Introduction:

All ANA tests must be performed on patient's diluted serum so as to dilute out lots of nonspecific "reactivity" (standard is a 1:10 dilution of serum). The classical "ANA" (and one used in our lab) is an indirect fluorescent antiby (IFA) screening test visually performed using a fluorescent microscope (so, sometimes called an F-ANA test) and is an "intellect intensive" IFA test. F-ANA usually looks for ANAs in the IgG class (not in IgA or IgM). The test substrate which provides the nuclei is either made of histological sections of tissue (histological cross-sections of nuclei) or cell culture monolayer sheets which have whole nuclei (such as HEp-2...which our lab uses). Depending on source or method of substrate production, cells may contain typical or scant amounts of the various currently known-to-be-significant antigens upon which any patient autoantibody might react. So, F-ANA is more or less a "global" ANA test. Inova reaction patterns charts & decision trees for the many varieties of ANA, ANCA, and liver HERE

Many labs use the automated, more "chemical", cheaper, non-global, non-visual (ELISA) test methods for ANA, and such methods do not reveal visual patterns (a machine "reads" positivity) and will ALWAYS miss the less prevelant but potentially highly important positive reactions. 

Interpretation conventions or cautions:

When  a screening ANA by any method is positive, it ought to be considered a non-specific positive (viral infections and some other not-primarily-auto-immune disorders can cause positivity). Of the great many possible types of autoantibodies, this ANA group of antibodies attaches to components of cell nuclei. The positive ANAs which also have "specificity" for DNA (non-soluble) or various ENAs (soluble, extractible nuclear [protein] antigens] are the ones more likely to herald or be associated with "lupus" or other diseases listed below). "Negative"...not necessarily meaning the patient is negative for autoimmune disease...is failure of the test to react positively (non-reactive [NR]) at a standard 1:10 dilution of serum. [autoimmune disease]

Our LML uses an indirect fluorescent test (IFA, F-ANA) in which patient's antibody-containing serum is mixed with a human HEp-2 nucleus-containing substrate [ warning]. A variety of possible positive HEp2 nuclear staining patterns⁴ can be visualized with the fluorescent microscope, as follows (their significance, a table):

HEp-2 nuclear patterns:

• homogeneous: positivity evenly involving all of nucleus.
• homogeneous with nuclear rim (homo/peripheral)
• nuclear membranous-linear: reacts to laminin antigens.
• nuclear membranous-pores
• mixed homogeneous & speckled
• coarse speckled
• large speckled (nuclear matrix)
• fine speckled
• pleomorphic speckled
• discrete speckled (centromere)
• few nuclear dots (scantily speckled)
• multiple nuclear dots
• nucleolar homogeneous
• nucleolar clumpy
• nucleolar speckled
• nucleolar speckled with mitotic dots
• tubulin (mitotic spindle)
• centosome (centriole)
• nuclear mitotic apparatus (NuMA or MSA-1)
• midbody (MSA-2)
• mitotic spindle antigen (MSA-3)

HEp-2 cytoplasmic patterns:

• fine -speckled Jo-1 (cytoplasmic, condensed around nucleus) [may reflect a number of tRNA synthetase auto-Abs]
• ribosomal (very fine speckled plus nucleolar)
• mitochondrial (granular cytoplasmic)
• signal recognition particle (like ribosomal but negative nucleolar)
• endoplasmic reticulum (as LKM-1 in rodent tissue)
• lysosomal (large irregular speckles)
• peroxisomes (in polymyalgia type situations)
• Golgi complex

A negative ANA screening test of any type does not rule out an auto-immune disorder! IFA "homogeneous" pattern is anti-DNA-directed & is when (1) the resting cell nucleus stains thoroughly PLUS (2) the mitotic figures have positive chromosome staining and negative nucleoplasm. A "speckled" pattern is anti-ENA-directed & is when (1) resting nuclei with intranuclear positive granules PLUS (2) mitoses with chromosome negativity and nucleoplasm positivity...a sort of morphological double-check. When this IFA screening test is positive (a "non-specific positive"), one must then use other tests and test methods to identify the specific auto-antibody positivity (which Ab in the group?) in order to determine if there is an actual, named, auto-immune-disease associated specific antibody present in the patient.

We now know that positive ANAs can precede the onset (by many years) of actual signs and symptoms of autoimmune disease. But, when a positive ANA is first detected in a person, an expert speculative estimate is that only one in 80 such situations will actually eventually become a case of auto-immune disease³, especially being a possible herald if titer is 1:120 or higher³.

situations associated with undetected levels of Ab:

• "negative" is failure of the test to react positively (non-reactive [NR]) at a standard 1:10 dilution of serum & could be (1) a true negative or (2) a false negative due to sample degradation or poor test reagents or test errors in a poorly run lab.
• "negative" may be where Ab has not yet risen to detectability; patient may have auto-immune disease but so early in its evolution that the antibodies are not detectible (so-called serologically sub-clinical disease)
• "negative" may be treated disease in which antibody level (titer) has dropped back below the reactive level of detectability.
• test may be negative, but patient might have other auto-immune antibody elevations which target some biological component other than cell nuclei
• Negative because no auto-immune disease

• a titer greater than 1:80 is positive.
• viral infections
• positivity due to some medications
• some normal, apparently perfectly healthy people who never manifest disease
• some presently normal, apparently perfectly healthy people who manifest disease years later
• some  types of hepatitis
  • 1:80 or higher in autoimmune hepatitis (AIH) (AJCP 114:705-711, 11/2000)
  • scantly speckled (0-6 dots) ANA with "dotted nuclear pattern" typical for primary biliary cirrhosis (PBC) & also seen in autoimmune & viral & liver diseases & rarely in collagen vascular & autoimmune disorders.
• rheumatoid arthritis
• Sjogren's syndrome
• polymyositis
• "musculoskeletal symptoms with positive ANA"
• scleroderma (systemic sclerosis)
• multiple sclerosis (MS)
• dermatomyositis
• SLE (systemic lupus erythematosus)
• discoid lupus
• MCTD (mixed connective tissue disease)

Other names for this exact or approximate agent are:   

• ANA, FANA, F-ANA

References:

1. Interpretation of Diagnostic Tests, Wallach, 2000, 7th Ed.
2. Arbuckle MR, James J, et. al., NEJM 349:1526-1533, 2003.
3. Judith James, MD, Oklahoma City, e-mail to JBC 15 Oct. 2004 (and the 11/04 LMC Lab issue of NewsPath.
4. Bradwell AR, et. al., Atlas of HEp-2 Patterns, 118 pages, 1995 (LML).

(posted about 2003; adjustments 25 June 2011)