Autoimmune Disease and the Laboratory

[Guest guest]

Publication: ADVANCE for Medical Laboratory Professionals

Issue Date: 10/4/1999

Search String: HEPATITIS

http://www.advanceformlp.com/editorial/mt/10-4-1999/p14.html?pub=ADVANCE+for

+M

edical+Laboratory+Professionals & issuedate=10%2F4%2F1999 & searchstring=HEPATIT

IS

----------------------------------------------------------------------------

--

--

Autoimmune Disease and the Laboratory

ANA, Other Diagnostic Tests Helpful but Not Perfect

By Weaver, MD

Many of the different inflammatory diseases treated by doctors today have as

a cause an abnormality of the immune system. Immune factors have been

implicated in the pathogenesis of an increasing number of illnesses. Some

examples of autoimmune disease include rheumatoid arthritis, systemic lupus

erythematosus (SLE) and inflammation of the blood vessels, which is known as

vasculitis.

The immune system is a complex interaction of different cell types, cell

receptors and soluble cell mediators. This intricate system is designed to

protect the host from microorganisms, but in autoimmune diseases seems to

react against the host itself.

Because manifestations of autoimmune disease can be very nonspecific, the

diagnosis is often difficult. Several different laboratory tests are used to

identify autoimmune disease. They in-clude antinuclear antibodies (ANA),

rheumatoid factors (RF), erythrocyte sedimentation rates (ESR) or C reactive

protein (CRP), and anticytoplasmic nuclear antibodies (ANCA).

Antinuclear Antibody Assay

The ANA is one of the most commonly ordered rheumatologic tests.

Immunofluorescent techniques are used, utilizing HEp2 cells that are human

epithelial cells in most laboratories. HEp2 cells provide some advantages

over murine tissue, which was previously used. They provide a standard

substrate and also have better sensitivity for certain antigens, such as Ro

and centromere. Titers with Hep2 cells also are generally higher than from

those measured with murine substrates. After serum from the patient is

added,

antibodies to nuclear antigens will stick to the nuclei of the cell. An

antibody to human IgG, which is conjugated to a fluorescein tag, is added. A

light source then excites fluorescein to fluoresce.

When the slide is viewed under the microscope, the antinuclear antibodies

can

be identified by their green fluorescence. The pattern of staining and

dilution at which the fluorescence disappears is noted.

The ANA is then reported by both titer and pattern. In most labs, a titer of

less than 1:40 is considered to be a negative test. The titer is important

because a low titer or borderline positive test is less likely to be due to

rheumatic disease and more likely to be a false positive test or due to

other

non-rheumatic causes.

A positive ANA also can be seen with advanced age, thyroid disease, chronic

infection, recent viral infection or even after recent vaccination for

influenza. Positive ANAs also can be present in a normal, healthy

population.

In one study where 125 healthy individuals were tested, 32 percent of the

samples tested positive in a titer of greater than 1:40, 13 percent had

titers greater than 1:80, and 3 percent were greater than 1:320.1

Autoimmune diseases that are limited to one organ also can produce a

positive

ANA. Examples of this include autoimmune hepatitis, Hashimoto's thyroiditis,

Grave's disease or primary biliary cirrhosis.

A very high titer ANA can be helpful as well in identifying certain

diseases.

Titers of greater than 1:640 suggest an underlying autoimmune disorder. On

the other hand, a low titer positive ANA in the absence of any other signs

or

symptoms of rheumatic disease is unlikely to be associated with autoimmune

disease.

The pattern of the ANA can also help to identify which nuclear antigens and,

therefore, which rheumatic disease are most likely to be present. A

homogeneous or diffuse pattern is produced by antibodies to the DNA-histone

complex, also referred to as the nucleosome. These antibodies are believed

to

be responsible for the LE cell phenomenon. A peripheral or rim pattern is

associated with double-stranded DNA (DS DNA) antibodies. The speckled

pattern

is produced by antibodies to (Sm), ribonucleoprotein (RNP), Ro/SSA,

La/SSB, Scl-70, centromere, pericytoplasmic nuclear antigen (PCNA), as well

as other antigens. An anticentromere pattern is produced by antibodies to

the

centromere. A nucleolar pattern is associated with fibillarin, nucleolin,

nucleophosmin and RNA polmerase I.

Different serum dilutions can produce different nuclear patterns. One

nuclear

pattern can also obscure another pattern if two or more patterns are

simultaneously present. For instance, a diffuse pattern may overlay a

speckled or rim pattern and make this second pattern difficult to identify.

Some antibodies, such as the anticentromere antibody, can be identified from

the ANA alone. Other specific antibodies require further testing with ELISA,

immunodiffusion or immunoblotting. Several different diseases can produce a

similar nuclear pattern, so antibody testing for specific antinuclear

antigens provides a more specific and sensitive testing for autoimmune

disease (see Table 1).

Moderate to high titers of antibodies directed toward DS DNA or Sm are very

specific for SLE. Titers to DS DNA can also fluctuate with disease activity

in SLE.2

Antibodies to RNP are associated with mixed connective tissue disease, an

autoimmune disease with overlap features of SLE, inflammatory myositis and

scleroderma. Antibodies to centromere are seen with CREST syndrome, which is

manifested by skin hardening over the fingers, Raynaud's phenomenon,

esophageal problems, telangiectasias and calcium deposits over the

fingertips. Scl-70 (topoisomerase I, and enzyme involved in DNA replication)

antibodies are associated with scleroderma, which causes progressive skin

hardening and also may affect multiple organs such as kidneys or lungs.

Rheumatoid Factors

The RF is also a commonly performed test in patients who present with

symptoms suggestive of an autoimmune process. The disease process that this

test is most often seen with is rheumatoid arthritis, although it can occur

in other rheumatic diseases as well. A RF is an antibody to the Fc portion

of

IgG.

The origin of the RF is not completely understood. Due to an unknown

antigenic stimulation, an abnormal immune response occurs, resulting in

production of antibodies against the IgG antibody. Just as the ANA is not

specific only for certain rheumatic disease, the RF also is seen in a

variety

of disease processes. A positive RF is seen in about two-thirds of patients

with rheumatoid arthritis. It can be seen in other rheumatic disorders such

as Sjögren's syndrome, which causes chronic dry eyes and mouth, SLE,

inflammatory myositis and mixed cryoglobulinemia. Chronic infections such as

subacute bacterial endocarditis, hepatitis B or C, malignancy and

inflammatory pulmonary disorders also may be associated with a positive RF.

Five percent of young, healthy individuals may have a positive RF. Incidence

in elderly subjects without rheumatic disease ranges from 3 percent to 25

percent.3 In healthy individuals, the titer is generally low to moderate,

ranging from 1:40 to 1:160. The positive predictive value, the likelihood of

having a rheumatic disease if the test is positive, in the general

population

is low.

In consecutive tests performed on patients admitted to the Beth Israel

Hospital, the positive predictive value for rheumatoid arthritis was 24

percent and only 34 percent for any rheumatic disease.4 In patients with

rheumatoid arthritis, a positive RF can help to confirm the diagnosis and

also can identify patients who are more likely to have severe, erosive

disease or extraarticular manifestations.

RF is detected by a variety of techniques such as agglutination of

IgG-sensitized sheep red cells, bentonite or latex particles coated with

human IgG, radioimmunoassay, ELISA or nephelometry. Measure-ment of RF is

not

standardized in many laboratories and no one technique seems to have any

clear advantage over others. The RF tested for in clinical practice is IgM

RF, although other immunoglobulin types such as IgG and IgA also can occur.

In summary, a variety of tests are used to help identify, diagnose and treat

autoimmune or rheumatic diseases. And as can be seen from this discussion,

these tests are somewhat imperfect. A positive test can be seen in a patient

who does not have a rheumatic disease or who has no disease at all.

The reverse is also true. A small number of patients with a rheumatic

disease

also may not test positive.

In diagnosing autoimmune diseases, the clinical findings such as the

patient's symptoms and exam findings always must be considered before a

diagnosis can be made. Laboratory findings can help to predict prognosis and

support or confirm a diagnosis, but by themselves are not diagnostic in

these

diseases.

References

1. Tan, E.M., Feltkamp, T.E., Smolen, J.S., et al. Range of antinuclear

antibodies in 'Healthy' individuals. Arthritis Rheum. 1997; 40:1601.

2. Hahn, B.H. Mechanisms of disease: Antibodies to DNA. N. Engl. J. Med.

1998; 338:1359.

3. Litwin, S.D., Singer, J.M. Studies of the incidence and significance of

anti-gamma globulin factors in the aged. Arthritis Rheum. 1965; 8:538.

4. Shmerling, R.H., Delbanco, T.L. How useful is the rheumatoid factor? An

analysis of sensitivity, specificity and predictive value. Arch. Intern.

Med.

1992: 152:2417.

Dr. Weaver is a rheumatologist in private practice in Rapid City, SD, and an

associate clinical professor with the University of South Dakota Medical

School. She and her practice partner have been involved in many clinical

investigations of new rheumatic drugs.

Autoimmune Testing--Related Websites

GDS Links (click on links)

http://globaldx.com/

Immunopathology (Picture) Index

http://www.dml.georgetown.edu/webpath/IMMHTML/IMMIDX.html

Autoimmunity

http://www.igenex.com/autoopt2.htm

--------------------