Indirect immunofluorescence (ANA)

Principle

• Two-step indirect fluorescent antibody test for antinuclear antibodies on HEp-2 cells, a human epithelial cell line with large nuclei and mitotic cells.
• Patient autoantibodies bind nuclear or cytoplasmic antigens; bound antibody is detected with fluorescein (FITC)-labelled anti-human IgG and seen as apple-green fluorescence.

Specimen

• Serum; avoid haemolysed, lipaemic or contaminated samples.

Procedure

• Dilute serum in buffer (screening dilution commonly 1:80 or 1:160 for adults; local policy).
• Incubate on fixed HEp-2 slide in a moist chamber (about 20–30 minutes); wash with PBS.
• Add FITC-conjugated anti-human IgG, incubate in the dark, wash.
• Mount with buffered glycerol and coverslip; read with a fluorescence microscope.
• Positive samples are titrated to end point.

Results

• Report titre and pattern (ICAP nomenclature).
• Homogeneous: dsDNA, nucleosomes, histones (SLE, drug-induced lupus).
• Speckled: Sm, U1-RNP, SSA/Ro, SSB/La, Scl-70 (SLE, MCTD, Sjögren syndrome, systemic sclerosis).
• Dense fine speckled (DFS70): common in healthy people; less associated with systemic autoimmune disease.
• Centromere: discrete speckles aligned on chromosomes in metaphase (limited cutaneous systemic sclerosis/CREST).
• Nucleolar: systemic sclerosis.
• Cytoplasmic patterns (e.g. mitochondrial in primary biliary cholangitis) should also be reported.
• Mitotic cells help distinguish patterns.

Quality control and pitfalls

• Run negative, positive and pattern-specific controls on each slide.
• Low-titre ANA is common in healthy people, especially older women; interpret with clinical findings.
• Photobleaching, drying of slides and poor washing cause artefacts.
• Reading is subjective; automated imaging systems help standardise.

Clinical use

• Screening for systemic autoimmune rheumatic diseases; ANA ≥1:80 on HEp-2 is the entry criterion in 2019 EULAR/ACR SLE classification. Confirm specificity by ENA, anti-dsDNA (e.g. Crithidia luciliae IIF or ELISA).