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

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.