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Detection of Anti-Nuclear Antibodies and Rodent Organ Sections
The term "anti nuclear antibodies" (ANA) describes a variety of autoantibodies that react with constituents of a cell's nuclei including DNA, RNA and several proteins and ribonucleoproteins.¹ These autoantibodies occur with high frequency in patients with connective tissue or rheumatic diseases, especially systemic lupus erythematosus (SLE). In fact, virtually all SLE patients are ANA positive. This diagnostic sensitivity has led to the incorporation of ANA testing into the 1982 Revised Criteria for the Classification of Systemic Lupus Erythematosus by an American College of Rheumatology subcommittee.² While ANA testing is an excellent screening test for SLE (a negative result virtually rules out active SLE³), it is by no means a specific test.

Patients with other connective tissue diseases such as rheumatoid arthritis, scleroderma and dermatomyositis are frequently positive, and low ANA titers may also be observed in other disease states and they can even be found in the normal population.⁴ Positive ANA results can occur following severe burns or viral infection and have been reported in some normal, healthy people, especially in older individuals. Because of this lack of specificity, it is recommended that all ANA positive samples be titered to endpoint and that more specific testing for autoantibodies to double stranded DNA (dsDNA) and extractable nuclear antigen (ENA) autoantibodies be performed.^1-3

Indirect immunofluorescence is the recognized reference method for ANA testing. Common substrates include thin sections of rodent organs or various types of cell lines such as HEp-2 are commonly utilized. It is generally agreed that cell line substrates are preferable to organ sections since these rapidly dividing cells have higher levels of certain clinically relevant antigens such as centromere, SS A(Ro), Scl 70 and PCNA/Cyclin. However, rodent organ sections do have their place. This is certainly the case when looking for anti-mitochondrial antibodies (AMA), which are found throughout the cytoplasm, anti-smooth muscle antibodies (ASMA), which are found primarily in the lining of veins and especially in the arteries, and anti-gastric partial antibodies (GPA), which are found in stomach tissue. Detection of autoantibodies using cell line substrates can be tricky because AMA antibodies can be confused with the various speckled patterns that can be present with cell lines, ASMA reactivity is difficult to visualize on a cell line and GPA cannot be seen at all with a cell line substrate.

High levels of AMA are often detected in association with primary biliary cirrhosis, while low titers of AMA may be detected in other liver disorders such as chronic active hepatitis and cryptogenic cirrhosis.^5,6 ASMA is found in high titers in the serum of 70% of patients with chronic active hepatitis. In addition, 50% of these patients are also positive for ANA and 25% demonstrate low AMA titers. Low ASMA titers may be present in viral infections, malignancies and normal individuals.^5,7

GPA occurs in the serum of 90% of patients with pernicious anemia. Along with other clinical and laboratory data, a positive GPA result helps to distinguish autoimmune pernicious anemia from other megaloblastic anemias. Although detected in less than 2% of the normal population under 20 years of age, the incidence of GPA increases in women over the age of 40 and may be present in up to 16% of the normal population over 60 years of age.^8,9

1. Tan EM: Autoantibodies to nuclear antigens (ANA): Their immunobiology and medicine. Advances in Immunology 33:167 239, 1982.

2. Tan EM, et al.: The 1982 Revised criteria for the classification of systemic lupus erythematosus. Arthritis and Rheumatism 25:1271 1277, 1982.

3. Casalo SP, Friou GJ, Myers LL: Significance of antibody to DNA in systemic lupus erythematosus. Arthritis and Rheumatism 7:379 390, 1964.

4. Wiik A: Antinuclear factors in sera from healthy blood donors. Acta Path Microbiol Scand. 84:215 220, 1976.

5. Peter JB, Dawkins RL: Evaluating autoimmune diseases. Diagnostic Medicine: 68 76, September October, 1979.

6. Doniach D, Roitt IM, Walker JG, Sherlock S: Tissue antibodies in primary biliary cirrhosis, active chronic (lupoid) hepatitis, cryptogenic cirrhosis and other liver diseases and their clinical implications. Clinical Experimental Immunology 1:237 262, 1966.

7. Toh BH: Smooth muscle autoantibodies and autoantigens. Clinical Experimental Immunology 38:621 628, 1979.

8. Cavallaro JJ, Palmen DF, Bigazzi PE: Immunofluorescent detection of Autoimmune Diseases, Immunology Series No. 7. Center for Disease Control, Atlanta GA, 1976.

9. Loveridge N, Bitensky L, Chayen J, Hausamen TU, Fisher JM, Taylor KB, Gardner JD, Bottazzo GF, Doniach D: Inhibition of parietal cell function by human gammaglobulin containing gastric parietal cell antibodies. Clinical and Experimental Immunology 41:264 270, 1980.