anti-nuclear antibody (ANA) in serum

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Introduction

Antinuclear Antibody (ANA)

Etiology

Reference interval

The expected value in the normal population is negative.

However, apparently healthy individuals may contain low titers of ANA.

This percentage increases with age, particularly in the 7th decade of life.

qualitative quantitative
Low titer: 1:40 to 1:80
Medium titer: 1:160 to 1:320
High titer: 1:640 or greater

Principle

A test serum is incubated on prepared slides that have Hep-2 human epithelial pharyngeal carcinoma tissue cell culture substrate affixed to them. The advantage to this substrate is the large number of cells in mitosis, so that the test detects ANAs reactive with nuclear antigens expressed during all phases of the cell cycle. If the test sample contains ANA, the auto- antibody will bind to the antigen in the substrate. The excess serum is washed away & the slide is incubated with fluorescein isothiocyanate (FITC) conjugated rabbit anti-human gamma globulin (polyvalent). The FITC conjugate will attach to the patient's antibody, if present, that is bound to the substrate. Excess conjugate is washed away, & the slide is viewed under a fluorescent microscope for a specific pattern of fluorescence.

Clinical significance

Antinuclear antibody (ANA) is a general term used to describe autoantibodies against various cell nuclear proteins. Early studies of these autoantibodies, using immunofluorescent techniques, revealed a select few nuclear protein specificities. Because of the high correlation of positive ANA with systemic lupus erythematosus (SLE), a negative ANA essentially rules out the disease.

Although antibodies specific to DNA continue to show a high disease correlation with SLE, in the last decade a number of nuclear & cytoplasmic macromolecules have been detected and associated with other connective tissue diseases, including progressive systemic sclerosis, mixed connective tissue disease, Sjogren's syndrome, polymyositis, & rheumatoid arthritis. ANA testing is now recognized as a general screening tool for connective tissue disease.

Up to 14% of normal elderly individuals may be positive for ANA.

ANA is not useful for evaluation of fatigue or myalgia or in patients with fibromyalgia[5]

Pattern Detection

    • Synonyms: Diffuse, solid.

b) Nuclear antigens: dsDNA, nDNA, RNP, histone, Scl-70, Ku, PM/Scl.

c) Disease association: High titers suggesitve of SLE; lower titers suggestive of SLE or other connective tissue diseases.

    • Synonyms: Rim, shaggy, membranous.
    • Synonyms: ACA, discrete speckled.

Specimen

No special patient preparation is required. At least 0.5 mL of unhemolyzed, cell-free serum is the recommended sample for this procedure.

Serum samples may be stored for up to two (2) days at 2 degrees C. For longer storage, freeze samples at -20 degrees C or colder. Repeated freezing & thawing may cause deterioration of the test specimen.

Interpretation

Negative A serum is generally considered negative for antinuclear antibodies if nuclear fluorescence is less than or equal to the negative control well. Caution: Some sera may demonstrate nuclear & cytoplasmic fluorescence with no apparent nuclear staining pattern. This phenomenon is generally due to heterophile antibodies & should be reported negative.

Positive A serum is considered positive if the nucleus shows fluorescence greater than the negative control well & a clearly discernible pattern of fluorescence is detectable.

Screening: Results should be reported as to the type of nuclear staining pattern observed as well as the fluorescent intensity.

Titering: Results should be reported as to the last serial dilution in which a 1+ fluorescent intensity, with a clearly discernible pattern of fluorescence, is detected. Results with fluorescent intensities greater than 1+ at 1:2560 dilution should be reported as greater than 1:2560 along with the fluorescent intensity.

Fluorescent Intensity

Fluorescent intensity may be semi-quantitated by following the guidelines for fluorescent antibody reagents established by the DHEW-USPHS Center for Disease Control, Atlanta, Georgia.

4+ Brilliant yellow-green (maximal fluorescence): clear-cut cell outline; sharply defined cell center.

3+ Less brilliant yellow-green fluorescence: clear-cut cell outline; sharply defined cell center.

2+ Definite cell pattern but dim fluorescence: cell outline less well defined.

1+ Very subdued fluorescence: cell outline almost indistinguishable from cell center in most instances.

Mitotic Cells

Detection: Mitotic cells should be visible on every field when viewed at 250x magnification or lower. To verify whether a cell is in mitosis, view at 400x magnification or greater. Mitotic cells show a characteristic round cell shape with no detectable nuclear membrane. The chromosome region of mitotic cells will generally show an irregular shape within the cell, due to the lack of nuclear membrane, & extreme constriction of the chromosomes.

Sera positive for DNA and/or RNP and/or histone (such as the homogeneous positive control) will show bright staining of the chromosome region of these cells. In negative samples for DNA and/or RNP and/or histone (such as the speckled positive control or nucleolar positive control), the mitotic cells will not show chromosome staining & may be difficult to see.

Discerning Patterns

Speckled vs. Homogeneous Antibody: Fine speckled samples are often difficult to differentiate from homogeneous staining samples. If the serum is homogeneous, there will be a solid staining of the chromosomes of mitotic cells. If the sample is strictly speckled, the entire mitotic cell (or the region outside of the chromosomes alone) will show a fine speckled, staining reaction.

* If fine speckling of the entire mitotic cell occurs along with solid staining of the chromosome region, it is highly probable that two or more antibodies are present. Report the screening dilution as speckled/homogeneous & titer each antibody to endpoint.

Peripheral vs. Nuclear Membrane Antibody: Peripheral antibody is generally associated with DNA/RNP nuclear antigens with high titers suggestive of SLE. In substrates not using mitotic cells, this staining pattern can be difficult to distinguish from nuclear membrane antibody. In order to verify peripheral antibody, the chromosome region of mitotic cells should stain brightly. If the chromosome region does not stain, the sample is likely to be nuclear membrane antibody, with no DNA/RNP specificity & no association with SLE.

Anti-Centromere Antibody (ACA) vs. Atypical speckled antibody resembling Centromere: In order to verify centromere antibody, the chromosome region of mitotic cells should stain brightly with similar discrete speckles. If the chromosome region does not stain, the sample is likely to be pseudo-centromere (NSpI or NSpII) with no kinetochore specificity & no association with the CREST variant of scleroderma.

Cytoplasmic Fluorescence

Although autoantibodies to cytoplasmic antigens are not commonly associated with connective tissue disease, high- titered sera may be detected with epithelial tissue culture cell substrates. Mitochondrial & smooth muscle antibodies are the two most commonly detected antibodies & are generally associated with mononucleosis, chronic active hepatitis, & liver disease. Smooth muscle antibody has also been demonstrated in patients with warts, when the HEp-2 cell substrate is used.

Anti-Mitochondrial Antibody (AMA): Discrete speckles concentrated in the perinuclear region of the cell & extended in lower density to the outer regions of the cytoplasm. This should be distinguished from GOLGI antibody, which generally stains only one side of the perinuclear region, & from ribosomal antibody, which demonstrates finer speckles with a strandlike appearance consistent with the location of the endoplasmic reticulum within the cell.

* Perinuclear speckles can most easily be distinguished from peripheral nuclear staining by noting that the mitochondrial speckles form an interrupted speckled staining around the outside of the nuclear membrane, while peripheral sera form a solid smooth staining inside the nuclear membrane.

REPORT SERA AS NEGATIVE FOR ANTINUCLEAR ANTIBODY.

Anti-Smooth Muscle Antibody (ASMA): Very fine fibrous staining over the entire cytoplasm of cells with a 'spiderweb' appearance. Unlike mitochondrial antibody, smooth muscle antibody staining is uniform over the entire cytoplasm & may also extend over the nucleus. Mitotic cells generally show large discrete speckles outside the chromosome region.

REPORT SERA AS NEGATIVE FOR ANTINUCLEAR ANTIBODY.

Reporting Results: Results are recorded on the worksheet as the highest titer showing a 1+ positive reaction. If, at the screening titer of 1:40 there is no nuclear staining or it is less than or equal to the negative control, then it is reported as negative. If, at the screening titer of 1:40 there is nuclear staining greater than the negative control, it is reported as positive on the worksheet & the intensity & pattern recorded. However, before a result can be entered in the computer, the serum must be titered & read for the highest dilution producing a 1+ nuclear staining with a discernible pattern.

A small percentage of patients with SLE may not demonstrate ANAs by indirect immunofluorescence but may have ANAs by other techniques.

Although a high-titered ANA may be highly suggestive of connective tissue disease, it should not be considered diagnostic but rather viewed as a part of the overall clinical history of a patient.

Positive ANAs are also seen in a small percentage of patients with infectious and/or neoplastic diseases.

Interferences

More general terms

More specific terms

Component of

References

  1. ↑ Reference Procedure for ANA HEp-2 Cell Culture IFA Test System. Zeus Scientific, Inc., Series 2500, 1987.
  2. ↑ Stites, Daniel P., Stobo, John D., Wells, J. Vivian, Basic & Clinical Immunology, 6th Edition, Appleton & Lange, Norwalk, Connecticut, 1987, p. 359.
  3. ↑ Henry, John Bernard, Clinical Diagnosis & Management by Laboratory Methods, 17th Edition, 1982, p. 926-931.
  4. ↑ Rose, Noel, R. & Friedman, Herman, Basic & Clinical Immunology, 2nd Edition, 1980. p.853-857.
  5. ↑ 5.0 5.1 Medical Knowledge Self Assessment Program (MKSAP) 11,14,16,17,18. American College of Physicians, Philadelphia 1998,2006,2012, 2015,2018
  6. ↑ ARUP Consult: Antinuclear Antibody Disease Testing Algorithm https://arupconsult.com/algorithm/antinuclear-antibody-testing-algorithm
    Antinuclear Antibody (ANA) with HEp-2 Substrate https://arupconsult.com/ati/antinuclear-antibody-ana-hep-2-substrate

Patient information

antinuclear antibody (ANA) in serum patient information

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