Antinuclear antibodies (ANA) are autoantibodies that target nuclear components in cells. They can be used in screening tests for certain autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren’s syndrome (SS), systemic sclerosis, dermatomyositis/polymyositis, and mixed connective tissue disease [1]. In most laboratories, indirect immunofluorescence assay (IFA) using human epithelial type-2 cells as substrates is used to detect ANA [2]. In 2014, 29 ANA interpretation patterns were identified by the International Consensus on Antinuclear Antibody Patterns [3]. This describes, defines, and classifies each pattern into three major groups: nuclear, cytoplasmic, and mitotic. However, it is important to note that a positive ANA test result is not definitive and must be combined with other clinical and laboratory information for proper diagnosis [4].

Unlike the typical nuclear speckled pattern or nuclear homogenous pattern in ANA interpretation, nuclear dense fine speckled (DFS) patterns are fluorescently stained in the nuclei of interphase cells, and strong positive reactions are observed in chromosomal regions of metaphase cells [3]. The antigen targeted by the antibody responsible for formation of the DFS pattern was identified as a 70 kDa protein (DFS70). It was found that DFS70 is the same protein as two other proteins namely DNA binding transcriptional coactivator p75 (p75) and lens epithelial growth factor (LEDGF) [5, 6]. DFS70 is a transcription co-activator regulating the expression of genes involved in oxidative stress, inflammation, and antioxidant properties [7]. Antibodies against the DFS antigen are referred to as anti-DFS70 antibodies (anti-DFS70). Anti-DFS70 was reported in patients with interstitial cystitis, atopic dermatitis, asthma, and various systemic autoimmune diseases [5]. The prevalence of a positive DFS pattern in healthy individuals has been variously reported at 7.8–20% [8-10]. Therefore, anti-DFS70 are considered to be natural autoantibodies present in patients with chronic inflammatory diseases and tumors as well as healthy individuals rather than specific autoantibodies associated with systemic autoimmune diseases [6, 11]. However, the clinical and biological significance of DFS patterns remains unclear [12]. According to a recent report, a positive DFS pattern may be a result of antibodies other than anti-DFS70 and may be a marker reflecting the proinflammatory status [13]. Most studies have investigated the association between DFS patterns or anti-DFS70 and diseases; however, the association of the DFS pattern with blood inflammatory markers has not been analyzed.

Herein, we sought to analyze the association between DFS patterns and inflammatory markers as well as various diseases.

1. Study subjects

This study included 862 subjects tested for ANA in regular health checkups or in the outpatient clinic at the Kangbuk Samsung Hospital between January 2019 and June 2022. A total of 436 subjects showed positive DFS patterns in IFA results during this period, and 426 DFS-negative subjects matching the age and sex of the DFS-positive group were randomly selected. There were no exclusion criteria for either group. Medical records of subjects were de-identified by assigning unique research identification numbers to maintain patient privacy. The following medical information was collected: demographic factors (age, sex, height, and weight); history of diseases including allergic, autoimmune, and dermatologic diseases; results of ANA tests; total IgE levels; vitamin D levels; rapid plasma regain (RPR) test results; inflammatory markers such as total white blood cell (WBC) counts with neutrophil, lymphocyte, monocyte, and eosinophil proportions, erythrocyte sedimentation rates (ESR), ferritin and C-reactive protein (CRP) levels; and autoimmune-related markers such as rheumatoid factor (RF), anti-Ro/SSA antibody (Ab), and anti-La/SSB Ab. For subjects who underwent regular health checkups, self-reported history of diseases was collected.

This study was approved by the Institutional Review Board of Kangbuk Samsung Hospital (2022-07-048).

2. Statistical analysis

All statistical analyses were performed using SPSS Version 28.0 for Windows (IBM Corp., Armonk, NY, USA). Continuous variables are presented as mean±2 standard deviations (SD) or median with interquartile range (IQR), while categorical variables were presented as numbers and percentages. For subjects with various diseases, cases that were not self-reported or with no diagnosis in the electronic medical records were classified as missing. Each variable was initially evaluated using the chi-square test, independent t-test, or Mann-Whitney U test. To analyze the association between inflammatory markers (WBC counts, CRP levels, ferritin levels, and proportions of neutrophils, lymphocytes, and monocytes) and DFS positivity in quartile or tertile groups, multivariate logistic regression models were used to estimate odds ratios (ORs) with 95% confidence intervals (CIs). Model 1 was not adjusted, and model 2 was adjusted for age, body mass index (BMI), and presence of allergic rhinitis, chronic urticaria, and atopic dermatitis. Statistical significance was defined as P value <0.05.

Characteristics of the 862 study participants according to DFS positivity are presented in Table 1. The median age of all subjects was 37 years (range, 2–92 years). The distribution of DFS titers in the DFS-positive group (N=436) was as follows: 1:80, 152 subjects; 1:160, 80 subjects; 1:320, 86 subjects; 1:640, 81 subjects; no titer, 37 subjects.

Table 1 . Characteristics of subjects according to DFS positivity

Variables	No. total cases	No. missing cases	Values*	DFS Pattern		P value†
				Negative	Positive
Number of subjects, N (%)	862		862 (100)	426 (49.4)	436 (50.6)
Age (yr) (mean ± SD)	862	0	38.7 ± 14.4	39.6 ± 12.8	37.8 ± 15. 7	0.053
Sex	862	0				0.604
Female, N (%)			612 (71)	299 (70.2)	313 (71.8)
Male, N (%)			250 (29)	127 (29.8)	123 (28.2)
BMI, kg/m2 (mean ± SD)	779	83	22.8 ± 3.6	23.0 ± 3.4	22.5 ± 3.8	0.053
BMI Category, N (%)
< 18.5			63 (7.3)	25 (5.9)	38 (8.7)
18.5–23			367 (42.6)	201 (47.3)	166 (38.1)
23–25			170 (19.7)	92 (21.6)	78 (17.9)
> 25			179 (20.8)	108 (25.4)	71 (16.3)
Unknown			83 (9.6)	0 (0)	83 (19.0)
Blood test results
WBC (/μL), median (IQR)	861	1	5,640 (4,680–6,900)	5,485 (4,630–6,520)	5,790 (4,700–7,220)	0.004
Platelet (103/μL), median (IQR)	861	1	255 (218–296)	254 (221–292)	255 (215–300)	0.746
Neutrophils (%), median (IQR)	861	1	55.2 (48.7–61.2)	54.25 (47.5–59.9)	56 (49.5–62.8)	0.001
Lymphocytes (%), median (IQR)	861	1	34.6 (28.7–40.3)	35.8 (30–41.2)	33.8 (27.7–39.4)	< 0.001
Monocytes (%), median (IQR)	861	1	7 (6–8.4)	7.2 (6.1–8.4)	6.8 (5.8–8.3)	0.008
Eosinophils (%), median (IQR)	861	1	2 (1.2–3.3)	2.05 (1.3–3.3)	1.9 (1.2–3.3)	0.205
CRP (mg/dL), median (IQR)	856	6	0.04 (0–0.1)	0.04 (0–0.09)	0.05 (0–0.12)	0.294
Ferritin (ng/dL), median (IQR)	740	164	87.8 (38.5–188)	90.3 (41.8–186)	83.6 (32.8–204)	0.603
Vitamin D (ng/dL), median (IQR)	740	122	20.2 (13.9–28.3)	20 (14–28.1)	20.5 (13.6–28.3)	0.681
Rheumatoid factor positive, N (%)	814	48	112 (13.8)	60 (14.1)	52 (13.4)	0.778
Anti-Ro/SSA Ab positive, N (%)	500	362	17 (3.4)	0 (0)	17 (10.3)	< 0.001
Anti-La/SSB Ab positive, N (%)	499	363	6 (1.2)	0 (0)	6 (3.7)	< 0.001
Various diseases
Rheumatoid Arthritis, presence	45	817	45 (5.2)	29 (6.8)	16 (3.7)	0.038
Allergic rhinitis, presence	64	798	64 (7.4)	8 (1.9)	56 (12.8)	< 0.001
Chronic urticaria, presence	35	827	35 (4.1)	29 (6.8)	6 (1.4)	< 0.001
Atopic dermatitis, presence	62	800	62 (7.2)	46 (10.8)	16 (3.7)	< 0.001
Other allergic disease, presence	325	537	325 (37.7)	219 (51.4)	106 (24.3)	< 0.001
Alopecia, presence	81	781	81 (9.4)	50 (11.7)	31 (7.1)	0.02
Psoriasis, presence	51	811	51 (5.9)	40 (9.4)	11 (2.5)	< 0.001

*Values are presented as N (%) or medians with IQR or mean±SD.

^†Each variable was evaluated using the chi-square test, independent t-test, or Mann-Whitney U test, according to DFS positivity. P values of <0.05 are statistically significant.

Abbreviations: N, number; SD, standard deviation; IQR, interquartile range; DFS, dense fine speckled; BMI, body mass index.

The following variables were significantly different between the DFS-positive and DFS-negative groups: WBC counts; proportion of neutrophils, lymphocytes, and monocytes; positive tests for the anti-Ro/SSA Ab; positive tests for the anti-La/SSB Ab; and presence of allergic rhinitis, chronic urticaria, rheumatoid arthritis, atopic dermatitis, other allergic diseases, alopecia, and psoriasis. However, age, BMI, platelet count, proportion of eosinophils, CRP, ferritin, and vitamin D levels, and results for positive tests for the rheumatoid factor were not significantly different between the two groups. Data for several subjects were missing for total IgE levels, ESR, and RPR test results to analyze the association.

The ORs of the positive DFS results according to the quartiles or tertiles (CRP) of inflammatory markers are shown in Table 2. In Model 1, the ORs were significant in the highest-value group (Q4 or T3) compared to those of the reference group for WBC counts, CRP levels, and the proportions of neutrophils, lymphocytes, and monocytes without adjustment for variables. In Model 2, after adjusting for variables, significant ORs for Q4 or T3 of WBC counts, CRP levels, and the proportions of neutrophils and lymphocytes were maintained. Median values of Q4 for WBC counts and those of T3 for CRP levels were 7,895/μL (range: 6,900–19,360/μL) and 0.2 mg/dL (range: 0.1–25.7 mg/dL), respectively, and both were below the inflammatory thresholds. The higher the proportions of lymphocytes and monocytes, the lower the likelihood of DFS positivity in both models. Ferritin levels were not found to be significant in any of the models.

Table 2 . Association between inflammatory markers and DFS positivity

Variables	Model 1		Model 2
	Odds ratio (95% CI)	P value	Odds ratio (95% CI)	P value
WBC counts
Q1	1 (reference)		1 (reference)
Q2	0.83 (0.57–1.21)	0.337	0.79 (0.52–1.22)	0.291
Q3	0.95 (0.65–1.39)	0.811	1.22 (0.8–1.87)	0.349
Q4	1.73 (1.18–2.54)	0.005	1.82 (1.17–2.81)	0.007
CRP
T1	1 (reference)		1 (reference)
T2	0.84 (0.61–1.14)	0.264	0.86 (0.6–1.23)	0.394
T3	1.42 (1.01–2)	0.041	1.62 (1.08–2.44)	0.02
Ferritin
Q1	1 (reference)		1 (reference)
Q2	0.71 (0.46–1.09)	0.117	0.73 (0.45–1.17)	0.189
Q3	0.61 (0.4–0.94)	0.026	0.63 (0.39–1.02)	0.06
Q4	0.87 (0.57–1.33)	0.516	1.04 (0.64–1.68)	0.887
Neutrophils %
Q1	1 (reference)		1 (reference)
Q2	1.21 (0.83–1.77)	0.333	1.24 (0.8–1.9)	0.334
Q3	1.33 (0.91–1.94)	0.147	1.45 (0.95–2.24)	0.088
Q4	1.8 (1.23–2.63)	0.003	2.04 (1.34–3.11)	0.001
Lymphocytes %
Q1	1 (reference)		1 (reference)
Q2	0.75 (0.51–1.1)	0.14	0.78 (0.51–1.2)	0.26
Q3	0.51 (0.35–0.75)	0.001	0.47 (0.31–0.73)	0.001
Q4	0.52 (0.35–0.76)	0.001	0.46 (0.3–0.71)	< 0.001
Monocytes %
Q1	1 (reference)		1 (reference)
Q2	0.77 (0.52–1.15)	0.201	0.78 (0.5–1.21)	0.265
Q3	0.59 (0.4–0.87)	0.008	0.57 (0.37–0.88)	0.011
Q4	0.66 (0.44–0.97)	0.035	0.71 (0.46–1.1)	0.125

Model 1: not adjusted; Model 2: adjusted for age, BMI, allergic rhinitis, chronic urticaria, and atopic dermatitis.

Abbreviations: Q, quartile; T, tertile.

This study showed that, the higher the CRP levels, WBC counts, and the proportion of neutrophils, the higher the likelihood of DFS positivity, whereas the proportion of lymphocytes and monocytes showed an inverse relationship with DFS positivity. Ludgren et al. reported the association between high frequency of symptomatic patients and diseases with immunologic or inflammatory process and thus suggested that a positive DFS pattern may indicate a proinflammatory microenvironment and potential autoimmune diseases [13]. Our results also supported a possible association between inflammatory states and DFS positivity; however, even the values belonging to the highest value groups of CRP levels and WBC counts were mostly below the inflammatory threshold. Among inflammatory-based diseases other than autoimmune diseases, only allergic rhinitis showed a significant association with DFS positivity, whereas other allergic diseases (atopic dermatitis and chronic urticaria) and dermatologic diseases (atopic dermatitis, alopecia, and psoriasis) were associated with a negative DFS pattern.

There has been no previous study regarding allergic rhinitis and DFS positivity, and a low rate of ANA positivity in allergic rhinitis was reported [14]. The relation between atopic dermatitis with DFS negativity observed in this study is contrary to that reported in other reports [5, 13]. Although chronic urticaria could be a manifestation of autoimmune diseases, a report stated that positive results for ANA in chronic urticarial patients could not indicate the differences in the main disease characteristics between them and ANA-negative patients [15]; therefore, the clinical significance of DFS negativity in chronic urticaria is unclear. Ochs et al. explained that environmental stressors, including allergens, can trigger oxidative events and inflammation in certain tissues, resulting in a response characterized by an increase in the DFS70 antigen levels, thus elucidating the relationship between allergy-related disease and DFS positivity [7]. Togay et al. also reported that both allergen-specific IgE levels and DFS70 positivity were statistically significant in younger age groups and proposed that the allergen-sIgE-mediated allergic disease should be considered in patients with isolated anti-DSF70 [16]. Among dermatological disorders, alopecia and psoriasis were significantly associated with DFS negativity. A previous study reported that there was no difference in DFS positivity according to dermatological disorders; however, the number of patients participating in the study with each disorder was too low to provide reliable data [8]. In a retrospective study, rash or skin lesions and hair loss were reported as 12.1% and 4%, respectively, among 425 patients with positive DFS patterns [12]. Inconsistencies in results of this study with respect to the association of DFS status with various diseases support the possible explanation that the weak association between anti-DFS70 and particular diseases may be due to the high relative prevalence of anti-DFS70 in the general population [17]. Therefore, further studies with more subjects are necessary to examine the association of DFS positivity with allergic and dermatologic diseases.

The presence of anti-Ro/SSA and anti-La/SSB Abs showed a high likelihood of DFS positivity with autoimmune diseases, wher eas the presence of RA was associated with a negative DFS pattern and positive RF showed no significant association with DFS positivity. Although the number of patients with RA was small, this result is consistent with previous reports [5, 6, 18-20]. The anti-Ro/SSA Ab is commonly found in the SLE, SS, SS/SLE overlap syndromes, and subacute cutaneous lupus erythematosus, and the anti-La/SSB Ab is more closely linked to SS [21-23]. Among 17 subjects positive for anti-Ro/SSA or anti-La/SSB Ab, all except two had autoimmune diseases including SS, RA, systemic sclerosis, and ankylosing spondylitis, and other two had thyroid conditions. All 17 subjects, except one with no results, showed elevated ESR and/or ferritin levels; however, increase in CRP levels and WBC counts was not consistent. Recent studies have shown that only a small proportion of individuals with a positive DFS pattern are positive for anti-DFS70. Other antibodies producing a positive DFS pattern besides anti-DFS70 are specified in previous studies [13, 24, 25], and anti-Ro/SSA Ab and/or anti-La/SSB Ab may produce a positive DFS pattern, based on our results. A positive DFS pattern in IFA but negative anti-DFS70 test can also be caused by differences in experience of individuals reading IFA data and in selection of the anti-DFS70 assay depending on the laboratory where the experiments were performed [17]. The relationship between anti-Ro/SSA and anti-La/SSB antibodies and DFS positivity requires further evaluation in additional subjects with positive anti-RO/SSA and/or anti-La/SSB antibodies.

There was no significant association between DFS positivity with age and sex in our study. Dinse et al. reported that age was not associated with anti-DFS70, as our results showed; however, the likelihood of having the anti-DFS70 antibody were three times higher in females than in males [17]. As the number of Asians included in Dinse et al.’s study was very low, differences in DFS positivity based on sex may vary by race. Meanwhile, a study from a Chinese institution revealed that DFS positivity was maximum in younger patients aged 21–40 years, whereas ANA positivity along with DFS negativity was predominantly observed in patients aged 41–60 years [26, 27]. Additionally, BMI was not significantly associated with DFS positivity in this study, and this finding was the same as that of another study [17]. However, BMI values were missing for 83 subjects with a positive DFS pattern only, which could have led to non-significant results.

We included a larger number of subjects compared to that in other studies, and we balanced the age and sex ratios between the DFS-positive and DFS-negative groups in this study. On the other hand, some of our results might differ from those of previous studies because we collected self-reported history of diseases from subjects who underwent regular health checkups, which may have resulted in inaccurate or missing data. As this was a retrospective study, we could not verify the presence of anti-DFS70 in the DFS-positive group.

In conclusion, inflammatory markers such as CRP levels, WBC counts, and the proportions of neutrophils were significantly associated with DFS positivity; however, most values of markers in the DFS-positive group were below the inflammatory threshold. Considering inconsistent association of inflammatory-based diseases with DFS positivity, the link between inflammatory states and DFS positivity is controversial and further studies are required to understand the clinical significance of the association between inflammatory states and DFS.

None declared.