Advances and pitfalls in anti-double-stranded DNA autoantibody detection and interpretation

Abstract

Anti-double-stranded DNA (anti-dsDNA) autoantibodies are detected in 20%-90% of patients with systemic lupus erythematosus (SLE), with prevalence varying according to disease activity, patient population, and assay methodology. Traditionally, four categories of anti-DNA autoantibodies have been described. The first recognizes only native dsDNA; the second recognizes both dsDNA and single-stranded DNA (ssDNA); the third reacts predominantly with denatured or ssDNA; and the fourth comprises anti-nucleosome autoantibodies, which recognize DNA-histone complexes and may yield results that differ from those obtained with assays specific for native dsDNA. Combining the Crithidia luciliae indirect immunofluorescence test with solid-phase immunoassays improves the diagnostic accuracy for anti-dsDNA antibody screening in patients with SLE. However, several limitations remain in the performance and interpretation of anti-dsDNA immassays, potentially leading to diagnostic errors. These pitfalls may arise mainly from analytical variability, assay design constraints, and a lack of standardized interpretation. Recent advances indicate a growing shift toward solid-phase multi-analyte arrays coupled with algorithm-based analysis, which may help reduce the seronegative gap in SLE while improving the harmonization of anti-dsDNA testing. This minireview critically evaluates current immunoassays for anti-dsDNA antibody testing, with particular emphasis on their analytical limitations, diagnostic pitfalls, and emerging advances.

Key Words: Anti-double-stranded DNA autoantibodies; Systemic lupus erythematosus; Crithidia luciliae indirect immunofluorescence; Solid-phase immunoassays; Pitfalls; Limitations

Core Tip: Crithidia luciliae indirect immunofluorescence testing provides excellent specificity by preferentially detecting high-avidity anti-double-stranded DNA autoantibodies, whereas solid-phase immunoassays offer greater sensitivity at the expense of specificity. Major analytical sources of variability include differences in substrate preparation, serum dilution protocols, conjugate performance, subjective interpretation of fluorescence microscopy, and the lack of standardized calibration and harmonized reference materials. Artificial intelligence-assisted image analysis may further enable automated interpretation of indirect immunofluorescence patterns, reducing inter-observer variability and improving analytical consistency.

INTRODUCTION

Systemic lupus erythematosus (SLE) is a chronic, multisystem autoimmune disease characterized by the production of a wide array of autoantibodies and a broad spectrum of clinical manifestations that can affect virtually any organ system, resulting in highly heterogeneous disease presentations[1,2]. The diagnosis of SLE is based on the integration of clinical manifestations and immunological biomarkers after exclusion of alternative diagnoses. Given the wide heterogeneity of clinical manifestations, several sets of classification criteria have been developed over time for epidemiological and research purposes.

As one of the most characteristic antinuclear antibodies (ANA) specificities, anti-double-stranded DNA (anti-dsDNA) autoantibodies exhibit high specificity (approximately 96%) but only moderate diagnostic sensitivity (approximately 52%-70%) for SLE, as they are not present in all patients and may fluctuate throughout the disease course[2]. Therefore, these autoantibodies should not be used in isolation to diagnose SLE, and a negative anti-dsDNA autoantibody result does not exclude the disorder. Their interpretation should always be integrated with the clinical manifestations and other immunological biomarkers. Consistent with this principle, the 2019 European Alliance of Associations for Rheumatology/American College of Rheumatology classification criteria for SLE require that anti-dsDNA autoantibodies be detected using an immunoassay with a demonstrated specificity of at least 90% compared with relevant disease controls[3].

SLE disease activity is routinely evaluated using validated clinical indices, including the British Isles Lupus Assessment Group (BILAG-2004) index, the Physician Global Assessment, and the SLE Disease Activity Index (SLEDAI-2K) together with immunological biomarkers such as anti-dsDNA autoantibodies and serum complement C3 and C4 levels[4-6]. The prevalence of anti-dsDNA autoantibodies among patients with SLE ranges from 20% to 90%, depending on the patient population, disease activity, and assay employed[7]. Anti-dsDNA autoantibodies are associated with the development and activity of lupus nephritis (LN) and may predict renal flares[8].

In patients with LN, serial measurements of anti-dsDNA autoantibody titers, together with serum complement levels and appropriate urinary biomarkers, are primarily used to monitor kidney disease activity and therapeutic response rather than to establish the diagnosis of SLE. Anti-dsDNA autoantibody titers can fluctuate over time[9]. They can disappear during treatment and return during a flare, especially in active LN[10].

Anti-dsDNA autoantibodies constitute a diverse group of ANA that target multiple DNA components, including B-DNA, single-stranded DNA (ssDNA), RNA-DNA hybrids, Z-DNA, and various DNA-protein complexes[11]. This antigenic diversity contributes to the variability in their detection and the interpretation of their clinical significance. The quantification of anti-dsDNA autoantibodies is further complicated by variability among laboratory assays[12-15]. Recent advances in solid-phase multi-analyte assays, combined with artificial intelligence (AI) for automated image analysis of indirect immunofluorescence (IIF) patterns, offer novel opportunities to overcome these difficulties.

This minireview discusses current laboratory assays for screening and quantifying anti-dsDNA autoantibodies, depicting key pitfalls and limitations.

SEARCH STRATEGY

The data presented in this review were obtained through a comprehensive literature search of the PubMed database using the following keywords: Anti-dsDNA autoantibodies, double-stranded DNA autoantibodies, HLA, major histocompatibility complex, SLE, LN, pitfalls, ELISA, Crithidia luciliae, chemiluminescence immunoassay, fluoroenzyme immunoassay, multiplex immunoassay, Farr assay, biosensors, and AI. Original research articles, systematic reviews, meta-analyses, and relevant narrative reviews published in English were considered. The reference lists of eligible articles were also manually screened to identify additional relevant publications.

PATHOGENESIS OF IGG ANTI-DSDNA AUTOABS

HLA basis of immunoglobulin G anti-dsDNA autoAbs production

The pivotal role of high-affinity immunoglobulin G (IgG) anti-dsDNA autoantibodies in SLE reflects a requirement for cooperation between CD4+ T helper (Th) cells and B lymphocytes[16,17]. Activation and differentiation of dsDNA-specific B cells into long-term plasmocytes producing high-affinity IgG anti-dsDNA autoantibodies is driven by Th cells that depend on the presentation of chromatin-derived peptides or nucleosomes by HLA class II (HLA-II) molecules to T-cell receptors. This hypothesis is supported by several studies, which have identified specific HLA-II alleles as risk or protective factors for SLE. HLA-DRB1*03:01 and HLA-DRB1*15:01 are among the strongest HLA-II susceptibility alleles for SLE and have been associated with anti-dsDNA autoantibody production in several ethnic populations[18]. These molecules influence disease development by presenting self-antigens, especially nucleosomal peptides derived from apoptotic cell debris, to Th cells, thereby promoting B-cell activation and class switching toward pathogenic autoantibody production. Conversely, certain HLA-II alleles, including HLA-DRB1*04, -DRB1*11, and -DRB1*14 alleles in some populations, have been correlated with a reduced risk of SLE, suggesting a protective role against disease development[19]. Anti-dsDNA autoantibodies are heterogeneous in specificity[20], and their epitope-binding properties vary in affinity and cross-reactivity[21,22], contributing to differences in pathogenic potential. Therefore, HLA-II polymorphisms critically determine the likelihood of AI reaction severity in SLE and anti-dsDNA autoantibody development.

Pathogenic mechanisms

The generation and deposition of circulating DNA-IgG immune complexes (ICs) constitute a main pathogenic mechanism in SLE. After deposition in the glomeruli, these ICs activate the classical pathways, leading to amplification of inflammation, generation of lytic membrane attack complexes, and kidney injury[16,23,24]. Anti-dsDNA autoantibodies may also contribute to renal injury through cross-reactivity with glomerular antigens such as alpha-actinin and heparan sulfate, thereby inducing in situ IC formation[25-27]. In addition, their physicochemical properties, such as high-affinity and polyreactive subsets, are more strongly associated with organ involvement and disease activity[16,28]. Beyond kidney injury, a subset of anti-dsDNA autoantibodies can cross-react with non-DNA tissue antigens, including the N-methyl-D-aspartate receptor and skin lesions, leading to extrarenal manifestations of SLE[29-32]. Interestingly, circulating low-affinity immunoglobulin M (IgM) anti-dsDNA autoantibodies may be detected in healthy individuals and transiently in patients with infections and cancers, highlighting their context-dependent pathogenicity[22,33].

CLINICAL SIGNIFICANCE

While circulating anti-dsDNA autoantibodies are highly specific for SLE and are included in the current disease classification criteria[34], their sensitivity varies considerably. The correlation between SLE disease activity and anti-dsDNA levels is assay-dependent and is influenced by patient-specific factors, including organ involvement, disease duration, and treatment status. A major contributor to the inter-assay variability in clinical practice among anti-dsDNA immunoassays is the antigenic heterogeneity of anti-DNA autoantibodies.

Anti-DNA autoantibodies have been classified into four categories. The first recognizes conformational epitopes that depend on the intact double-helical structure of DNA. These are typically high-affinity IgG antibodies and are most strongly associated with SLE[35]. Increasing anti-dsDNA autoantibody titers may precede or accompany disease flares in some patients, particularly those with LN. However, this relationship is not universal and should be interpreted in the clinical context. The second binds both dsDNA and ssDNA, primarily through recognition of the sugar-phosphate backbone common to both DNA forms[35]. The third preferentially recognizes denatured or ssDNA by binding exposed purine and pyrimidine bases[35]. Anti-ssDNA autoantibodies are less specific for SLE and may also be detected in other AI disorders, infections, and drug-induced SLE[35]. Consequently, these autoantibodies have limited diagnostic utility compared with anti-dsDNA autoantibodies. The fourth category comprises anti-nucleosome autoantibodies, which recognize DNA-histone complexes and may yield results that differ from those obtained with assays specific for native dsDNA[20].

Available assays differ in immunoglobulin class detection, avidity profiles, complement-fixing capacity, and susceptibility to cross-reactivity, all of which contribute to significant inter-assay variability[36,37]. Most clinical assays primarily detect IgG anti-dsDNA autoantibodies, whereas IgM and immunoglobulin A anti-dsDNA autoantibodies are less frequently measured and their clinical significance remains less well established.

Taken together, these immunological characteristics influence antibody pathogenicity, assay performance, and the clinical significance of test results, thereby contributing to inter-assay variability and differences in their association with disease manifestations[21,22].

LIMITATIONS AND PITFALLS OF ANTI-dsDNA AUTOANTIBODIES

Anti-dsDNA autoantibody testing is performed using several platforms, including Crithidia luciliae IIF test (CLIFT) and multiple solid-phase immunoassays. These assays are based on different analytical principles and are used for diagnostic, classification, and monitoring purposes in SLE. Despite technological advances, no single assay is universally accepted as the optimal method for all clinical purposes, including screening, diagnosis, classification, and disease monitoring[38]. Furthermore, SLE classification criteria require assays with high specificity (≥ 90%)[34]. Different assay platforms employ distinct antigen sources, DNA conformations, and detection principles, resulting in differences in antibody recognition and clinical interpretation.

Discordance between human epithelial type 2 IIF and anti-dsDNA immunoassays

In the human epithelial type 2 (HEp-2) IIF assay, anti-dsDNA autoantibodies are typically associated with a homogeneous nuclear pattern[39,40]. However, discordance between anti-dsDNA immunoassays and ANA-IIF may occur. Anti-dsDNA immunoassays using purified or synthetic DNA can yield positive results despite negative antinuclear antibody IIF findings[41]. In HEp-2 cells, dsDNA is bound by histones and other proteins, while fixation procedures may further modify antigen accessibility[42-44]. Thus, these methodologies may recognize distinct antibody populations and exhibit incomplete concordance.

CLIFT assay

The CLIFT assay uses the kinetoplast of Crithidia luciliae, which contains circular dsDNA devoid of histones (Figure 1). High-avidity anti-dsDNA autoantibodies detected by CLIFT are highly specific for SLE and have been associated with the development of LN. The assay primarily detects medium- to high-avidity IgG anti-dsDNA autoantibodies and demonstrates relatively low sensitivity (47%-55%) but excellent specificity (98%-100%) (Table 1)[45]. Therefore, CLIFT is not recommended as a standalone screening or disease-monitoring assay.

Figure 1 Schematic representation of Crithidia luciliae showing cellular structures relevant to Crithidia luciliae immunofluorescence test interpretation. A positive Crithidia luciliae immunofluorescence test result is defined by specific fluorescence of the kinetoplast, which contains pure double-stranded DNA (dsDNA) devoid of histones. Nuclear fluorescence may be present but is not required for a positive result. Isolated nuclear fluorescence without kinetoplast staining should be interpreted as negative for anti-dsDNA antibodies and may indicate antibodies to other nuclear antigens.

Table 1 Performance characteristics, limitations, and pitfalls of anti-dsDNA autoantibody testing.

Assay	Study populations	Control groups	Assay manufacturers	Reference standards	Cutoffs	Sensitivity, %	Specificity, %	ROCAUC1	Limitations and pitfalls	Ref.
CLIFT	SLE patients, lupus nephritis, ANA-positive connective tissue diseases	Healthy controls, disease controls (RA, Sjögren syndrome, SSc, MCTD, infections)	Euroimmun, Bio-Rad, Immuno Concepts, Inova, NOVA Lite	ACR/EULAR SLE classification criteria, clinical diagnosis, comparator anti-dsDNA assays	Manufacturer-defined serum dilution (typically 1:10-1:20)	47-70	95-100	0.80-0.90	Low sensitivity; subjectivity and operator dependence; technical variability: Affected by serum dilution, substrate prep, microscope systems, and conjugate performance; limits reproducibility (though AI automated image analysis is emerging to help)	[49,77-79]
Farr RIA	SLE patients, lupus nephritis	Healthy controls; autoimmune disease controls	Trinity biotech; laboratory-developed assays	ACR/EULAR criteria, clinical diagnosis, comparator assays	Manufacturer-defined (IU/mL or cpm)	70-85	95-100	0.75-0.95	The short shelf-life of reagents and requirement for handling radioactive tracers	[80-85]
ELISA	SLE patients, lupus nephritis, autoimmune diseases	Healthy controls; autoimmune disease controls	UROIMMUN, Thermo Fisher (Phadia), Inova, Bio-Rad, AESKU, Orgentec, Trinity	ACR/EULAR criteria, clinical diagnosis, CLIFT/Farr comparison	Manufacturer-specific (IU/mL)	70-90	80-95	0.84-0.90	Lower specificity: Detects both low- and high-avidity IgG autoantibodies Susceptibility to cross-reactivity (e.g., with anti-ssDNA or other nuclear antigens); performance variability: Highly influenced by the specific antigen source, coating conditions, and manufacturer	[49,78,82]
CLIA/FEIA	SLE patients, lupus nephritis; systemic autoimmune rheumatic diseases	Healthy controls; autoimmune disease controls	Werfen (BIO-FLASH), Thermo Fisher (EliA), Fujirebio	Clinical diagnosis, CLIFT/Farr comparison	Manufacturer-specific (IU/mL)	75-90	90-98	0.88-0.95	The lack of inter-laboratory standardization and harmonization	[49,78,82]
MFI	SLE patients; systemic autoimmune rheumatic diseases	Healthy controls; autoimmune disease controls	BioPlex®, FIDIS®, Luminex® platforms	Clinical diagnosis, comparator immunoassays	Manufacturer-specific fluorescence units	70-90	90-98	0.80-0.95	Complexity: Requires complex instrumentation and specialized bioinformatics and data interpretation; bead-based variability: Analytical performance can vary based on how well the fluid-phase antigen interactions preserve native antigen conformation; high cost	[45,68,78,86,87]
Emerging Platforms (DLCM, Biosensors, Microarrays)	Primarily research cohorts of SLE patients	Healthy controls; limited disease controls	Research-use platforms	Comparator immunoassays (ELISA, CLIA, CLIFT)	Not standardized	-	-	> 0.90	-	[88-90]

¹Sensitivity, specificity, and receiver operating characteristic area under the curve values represent approximate ranges reported across comparative studies and meta-analyses. Performance varies according to assay manufacturer, antigen source and conformation, calibration, cutoff selection, study population, and reference standard.

CLIFT: Crithidia luciliae indirect immunofluorescence test; RIA: Radioimmunoassay; ELISA: Enzyme-linked immunosorbent assay; CLIA: Chemiluminescence immunoassay; FEIA: Fluoroenzyme immunoassay; MFI: Multiplex flow immunoassay; ROCAUC: Receiver operating characteristic area under the curve; SLE: Systemic lupus erythematosus; RA: Rheumatoid arthritis; SSc: Systemic sclerosis; MCTD: Mixed connective tissue disease; anti-dsDNA: Anti-double-stranded DNA; ANA: Antinuclear antibodies; AI: Artificial intelligence; IgG: Immunoglobulin G; anti-ssDNA: Anti-single stranded DNA; ACR: American College of Rheumatology; EULAR: European Alliance of Associations for Rheumatology.

The CLIFT assay is subject to several limitations. Although it is generally less susceptible to pre-analytical variables than many automated immunoassays, appropriate sample collection, handling, storage, and avoidance of repeated freeze-thaw cycles remain essential to preserve antibody integrity. Major analytical sources of variability include differences in substrate preparation, serum dilution protocols, conjugate performance, subjective interpretation of fluorescence microscopy, and semiquantitative result reporting[46]. In addition, the assay is labor-intensive, time-consuming, and associated with a relatively long turnaround time. When CLIFT is performed manually, the procedure requires approximately 60 minutes. Anti-dsDNA autoantibodies exhibit different binding characteristics across assay platforms because each method presents DNA in a distinct structural conformation. CLIFT preferentially detects antibodies recognizing the bent circular B-form DNA present in the kinetoplast of Crithidia luciliae, which resembles plasmid-like DNA[47]. Furthermore, cutoff selection is not universally standardized and depends on visual interpretation criteria and the screening serum dilution, both of which influence assay specificity, sensitivity, and inter-laboratory comparability[48,49]. Thus, these factors limit reproducibility and assay standardization.

Solid-phase immunoassays

Solid-phase immunoassays for anti-dsDNA detection include enzyme-linked immunosorbent assays (ELISA), Farr radioimmunoassay (RIA), multiplex flow immunoassays, fluoroenzymatic immunoassay (FEIA), chemiluminescence immunoassay (CLIA), microdot arrays, and particle-based multiplex systems[49,50]. These assays differ in detection principles and antigen presentation, resulting in considerable variability in analytical sensitivity and specificity. A comparative study of eight anti-dsDNA immunoassays demonstrated that, using manufacturer-recommended cutoffs, specificity ranged from 84% to 98%, whereas sensitivity ranged from 67% to 92%[49]. Moreover, quantitative results correlate poorly across methods, indicating that numerical values are not readily interchangeable between laboratories or assay platforms[49].

ELISA detects both low- and high-avidity IgG anti-dsDNA autoantibodies while providing quantitative results[51]. Owing to its high sensitivity, simplicity, and relatively low cost, ELISA remains one of the most widely used methods in clinical laboratories. It is also more sensitive than CLIFT[52], making it particularly useful as a screening assay for anti-dsDNA autoantibodies in patients with SLE. However, its performance is influenced by several factors, including antigen source, coating conditions, and potential cross-reactivity[53]. Depending on the antigen employed, ELISA may detect antibodies against multiple DNA conformations, including elongated linear B-DNA, cruciform DNA, and ssDNA associated with transcriptionally active chromatin[47]. In addition, contamination with ssDNA may lead to overestimation of anti-dsDNA antibody levels because anti-ssDNA autoantibodies lack specificity for SLE[54,55]. Calibration curves in ELISA may vary between reagent lots and are not stable over prolonged periods; consequently, a new calibration curve is generally required for each reagent lot[48].

Although the Farr RIA has historically been regarded as a reference method, it primarily detects high-avidity IgG anti-dsDNA autoantibodies and provides quantitative results. The Farr assay preferentially detects antibodies recognizing Z-DNA or high-avidity interactions under high-salt conditions[47]. In active LN, a recent study comparing Farr RIA, ELISA, FEIA, and CLIA demonstrated 95% agreement between the Farr assay and FEIA. With respect to SLE activity, Farr and FEIA showed similar behaviour[56]. However, its clinical use has declined because it requires radioactive tracers[57] (Table 1).

FEIA and CLIA are fully automated immunoassays used to detect and quantify anti-dsDNA autoantibodies. These methods use stable, non-radioactive reagents, offering improved laboratory safety, high throughput, and standardized performance compared with older Farr RIA and ELISA[41,58-60]. With respect to the substrate, CLIA uses synthetic dsDNA immobilized on magnetic or paramagnetic particles[61,62], whereas FEIA uses synthetic or purified dsDNA immobilized on micro-wells[63]. Compared with CLIA, FEIA generally has a longer analysis time.

Although conventional enzymatic and chemiluminescent immunoassays exhibit high sensitivity, clinical laboratories are increasingly adopting multiplex bead-based assays because they enable the simultaneous detection of multiple autoantibodies[64]. DLCM has shown significant agreement with single-plex assays such as CLIA[65]. Likewise, ratiometric electrochemical biosensors have shown excellent analytical sensitivity and potential clinical utility, particularly for correlating anti-dsDNA autoantibody levels with LN[66]. However, their widespread implementation remains limited by cost, especially in resource-constrained settings.

Overall, discordance among anti-dsDNA immunoassays is common because assay performance is influenced by differences in antigen source, DNA conformation, and antibody avidity. CLIFT preferentially detects high-avidity anti-dsDNA autoantibodies and therefore provides excellent specificity, whereas highly sensitive solid-phase assays may detect additional low-level or lower-avidity autoantibodies, thereby increasing sensitivity at the expense of specificity. Accordingly, low-level positive results should be interpreted with caution, particularly in patients with a low pre-test probability of SLE, because they may not represent clinically significant disease. Combining a highly specific assay, such as CLIFT, with a highly sensitive immunoassay (e.g., ELISA or CLIA) is therefore recommended to optimize the diagnosis and monitoring of SLE[48,49]. Furthermore, the lack of a universally accepted international reference standard for calibration hinders harmonization of quantitative anti-dsDNA autoantibody measurements across platforms, contributing to persistent inter-laboratory variability.

ADVANCES IN ANTI-DSDNA ANTIBODY DETECTION AND INTERPRETATION

Automated HEp-2 IIF pattern recognition

AI tools, including deep learning (DL) and machine learning (ML), are increasingly being integrated into ANA-IIF assays to improve diagnostic accuracy[67]. These methods allow automated interpretation of IIF patterns and acquisition[68,69]. Specifically, computer-aided systems such as EUROPattern and other automated ANA readers have shown promising performance in reducing inter-observer variability and improving workflow efficiency[69-74]. However, these approaches remain platform-dependent and require validation in diverse populations and routine clinical settings.

Multimodal ML models

ML and DL algorithms have significantly improved diagnostic precision and management of rheumatic disorders[75,76]. This strategy is being investigated for integrating clinical data, various immunological biomarkers, and multi-omics data to support diagnosis and monitoring in SLE[77,78]. Orme et al[79] show the utility of ML algorithms using anti-dsDNA autoantibody signatures to predict kidney injury in SLE. Despite considerable potential to assist clinicians in diagnosing AI diseases with faster and more accurate results[80], current AI applications face important limitations: (1) Limited external validation across diverse populations and healthcare settings; (2) Platform dependence and lack of standardization; (3) Insufficient evidence that automated interpretation improves treatment decisions or patient outcomes; (4) Requirement for technical infrastructure and expertise; and (5) Regulatory and reimbursement challenges. Further prospective validation is required before routine clinical implementation.

STRATEGIES FOR ANTI-DSDNA AUTOANTIBODY TESTING

Initial diagnosis

A practical testing strategy for anti-dsDNA autoantibodies should be tailored to the specific clinical objective, whether diagnosis, disease classification, or longitudinal monitoring (Figure 2). For the initial diagnosis or classification of SLE, a three-step approach is recommended, beginning with clinical suspicion of SLE and a positive ANA-IIF with a homogeneous nuclear pattern, followed by measurement of anti-dsDNA autoantibodies using a solid-phase immunoassay (CLIA, FEIA, or ELISA), with confirmation of positive results using the highly specific CLIFT[81]. In general, blotting is not recommended for anti-dsDNA testing[82]. Final interpretation should always integrate laboratory findings with clinical manifestations, ANA profile, low complement levels, and other disease-specific autoantibodies (e.g., anti-Smith antibodies, anti-phospholipid antibodies). When screening assays and CLIFT yield discordant results, careful interpretation is required[81]. A positive solid-phase assay with a negative CLIFT result may reflect low-avidity anti-dsDNA autoantibodies, cross-reactivity with other nuclear antigens, assay-specific analytical differences, or early-stage disease. When clinical suspicion of SLE remains high, repeat testing after 3-6 months is recommended to assess serological evolution. Conversely, a negative screening assay with a positive CLIFT result, although uncommon, should prompt repeat testing to exclude technical or analytical interference. If diagnostic uncertainty persists despite discordant findings, assessment of anti-nucleosome or anti-chromatin antibodies may provide complementary diagnostic information[83].

Figure 2 Proposed testing strategy for anti-double-stranded DNA autoantibodies. For the initial diagnosis of systemic lupus erythematosus (SLE), a three-step approach is recommended, beginning with clinical suspicion of SLE and a positive antinuclear antibodies (ANA) - indirect immunofluorescence with a homogeneous nuclear pattern, followed by measurement of anti-double-stranded DNA (anti-dsDNA) autoantibodies using a solid-phase immunoassay (chemiluminescence immunoassay, fluoroenzyme immunoassay, or enzyme-linked immunosorbent assay), with confirmation of positive results using the highly specific Crithidia luciliae indirect immunofluorescence test (CLIFT). Final interpretation should always integrate the clinical manifestations, ANA profile, and C3 and C4 complement levels. In patients with persistent clinical suspicion, a positive solid-phase assay with a negative CLIFT result may warrant repeat testing after 3-6 months. In patients with established SLE, disease monitoring should preferably be performed using the same assay platform. Anti-dsDNA results should always be interpreted in conjunction with clinical disease activity, serum C3 and C4 concentrations, and organ-specific assessments, particularly in patients with suspected lupus nephritis. CLIFT: Crithidia luciliae indirect immunofluorescence test; ELISA: Enzyme-linked immunosorbent assay; CLIA: Chemiluminescence immunoassay; FEIA: Fluoroenzyme immunoassay; SLE: Systemic lupus erythematosus; anti-dsDNA: Anti-double-stranded DNA; LN: Lupus nephritis; ANA: Antinuclear antibodies; IIF: Indirect immunofluorescence.

Disease monitoring

In patients with established SLE, disease monitoring should preferably be performed using the same assay platform to minimize inter-assay variability, with greater emphasis placed on longitudinal changes in antibody titers than on isolated absolute values[81]. Anti-dsDNA antibody results should always be interpreted in conjunction with clinical disease activity, serum complement concentrations, and organ-specific assessments, particularly in patients with suspected LN[4]. Routine serial measurement of anti-dsDNA autoantibodies is generally not recommended in clinically stable patients without evidence of disease activity[4]. Ultimately, the optimal testing strategy should be individualized according to the clinical scenario, the diagnostic or monitoring objective, laboratory expertise, assay availability, and local healthcare resources.

CONCLUSION

The development of rapid, fully automated, sensitive, and specific labelled immunoassays to identify anti-dsDNA autoantibodies ushered in a new era of testing in the clinical laboratories. To optimise assay performance, the tendency in the laboratory today is toward the use of a three-step strategy, including HEp-2 IIF assay and sensitive solid-phase assays such as CLIA or FEIA for initial screening and a highly specific CLIFT for confirmation. This strategy can significantly improve diagnostic performance for anti-dsDNA autoantibody screening in SLE. AI-assisted approaches further allow automated acquisition and interpretation of CLIFT and ANA-IIF patterns, thereby reducing inter-observer variability. Such an approach could improve the prediction of kidney injury and enable effective monitoring of immunosuppressive therapy.