterns were analyzed in a healthy population (age≥18) from the Renji Hospital Physical Examination Center for routine physical examination (RJ-HEALTH cohort, n=4,234) from July to September 2019. This cohort according to a clinical questionnaire had no history of SARD, malignancies or chronic infections, and were considered healthy by judgment of the chief physician based on physical examination reports. This healthy cohort was compared to the adults in RJ-PA cohort, which consisted of adult (≥18 years, n=20,293) and pediatric (<18 years, n=756) patients undergoing routine HEp-2 IFA screening in the same hospital during the same period (RJ-PA cohort, n=21,049). The percentage of SARD patients in RJ-PA cohort were also investigated according to medical record. All serum specimens collected in this study were approved by the Institutional Review Board of Renji Hospital (No. KY[2019]121). No consent was required for this study.

ANA Screening
Of the 30 participating laboratories, including Renji Hospital, 25 of the laboratories performed ANA screening on HEp-2 slides from Euroimmun AG (Lübeck, Germany), 3 used slides from  Inova Diagnostics (San Diego, CA, USA), and 2 used HEp-2 IFA kits from two companies in mainland China (H&J NovoMed, Beijing and HOB, Suzhou).
All samples received by the organizing center at Renji Hospital were re-tested using HEp-2 slides from both Euroimmun and Inova Diagnostics. Euroimmun HEp-2 slides were prepared using the Sprinter XL automated IFT/ELISA analyzer (Euroimmun). Images were acquired with EUROPattern Suite version 3.4.24.0 with a cutoff of 80 AU per the manufacturer's instructions. NOVA Lite HEp-2 IgG ANA with DAPI slides were processed using the IFA-ELISA processor platform of the QUANTA-Lyser instrument (Inova Diagnostics) and scanned via the NOVA View automated microscope with software version 1.0.4.3, using a cutoff of 49 nuclear intensities (NI) per the manufacturer's instructions. Samples positive for any nuclear/cytoplasmic autoantibody by IFA were considered HEp-2 IFA positive. Anti-DFS70 reference material IS2726 ANA #24 (21) for ICAP pattern AC-2 was applied as the quality control for HEp-2 IFA.

Anti-DFS70 Antibodies Detection
Anti-DFS70 antibodies were detected by both line immunoblot assay (LIA) and enzyme-linked immunosorbent assay (ELISA). For LIA, the IMTEC-ANA-LIA XL assay (Human Worldwide, Weisbaden, Germany) was used. HumaScan was used to analyze and interpret the results according to the manufacturer's instructions. For ELISA, 96-well plates were coated with 0.5 µg/mL purified recombinant DFS70 antigen (DIARECT AG, Freiburg, Germany) overnight at 4°C. After blocking with 10 mg/mL gelatin, patient sera were diluted 1:200 in serum diluent (0.75 mg/mL bovine gamma globulin, 0.15 mg/mL bovine serum  Results were interpreted using a manual fluorescence microscope (Nikon Eclipse Ni, Tokyo, Japan). Anti-DFS70 antibodies were confirmed by bright staining of interphase nuclei with the corresponding DFS pattern in~10% of cells.

Questionnaire for HEp-2 IFA Screening in Participating Centers
The following were determined by questionnaire for each participating center: which commercial kits they used for routine HEp-2 IFA screening; whether the laboratory routinely reports the DFS pattern; the working experience of their research technicians; whether they apply third-party quality control for HEp-2 IFA. Moreover, the clinical diagnoses of sera with the DFS pattern, which were re-tested and confirmed by Renji Hospital, were retrospectively analyzed by reviewing the medical records including the age, gender, ethnicity and clinical diagnosis from each center.

Statistical Analysis
Statistical Package for Social Sciences (SPSS) (IBM-SPSS, Inc., Armonk, New York) was used to perform statistical analysis. Cohen's kappa was applied to analyze the interrater agreement between two commercial slides. Differences in the distribution of anti-extractable nuclear antigen (ENA) between SARD versus non-SARD were evaluated by the two-tailed Chi-squared (c 2 ) test or Fisher's exact test. The correlations between anti-DFS70 ELISA O.D. values and HEp-2 IFA nuclei fluorescence intensity read by NOVA View or titer by HEp-2 IFA were calculated by Spearman's rank correlation test. In our study, a two-sided t-test with a P-value <0.05 was considered significant.

Prevalence of ANA and DFS Pattern in China
The 30 participating laboratories consisted of one rheumatology specialist hospital in southern China, three traditional Chinese medicine hospitals in eastern and western China, two obstetrics and gynecology specialist hospitals in eastern China, and twentyfour general hospitals across mainland China. Each laboratory was assigned a unique hospital identification number for the study as listed in Table 1. Overall, the HEp-2 IFA positive rate was 36.2% (34,417/95,131) with some significant differences among the centers. For example, rheumatology specialist hospital H22 had the highest HEp-2 IFA positive rate (76.9%), while obstetrics and gynecology specialist hospital H3 had the lowest rate (8.9%, Table 1).
The prevalence of the DFS pattern was 0.6% (582/95,131) of total patients and 1.7% (582/34,417) in the HEp-2 IFA positive samples, respectively. Interestingly, an obstetrics and gynecology specialist hospital (H3) had the lowest overall HEp-2 IFA positive rate (8.9%), but the highest positive rate of DFS pattern (19.4%) among the participating centers. Note that this high percent of positive DFS pattern could not be accounted for by unusual local environmental exposure when compared to four neighboring centers in the same region (H1, H2, H31, and H32, Figure 1). Three centers, including another obstetrics and gynecology specialist hospital (H8), a Traditional Chinese Medicine hospital (H25), and a general hospital (H17), did not have any confirmed samples with DFS pattern.

Comparison of the Prevalence of the DFS Pattern in Adult and Pediatric Patients Versus Healthy Population Cohort
Quantitative comparisons between the RJ-PA and RJ-HEALTH cohorts, along with patient distributions in different departments and antibody titers, are shown in Table 2 and Supplementary  Table 1. It should be noted that the prevalence of the DFS pattern in HEp-2 IFA positive sera was significantly higher in pediatric (10.6%) compared to adult patients (3.3%, P<0.001) in the RJ-PA cohort, but no statistical difference was observed in the RJ-HEALTH cohort (7.8%, P=0.173). The same trend was observed when investigating association with gender in various populations in Table 2. DFS pattern was more common in females in the HEp-2 IFA screening cohort (females: 3.6%, 231/6,461, males: 1.6%, 16/1,029, P<0.0001), while no gender differences were observed in pediatric patients (females: 9.2%, 26/284, males: 17.2%, 11/64, P=0.060) or the healthy population (females: 6.3%, 17/268, males: 10.3%, 16/155, P=0.141). Moreover, the same distributions of DFS pattern titers were detected among adult and pediatric RJ-PA and RJ-HEALTH cohorts, as over 40% of titers were ≥1/640 (Supplementary Table 1).
In the RJ-PA cohort, the percentages of SARD patients with DFS pattern were compared respectively with HEp-2 IFA negative (AC-0) and three other common patterns including nuclear homogeneous (AC-1), fine speckled (AC-4), and large/ coarse speckled (AC-5), and remaining patterns ( Table 3). In the RJ-PA adult cohort, the percentage of SARD in each AC pattern was much lower in patients with DFS pattern compared to those with AC-1, AC-4, AC-5 or other remaining patterns, but higher than the HEp-2 IFA negatives. In contrast, the percentage of SARD in pediatric patients with DFS pattern was comparable to those with HEp-2 IFA negative or AC-1, lower than AC-4 or AC-5, and higher than other remaining patterns.

Consistency of Interpretation Rates of the DFS Pattern Between Renji Hospital and Participating Centers
At the onset of this study, only 14 of the 29 participating laboratories routinely reported the DFS pattern ( Table 1). Among the participating centers, the mean years of experience with reading ANA patterns was 4.4 ± 2.9 years. The interpretation consistency rates between Renji Hospital and other centers were low, as only 46.2% (298/645) of delivered sera were confirmed positive for the DFS pattern. Heterogeneity in medical resources might be a reason for the lack of expertise in the recognition of the DFS pattern in some centers. Figure 3A shows a relatively small change in interpretation consistency from 80.8% to 89.5% (P=0.535) in Shanghai centers from July to September, compared to a more obvious increase from 29.7% to 60.6% (P<0.0001) in other regions. An apparent improvement in DFS pattern interpretation was observed in many participating centers throughout the program ( Figure 3B).

Comparison of DFS Pattern Interpretation by Two Commercial HEp-2 IFA Kits
All 645 serum samples delivered from the participating centers were tested on two different commercial HEp-2 IFA slides to evaluate the agreement in identifying the DFS pattern. There was good agreement between the two brands (kappa=0.598) as 31.2% (201/645) of sera showed single or mixed DFS pattern on both slides, while 4.3% (28/645) and 15.3% (99/645) exhibited the DFS pattern only in Inova Diagnostics or Euroimmun slides, respectively. Some of the typical inconsistent cases are shown  Percentages of SARD patients in respective HEp-2 IFA patterns. c P value is the SARD patient positive rate in each AC pattern compared with that in AC-2 pattern. Bold characters indicate significant differences between two groups. in Supplementary Figure 1. Moreover, 49.1% (317/645) of sera did not show the DFS pattern on either type of slide. All the misinterpreted patterns are listed in Figure 3C, which shows that the nuclear homogeneous pattern (AC-1) was the most difficult pattern to distinguish from DFS. 18.1% and 12.4% of sera were misinterpreted as nuclear homogeneous on Inova and Euroimmun slides, respectively.

Clinical Phenotypes and Additional ENA Specificities in Anti-DFS70 Positive Sera
In total, 84.7% (546/645) of serum samples with the DFS pattern, including 517 samples from the HEp-2 IFA screening cohort and 29 sera from the RJ-HEALTH cohort, showed positive reactivity to DFS70 by both ELISA and LIA. Of these positive patients, 336 adult (89.4%) and 40 pediatric (10.6%) patients had traceable clinical phenotypes. The spectrum of disease and clinical conditions of adult and pediatric patients with positive reactivity to DFS70 by both ELISA and LIA were analyzed respectively in Table 4 and the disease groups were detailed in Supplementary Table 2. For SARD group, the most prevalent diseases among DFS70-positive adult and pediatric patients were spontaneous abortion (28.0%, 94/336) and juvenile idiopathic arthritis (JIA, 22.5%, 9/40), respectively (Supplementary Table  2  All 376 patients were further tested for additional anti-ENA antibodies by LIA. The prevalence of "monospecific" anti-DFS70 was higher in non-SARD patients (76.8%, 289/376) than in SARD patients (15.2%, 57/376; P<0.0001) as shown in Supplementary  Table 3 Table 4. Moreover, additional anti-ENAs were not detected in DFS sera from the RJ-HEALTH cohort.

DISCUSSION
A large-scale, short-term multi-center study was conducted with 30 laboratories from various regions in China, of which 16 started with no experience of reporting DFS pattern and 14 had begun reporting DFS pattern only one or two years prior to the study. After the DFS pattern training/re-orientation program, we found that: 1) the short-term training greatly improved DFS pattern recognition in many of the participating laboratories as the interpretation consistency increased from 29.7 to 60.6% in the areas other than Shanghai, 2) the pattern identification consistency was higher in areas with welldeveloped health facilities (i.e. the consistency rate in the four laboratories in Shanghai was 80.8 versus 29.7% for the other participating laboratories at the start of the program), 3) sera with DFS pattern confirmed by two commercial HEp-2 slides showed higher positive rates for anti-DFS70 antibodies than by only one slide brand (96.0 versus 56.2, or 75.0%).
To better understand the prevalence of DFS pattern in the routine HEp-2 IFA screening cohort of this study, we have summarized HEp-2 IFA positive rates from different reports worldwide in Supplementary Table 5. The reported HEp-2 IFA positive rates in Supplementary Table 5 based on routine screen test cohorts range from 11.6-82.0% with a median of 28.5%, which is close to our present study of 36.2%. However, our prevalence of the DFS pattern was 0.6% in the routine HEp-2 IFA screening cohort, which is relatively low compared with reports from other countries (Supplementary Table 5). There are a few possible explanations for this low frequency. First, ethnicity may have played a role in the prevalence of the DFS pattern/anti-DFS70. To put this into perspective, the highest prevalence of 27% was only reported in the United States (16) compared to 0.3-8.4% reported in other continents (Supplementary Table 5). In the present study, the HEp-2 IFA positive rate was significantly higher for Han Chinese than for Uygurs and Kazakhs, yet DFS pattern positive rates were higher among these minority populations. In contrast, the HEp-2 IFA positive rate was higher in Tibetans, Huis, and Mongolians than Han Chinese, while the DFS pattern positive rate in HEp-2 IFA positive sera showed no statistical difference. To our knowledge, this is the first report to compare HEp-2 IFA positive rates between national minority groups and Han Chinese in China. Second, reported DFS pattern positive rates can also be affected by the experience of IFA evaluators (16,23). In Supplementary  Table 5, the reported prevalence of the DFS pattern in Turkey varied widely from 0.3 to 8.1% (24). In North America, the prevalence of DFS has been reported as 27.0% in the United States (16) but only 1.6% in Canada (6). As the present study shows, improving the accuracy of DFS pattern interpretation in some of the participating centers, especially those in areas with relatively poor medical resources, will affect the overall reported anti-DFS70 prevalence. Finally, application of different commercial HEp-2 IFA kits may contribute to the discrepancy in prevalence of the DFS pattern (25,26). Due to the inevitable heterogeneity in performance of various slides, the consistency of the two kits used in our study only exhibited moderate agreement (kappa=0.598) in DFS pattern identification.
We further investigated the prevalence of the DFS pattern in pediatric patients. Schmeling et al. (27) reported a 4.5% positive rate (titer ≥1:80) of anti-DFS70 in pediatric patients referred for ANA testing, which is comparable to the 4.9% reported in the present study. Anti-DFS70 antibodies have been reported to be more prevalent in younger age groups (4,28). This may partly explain why the positive rate of DFS pattern was higher in pediatric versus adult patients in this study. However, no statistical difference between pediatric patients and adult healthy individuals was found. Notably, the DFS pattern was more prevalent in adult female patients (females 3.6%, males 1.6%), while no gender differences were observed among the pediatric patient and healthy populations. Therefore, the prevalence features of anti-DFS70 in pediatric patients were similar to those in the healthy adult population. To date, few studies have focused on anti-DFS70 in pediatric patients. The prevalence in healthy children should be further investigated and compared to pediatric patients in the same age category (29).
As accurate DFS pattern interpretation by IFA alone is challenging, additional objective tests such as ELISA, LIA, or chemiluminescence immunoassay (CLIA) are necessary to identify anti-DFS70 antibodies (25,(30)(31)(32). ELITE/DFS KO substrate also offers a unique possibility of evaluating anti-DFS70 antibodies at the IFA stage (23,33 1% monospecific DFS70 in anti-DFS70 positive sera from a HEp-2 IFA screening cohort which was lower than our findings of 92.0%. Monospecific anti-DFS70 is considered rare in SARD and may serve as an exclusion biomarker for SARD (5,7,34). In a multi-center study, Choi et al. reported 1.1% monospecific anti-DFS70 in a large cohort of SLE patients (34). In our study, the percentage of monospecific anti-DFS70 patients was much higher in non-SARD (76.8%) than in SARD (15.2%) patients in HEp-2 IFA screening cohort. This to some extent supports that monospecific anti-DFS70 is a reliable biomarker to rule out diagnosis of SARD (35). Moreover, anti-Ro52/TRIM21 and anti-SSA/Ro60 were the most commonly detected autoantibodies accompanying anti-DFS70 antibodies in our LIA ENA profiles, while SLE-specific antibodies including Sm and dsDNA were not observed. Choi et al. (34) also reported anti-SSA/Ro60 (34.6%) and anti-Ro52/ TRIM21 (27.2%) detected by addressable laser bead immunoassay array, as the most common autoantibodies found with anti-DFS70 antibodies in SLE patients, which is consistent with our results.
Regarding the clinical relevance of anti-DFS70 antibodies, there were some unexpected findings. First, the effect of DFS pattern in helping to rule out SARD in the routine HEp-2 IFA screening cohort was not in fact stronger than negative ANA in adults, while no statistical difference was observed between DFS pattern and negative ANA in pediatric patients. The latter can be explained partly to the high percentage of JIA patients (26.6%, 201/756) in the pediatric ANA screen cohort and the HEp-2 IFA positive rate of JIA was 21.9% (44/201) (data not shown). Moreover, it is still worth noting that the frequency of SARD in anti-DFS70 positive pediatric patients was unexpectedly as high as 50.0%. We consider this data significant because in this RJ-PA pediatric cohort, the percentage of SARD patients with DFS pattern was similar to those with AC-1 and much higher than those with other ANA patterns except AC-4 and AC-5. However, in the RJ-PA adult cohort, the percentage of SARD patients with DFS pattern was much lower than those with AC-1, AC-4, AC-5 or even those with other patterns. Together, this data strongly suggests that anti-DFS70 prevalence was different in pediatric and adults patients with SARD. Sperotto et al. tested four monospecific anti-DFS70 positive cases out of a population of 261 school-age children and found that three of the cases (75%) had a family history of autoimmune disease, but no disease symptoms (29). Moreover, none of the ANA-positive (anti-DFS70 positive/negative) subjects developed SARD in a three-year follow up (29). Therefore, future multi-center studies should focus on the underlying role of anti-DFS70 in pediatric cohorts. Second, the proportion of anti-DFS70 positive adult patients who had experienced spontaneous abortion from the ANA screening cohort was remarkably high at 28.0%. The highest rate of DFS pattern among the HEp-2 IFA positive population was observed in an obstetrics and gynecology hospital in Shanghai, China. For the first time, these data suggest that anti-DFS70 antibodies may be associated with female reproductive diseases. Furthermore, in an ongoing study in the Renji Hospital, the prevalence of DFS pattern in females with spontaneous abortion (3.1%, 94/2990) was much higher than age-matched healthy females (1.5%, 45/2990; P<0.0001, data not shown), which also support the association between them. Isolated studies have also reported anti-DFS70 in eye diseases, like sympathetic ophthalmia (9), Vogt-Koyanagi-Harada syndrome (11), and atopic dermatitis with cataracts (36), and as tumor-associated antibodies present in prostate cancer patients (8,13,37). Marlet et al. (38) was the first to report that anti-DFS70 antibodies may be correlated with thrombosis and obstetric complications. DFS70 has been shown to be upregulated by human papilloma virus (HPV) infection and its associated oncogenes E6/E7 (39) and has been implicated in autoimmune thyroiditis (40). Both diseases are also associated with several reproductive pathology (41). However, anti-DFS70 have previously been shown to be associated with young age and female sex (40). Therefore, to better investigate the association between spontaneous abortions and anti-DFS70, the prevalence of anti-DFS70 needs to be compared between the pregnant women who completed the pregnancy versus those with spontaneous abortions. Further studies are necessary to explore the clinical association between anti-DFS70 and reproductive diseases.
In addition to those mentioned above, UCTD and RA were the most common SARD in adults associated with anti-DFS70, while JIA, UCTD, and pediatric SLE were seen in anti-DFS70 positive pediatric patients. This is consistent with some previous reports that anti-DFS70 antibodies may be restricted to SARD patients without typical ANA-associated antibodies, and only rarely found in patients with ANA-associated rheumatic disease (6,7,42). Infantino et al. also reported high prevalence of anti-DFS70 antibodies in UCTD cases (13.3%) and suggested that anti-DFS70 antibodies could serve as an appropriate biomarker for the development of UCTD to CTD (28).
One limitation of this study was that we were unable to obtain some additional laboratory test results, including anti-thyroid peroxidase antibodies, serum free triiodothyronine, thyroxine, thyroid-stimulating hormone, and activated partial thromboplastin time for the anti-DFS70 positive cohort from the participating laboratories to further explore their relationship to anti-DFS70 positive patients.
In conclusion, DFS pattern interpretation can be a challenging task for many clinical laboratories. However, a short-term training course and inter-laboratory comparison of HEp-2 IFA results can improve IFA pattern reading accuracy. The prevalence of ANA and the DFS pattern may vary between different Chinese ethnic groups. The clinical usefulness of anti-DFS70 may help to exclude SARD in adult patients. The increased prevalence of spontaneous abortion and pediatric SARD in anti-DFS70 positive patients will require further follow-up studies.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
All serum specimens collected in this study were approved by the Institutional Review Board of Renji Hospital (No. KY[2019]121). No consent was required for this study.