Dense fine speckled immunofluorescence pattern in a Chinese population: Prevalence and clinical association

Abstract Objective To provide information on the prevalence and possible clinical association in a Chinese population for medical practice of the dense fine speckled pattern (DFS pattern). Methods A retrospective study was conducted with patients who had the DFS pattern from June 2018 to December 2019 in West China Hospital. Results A total of 469 patients (1.27% of patients with positive anti‐nuclear antibody indirect immunofluorescence (ANA IIF) test results) revealed the DFS pattern, of which 92.96% had isolated DFS pattern and 23.67% had titers above/equal to 1:320. The average age of patients with the DFS pattern was 43.45 years, and females accounted for 76.97% of them. Ten different kinds of diseases made up the vast majority of the disease spectrum, in which inflammatory or infectious diseases (46.11%), mental diseases (21.45%), and systemic autoimmune rheumatic diseases (SARDs) (18.23%) ranked in the top three. The most common SARDs were rheumatoid arthritis (RA), undifferentiated connective tissue disease (UCTD), and systemic lupus erythematosus (SLE). Forty‐six patients (10.55%) had positive or suspicious extractable nuclear antigen (ENA) antibodies test results and a higher risk of suffering from SARDs. Forty‐seven patients would be missed if the DFS pattern with negative ENA antibodies test result was considered as exclusion criterion of SARDs. Conclusions The DFS pattern is basically isolated and with low titer. It is unwise to exclude the diagnosis of SARDs only depending on the appearance of the DFS pattern. Autoimmune diseases‐related antibodies, clinical information of patients, and long‐term follow‐up are of great importance to avoid missed or delayed diagnosis of SARDs.


| INTRODUC TI ON
Autoantibodies directing against human organs, tissues, and cells have been considered as serological hallmarks of various autoimmune diseases. 1 Among the autoantibodies, anti-nuclear antibodies (ANAs) play an irreplaceable role in the diagnostic workup of SARDs. The indirect immunofluorescence (IIF) assay based on HEp-2 cell substrates is extensively used to detect ANA, 2 and there has been increasing appreciation of the ability of morphological patterns to direct further investigation of specific autoantibodies in recent years, 3 as reflected in orderly classifying and harmonizing the nomenclature of several relevant HEp-2 IIF patterns, including the DFS pattern, by The International Consensus on ANA patterns (ICAP). 3 The DFS pattern, characterized by a dense and heterogeneous speckled staining of both the nucleoplasm of interphase cells and the chromosomal plate of metaphase cells, 4 was first described in 1994 in interstitial cystitis and later on in a variety of autoimmune conditions, other non-autoimmune conditions, and even healthy donors. 5,6 Because sera with the DFS pattern were shown to bind a 70-kDa protein in immunoblots, the target autoantigen was designated DFS70. 7 To the best of our knowledge, the DFS pattern/anti-DFS70 antibodies can be found in a wide spectrum of clinical conditions, 8,9 but the precise clinical significance of them is still unclear. 10,11 In addition, due to their low prevalence in SARDs, whether and how can the DFS pattern/anti-DFS70 antibodies be used to exclude the diagnosis of SARDs remain controversial. Some authors suggested that isolated anti-DFS70 positivity could be used as exclusion biomarker in SARDs, 6,12 thus preventing unnecessary further testing, treatment, and distress to patients. 13 By contrast, other authors claimed that this proposal was difficult to support and found no differences emerged in terms of prevalence of anti-DFS70 positive samples between SARDs and non-SARDs groups. 5,14 Hence, further studies on the DFS pattern/anti-DFS70 antibodies are required. In this study, we analyzed data on the DFS pattern and investigated its prevalence and possible clinical association in a Chinese population for medical practice of the DFS pattern.

| Subjects
The study enrolled 115,185 patients who underwent the ANA IIF

| ANA IIF tests and criteria for determination of the DFS pattern
ANA tests were measured in sera by IIF substrated with HEp-2 cells (Euroimmun, Germany), using serial dilutions commencing at 1:100. 2 Slides were read by two qualified and experienced technologists, and would be read by a third one if they did not reach an agreement.
The DFS pattern should be carefully distinguished from other patterns such as the homogeneous pattern or the fine speckled pattern.
The DFS pattern was characterized by three morphologic fea-

| Other information
Other clinical and laboratory information such as demographic characteristics, diagnosis, and clinical serum index results was collected from the hospital information system and the hospital laboratory information system.

| Ethical statements
This study has gained the ethical approval and consent of West China Hospital Ethics Committee. As a retrospective analysis of F I G U R E 1 The dense fine speckled pattern (DFS pattern) routinely collected programmatic data, all patient information was de-identified and precluded the requirement of informed consent.

| Data analysis
The statistical software SPSS 19.0 was used for statistical analysis.
K-S test was used to judge whether the results were normally distributed. The continuous variables satisfying the normal distribution were expressed as "mean ± standard deviation," otherwise as "median (interquartile interval)." For quantitative data, t-test or variance analysis was performed if the data were in line with normal distribution and even variance, otherwise nonparametric test was used.
Chi-square test or Fisher's exact test was used for counting data.
The threshold for statistical significance was set at p = 0.05. The bar chart was made by Origin 2018. The Venn diagram was made on the website (http://www.ehbio.com/test/venn/#/).

| Relationship among disease spectrum, ENA antibodies test results, and titer of the DFS pattern
We investigated the relationship of the DFS pattern with diseases.
In all patients with DFS patterns, 373 had definitive clinical diagnosis (79.53%). Ten different kinds of diseases (as shown in Table 1) made up the vast majority of the disease spectrum, in which inflammatory or infectious diseases, mental diseases, and SARDs ranked in the top three, with proportions of 46.11%, 21.45%, and 18.23%, respectively. Figure  it seems that patients with titers above/equal to 1:320 were more likely to have hypertension (p = 0.04).
In order to illustrate the relationship among DFS pattern titers, SARDs, and ENA antibodies test results in patients with DFS patterns visually, we created a Venn diagram. As shown in Figure 4,

| Characteristics of patients with isolated and complex DFS patterns
We divided the 469 patients into two groups: isolated DFS pattern and complex DFS patterns groups. For clarification, isolated DFS pattern represented that patients only had DFS patterns in their ANA IIF test results, while complex DFS patterns indicated that other patterns were also identified. Inflammatory or infectious diseases, mental diseases, and SARDs ranked in the top three, no matter in which group. In isolated DFS pattern group and complex DFS patterns group, RA was responsible for more than half of all the SARDs. Other SARDs including SLE, SS, etc. were also found, but there was no significant difference in the prevalence of SARDs between these two groups. As for ENA antibodies, anti-SSA antibodies (5.73%) were the most common antibodies, followed by anti-U1RNP (2.06%) and anti-Scl-70 antibodies (1.83%). Almost the same situation was found in isolated and complex DFS patterns groups, and there was no significant difference in the prevalence of ENA antibodies between two groups. We also detected other antibodies (such as anti-DNA, AKA, etc.) and clinical serum indexes reflecting blood routine parameters, liver, renal and immunologic function, but unfortunately, no significant difference appeared between two groups except for gamma-glutamyl transferase (GGT) (p = 0.049). Detailed characteristics of these patients are shown in Table 2.

| DISCUSS ION
As one of the most commonly seen IIF patterns in routine diagnostic laboratories performing ANA test on HEp-2 substrates, 16 the DFS pattern was initially identified in a patient with interstitial cystitis, but later in various disease conditions. 16 Anti-DFS70 antibodies target the lens epithelium-derived growth factor (LEDGF) and react with conserved and conformational epitopes. 6 There is evidence to  Hence, other related antibodies, clinical information of patients, and long-term follow-up are also of great importance to avoid missed or delayed SARDs diagnosis.

ACK N OWLED G M ENT
We acknowledge other colleagues who took part in the routine tests but do not become authors.

CO N FLI C T O F I NTE R E S T S
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

AUTH O R CO NTR I B UTI O N S
Keyi Zhang, Chaojun Hu, and Bin Yang conceived the study concept and design. Zhenzhen Su and Jing Hu collected data. Zhuochun Huang performed statistical analysis. Keyi Zhang drafted the manuscript, and all authors significantly contributed to the revision of the manuscript and approved the submission.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.