Anti-Nuclear Antibody (ANA), Serum Test
INTRODUCTION: A POSITIVE TEST DOES NOT, ALONE, DIAGNOSE ANY DISEASE! Our protective serum antibodies search for various protein antigens and may attach (have an antigen-antibody reaction) to any that they find. In some cases, the antibodies may actually attack our own body proteins. If they do, they have become a part of an "autoimmune disease process"...of which there are a wide variety. Specialists dealing with these diseases are known as Rheumatologists (whose association, the ACR, organizes a lot of the thinking about these diseases and the tests for them).
IMPORTANT: the ANA test, regardless of the test method, is a generic 5 test with highly restricted specificity. That is, a "positive" test might mean something important or...at that point in time...be meaningless. More has to be done to discern the meaning of that ANA test result!
PUNCH LINE DECISION TREE/chart of possible disorders suggested by your f-ANA test result by itself (specific "positive" and "negative" have not yet been established!). There may be more testing pending or yet to come!
My e-mail layman explanation of the ANA test to a patient: Lisa:
(a) As to "self attacking self" (autoimmune attack): it does so in 3 possible ways = either with the person’s own (1) antibodies (chemical molecules) or (2) lymphocytes (cells) or (3) both. The fluorescent ANA test catches every possible cause of an ANA…even some things of as-yet unknown significance…because the target-containing cells (HEp-2 cells) contain every single antigen (antibody target). So, this test is often (but not everywhere) used as a first-step screening test. If positive, then some docs take at least one second step = ELISA test [or (d), below].
(b) The ELISA tests contain a very limited variety of antigen targets…just the targets that are considered most important.
(c) So if fluorescent ANA is positive but ELISA negative, then that is a piece of evidence that the “positivity” is NOT LIKELY important.
(d) Some (such as our Lexington system's rheumatologists) take the additional extra step and order the ds-DNA and the panel of 11 ENA tests (they order ds-DNA and ENAs). We may have an agreement to order these two by reflex...go to these two automatically. If those are negative, then that confirms the position taken in (c), and the patient will be “watched” as further testing and studies go on trying to figure out what might be wrong OTHER than “autoimmune”.
How test is officially done: All ANA tests must be performed on patient's diluted serum so as to dilute
out lots of nonspecific "reactivity" (our standard is a 1:40
dilution of serum with aqueous [water] diluent). The classical, visible-with-the-eye "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. That is, an educated human mind has to (1) determine at what dilution the test stopped "reacting" and (2) what pattern of reaction is observed (there may be as many as 20 nuclear or cytoplasmic "patterns"). Beginning in 2013, our lab became one of the first in the USA to work with an instrument which uses artificial intelligence to help assign the reactive (1) titer and (2) nuclear pattern (which must then be confirmed or corrected by the expert technologist and/or pathologist). [check INOVA's good over-view testing explanation, HERE].
What test is looking for: Considered a "global" ANA test, F-ANA usually looks
for ANAs in the IgG class (not in IgA, IgE, or IgM classes). If...in one test run...it could look for antibodies in all FOUR classes, it would be a truely global ANA test (checks every concievable type of ANA). However, in common lab lingo, it is considerred the "global ANA test". The test substrate which
provides the cells (cytoplasm & nuclei) is either made of histological sections of
tissue (histological cross-sections) or cell culture monolayer sheets
of cells 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.
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.
Official Interpretation conventions
Steps in "positive" vs. "negative": Step #1 is to see if there is an ANA "reaction" at a certain cut-off level or HIGHER. This step #1 is expressed in numbers so that the reaction can be mentally classified as weakly (1+) reactive (titer of 1:40-1:160), moderately (2+) reactive (1:320-1:640), or strongly (3+) reactive (higher than 1:640). Although positive titres of 1:160 or higher are strongly associated with autoimmune disorders, they are also found in 5% of healthy individuals (HERE). Step #2 = THEN, tests on the same serum sample must be performed for (1) the IgG antobody to double stranded DNA (ds-DNA) and (2) the Ig G antibodies to eleven (11) ENA antigens...we (in 2015) test by the blot method for both ds-DNA & the 11 ENAs (5 or less reaction units is negative; 6-10 units is borderline [(+)]; 11-25 units is mildly positive [1+]; 26-50 units is moderately positive [2+]; greater than 50 units is strongly [3+] positive).
When a screening ANA by any method is "positive", it ought
to be considered a non-specific positive (viral infections, medications, certain apparently normal persons,
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 protein components of cell nuclei (and sometimes to cytoplasmic components). The positive
ANAs which also have "specificity" for DNA (non-soluble)
or various protein ENAs (soluble, extractible nuclear
antigens] are the ones more likely to herald
or be associated with "lupus" or other autoimmune diseases listed
below). The ENAs are detected by "blot testing" and a densitometer "reads" the intensity of darkness of the test line and produces the number. "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 IgG indirect fluorescent test (IFA, F-ANA) in which patient's
antibody-containing serum is mixed with a human HEp-2 cell & 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 is evenly involving of all of nucleus.
- homogeneous with nuclear rim (homo/peripheral nuclear intensity)
- 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
endoplasmic reticulum (as LKM-1 in rodent tissue)
lysosomal (large irregular speckles)
peroxisomes (in polymyalgia type situations)
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
- "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
Causes of INCREASED Values/Levels (positive):
- a titer equal to or greater than 1:40 is "positive".
- positivity may be due to viral infections
- positivity may be due to some medications
- positive may be 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
- 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
- "musculoskeletal symptoms with positive
- scleroderma (systemic sclerosis)
- multiple sclerosis (MS)
- SLE (systemic lupus erythematosus)
- discoid lupus (DLE)
- MCTD (mixed connective tissue disease)
Other names for this exact or approximate agent are:
Interpretation of Diagnostic Tests, Wallach, 2000,
- Arbuckle MR, James J, et. al., NEJM 349:1526-1533, 2003.
- Judith James, MD, Oklahoma City, e-mail to JBC 15 Oct. 2004
(and the 11/04 LMC Lab issue of NewsPath.
- Bradwell AR, et. al., Atlas of HEp-2 Patterns, 118 pages, 1995
- Homberger Henry A. (of Mayo Clinic),"The Current Controversy Regarding Detection and Measurement of Antinuclear Antibodies", CAP meetiung course RT101, 11 Sept 2010.
(posted about 2003; adjustments 1 June 2015)