Highlights of the AACC 2005 Issues in Immunodiagnostics Symposium

James D. Faix, MD

Disclosures

December 01, 2005

In This Article

Immunofluorescence Is the Traditional Way to Measure Antinuclear Antibodies

The assay for ANA is almost as old as RF if one considers the lupus erythematosus (LE) cell assay. Neutrophils would phagocytose blood cell nuclei released in vitro because of the presence of antichromatin antibodies. In the 1950s, the indirect immunofluorescent test was developed and continues to be performed today. Originally, frozen sections of mouse or rat liver were used as substrate and, after incubating with patient serum, any ANA binding to the nuclei was identified by fluorescein-labeled antihuman immunoglobulin. Using this technique, more than 95% of SLE patients were positive -- but the test was not specific, as variable percentages of patients with other rheumatic (and nonrheumatic) disorders were also positive. Sensitivity for SLE was increased to essentially 100% when cultured human cells were used as substrate,[5] especially after transfecting cells with the gene for an important autoantigen called Ro.[6]

Although patients with SLE could have any of the various classic patterns described, certain patterns, especially using cultured human cells such as the Hep-2 line, are characteristic of certain disorders (see Figure 2).

ANA patterns, autoantibody, and disease associations. Homogeneous (diffuse) pattern is the most common one seen because it is associated with antibodies to single-stranded DNA, which are present in a variety of disorders. The peripheral (or rim) pattern seen using mouse liver and considered specific for anti-double-stranded DNA is not seen using Hep-2 cells. Speckled patterns include both coarse and fine speckling as well as atypical speckling, including the pattern seen with anticentromere antibodies (characteristic of the limited variant of progressive systemic sclerosis). Anti-RNP, an autoantibody commonly seen in SLE, refers to an autoantibody to a ribonucleoprotein complex distinct from anti-Sm.

The most commonly seen is the "diffuse" pattern, but the pattern with the greatest diversity of associated autoantibodies is the "speckled" pattern. Antibodies to Sm, a protein complexed with RNA that is part of the apparatus that splices out introns not needed for protein coding, produce coarse speckling; these antibodies are specific for SLE but only present in 20%-30% of patients. Antibodies to Ro and La, part of another RNA-protein complex, produce fine speckling; these are markers of Sjögren's syndrome.

Atypical speckling patterns are seen with other marker autoantibodies. Antibodies to Jo-1 (an enzyme that links a transfer RNA with its appropriate amino acid during protein synthesis) cause coarse nuclear and cytoplasmic staining. Antibodies to Mi-2 (an enzyme involved in the termination of transcription) cause fine nuclear staining. Anti-Jo-1 and anti-Mi-2 are markers of polymyositis and dermatomyositis, respectively. One of the most important differentiations to be made involves progressive systemic sclerosis (PSS) or scleroderma. Patients with the diffuse form of PSS (who will develop visceral vascular inflammation and have a poor prognosis) may have antibodies to Scl-70 (an enzyme that helps unwind DNA during transcription). On the other hand, patients with the limited variant of PSS usually have antibodies to centromeres; this pattern is best seen in dividing cells as the fluorescent centromeres line up along the dividing plate.

In addition to pattern, the level of ANA present is important in considering the clinical significance of the positive result. Most laboratories screen specimens by diluting them 1:40 with saline. If there is no nuclear fluorescence at this dilution, the result is reported as "negative." If there is bright green fluorescence, the level of ANA present is usually determined by repeating the test (often, the next day) after serially diluting the specimen 1:40, 1:80, 1:160, etc. Each individual dilution is tested and examined under the fluorescent microscope. The dilution at which no nuclear fluorescence is present is considered the "titer" of antibody present. Titers greater than 1:160 are significant, and titers greater than 1:640 almost always indicate some serious rheumatic disorder.

A recent advance has been the use of fluorimetry to quantify the level of fluorescence present in the screening specimen and comparing this to known standards (see Figure 3).

ANA: titer vs direct quantitation. The traditional approach to quantitation of ANA involves serial dilution of the specimen and retesting. By determining the level of fluorescence present in the screening sample and comparing this to calibrators, the titer can be predicted.

This approach has been validated[7] and is as accurate as the traditional one. Furthermore, it standardizes the procedure. Indirect immunofluorescence is subjective, and determining the degree of "brightness" (and, hence, the titer of ANA) is subject to significant interindividual variation. Using a quantitative assay ensures that titers reported over time will be more reproducible when following individual patients.

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