What method is used to detect antinuclear antibodies (ANA) in patient serum?

Detection of Antinuclear Antibodies

The detection of antinuclear antibodies involves the use of mouse cells or tissue culture cells incubated with patient serum to detect fluorescence in nuclei, on nuclear membranes, or in the cytoplasm of cells. 1

Method of ANA Detection

The indirect immunofluorescence assay (IIFA) is the reference method for detecting antinuclear antibodies (ANA). This technique involves:

1. Incubating patient serum with substrate cells (typically HEp-2 cells or variants)
2. Adding fluorochrome-labeled anti-human IgG secondary antibodies
3. Examining for fluorescence patterns in different cellular compartments

Key Components of the IIFA Method

• Substrate: HEp-2 cells (human epithelial cell line) or variants like HEp-2000 are most commonly used 1
• Detection: Fluorochrome-labeled anti-human IgG-specific secondary antibodies bind to patient antibodies 1
• Visualization: Fluorescence microscopy reveals patterns in:
  • Nuclei
  • Nuclear membranes
  • Cytoplasm
  • Mitotic spindle apparatus

Importance of Pattern Recognition

The IIFA method allows identification of specific patterns that correlate with different autoantibody specificities and associated diseases:

• Nuclear patterns (homogeneous, speckled, nucleolar)
• Cytoplasmic patterns
• Mitotic apparatus patterns 1

Why IIFA Is the Gold Standard

The international recommendations from the European Autoimmunity Standardization Initiative and other major organizations emphasize that IIFA is the reference method for ANA screening because:

1. It detects a broad spectrum of autoantibodies directed at various cellular compartments
2. It provides pattern information that helps identify specific autoantibody types
3. It has higher diagnostic sensitivity (96.8%) compared to other methods 2

Alternative Methods

While IIFA is the reference standard, alternative methods have been developed:

• ELISA (Enzyme-Linked Immunosorbent Assay): Uses purified or recombinant antigens
• Multiplex assays: Based on technologies like xMAP
• Automated platforms: AKLIDES, ALEGRIA, BioPlex 2200 2

However, these alternative methods have limitations:

• Lower sensitivity (79.8% for ELISA, 82.9% for multiplex assays) 2
• May miss certain autoantibodies detected by IIFA
• Should not be used alone as primary screening tests due to false-negative results in 17-20% of cases 2

Clinical Implications

The detection of ANA by IIFA is crucial for:

• Diagnosis of systemic autoimmune rheumatic diseases
• Classification of specific autoimmune conditions
• Providing guidance for further specific antibody testing 1, 3

When ANA is positive, further testing for specific antibodies (anti-dsDNA, anti-ENA) is recommended to better characterize the autoimmune condition 4.

Common Pitfalls and Caveats

1. Terminology confusion: The term "ANA" is technically outdated as it now encompasses antibodies directed at various cellular compartments, not just nuclear elements 1

2. Standardization issues: IIFA requires experienced technicians and standardization of:

   • Substrate quality
   • Screening dilution (typically 1:160)
   • Interpretation of patterns 1

3. False negatives with alternative methods: ELISA or multiplex assays alone may miss antibodies detectable by IIFA 2

4. Pattern reporting: Both nuclear and cytoplasmic patterns should be reported and specified according to standardized terminology 1

The detection of fluorescence patterns in cells is the correct method for ANA detection, not complement binding (option B), cell viability assessment (option C), or rheumatoid factor quantitation (option D).