Recognizing mitotic cells and the metaphase plate is an important step in learning to read and interpret HEp-2 IFA. The metaphase plate is like a special place in a cell where all the chromosomes line up perfectly along the middle, making sure everything is ready for the cell to divide, a phenomenon that occurs during the metaphase stage of the cell cycle. Therefore, the metaphase plate primarily contains the autoantigens that belong to chromosomes. The first step is to identify the cells in mitosis. If you use a conjugate that contains DAPI or Hoechst for DNA counterstain, you should be able to recognize metaphase cells using the appropriate filter, as shown in Figure 1.

Alternatively, you can train yourself in the recognition of cells in metaphase using well-characterized serum samples. For example, select a sample with anti-double-stranded DNA antibodies that produce a clear-cut homogeneous nuclear pattern (AC-1). You will see that the majority of cells will exhibit homogeneous staining within the nucleus during the interphase. Cells in metaphase, however, are less common and you might see only one in every 40X view. These cells look different because they have a specific intense staining of the metaphase plate in absence of staining of the rest of the cell's components. This staining phenomenon of the homogeneous pattern arises due to the integral presence of DNA within the chromosomes. In contrast, a sample with anti-U1RNP antibodies should yield the coarse speckled nuclear pattern (AC-5) in the interphase cells, whereas the metaphase cells present a dark transversal band (not stained) at the cell’s "equator". This is the negative metaphase plate (not stained), because U1RNP is not a chromosomal component. Figure 2 clearly illustrates positive and negative metaphase plates.

It is not uncommon that ANA (or HEp-2 IFA) positivity can change over time. This may be especially true when the first test showed weak reactivity, as it was in this case. Such fluctuations between positive and negative are common in cases with weak reactivity and can simply be the result of the variability of the IFA technique itself. The serum levels of anti-SS-A/Ro antibodies can also fluctuate over time (1).

It should be noted that anti-SS-A/Ro often includes two distinct autoantibodies that recognize 60kD (Ro60) and 52kD (Ro52) antigens. To ensure reactivity is detected in a sensitive manner, ideally solid phase specific assays for Ro52 and Ro60 should be included, next to HEp-2 IFA to complete the analysis.

The other issue relevant to the result for anti-SS-A/Ro that was positive last year is its clinical significance. Anti-SS-A/Ro antibodies occur in diseases, such as lupus and Sjögren´s syndrome. The prevalence of anti-Ro52 autoantibodies is similar to that of anti-Ro60 in every connective tissue disease cohort except for myositis, where anti-Ro52 is common but not anti-Ro60. Occasionally, anti-SS-A/Ro may also occur in people with no apparent disease.

It is very important to keep in mind that the autoantibodies, by themselves, are not sufficient for the diagnosis of any disease. It is important that the physician match the results of the autoantibody tests with the entire clinical picture of the patient to come to an appropriate diagnosis.

1.Faria AC, Barcellos KSA, Andrade LEC: Longitudinal oscillation of antibodies to extractable nuclear antigens in systemic lupus erythematosus. J Rheumatol 32: 1267-72, 2005.

The term "nuclear speckled" is an overarching competent- HEp-2 IFA pattern that includes both fine speckled (AC-4) and large/coarse speckled (AC-5). In some cases, it can be difficult to define precisely the speckled pattern a sample produces, being either the fine or large/coarse speckled pattern. Obviously, one may use the ICAP competent level terminology AC-4/5. In such case, one may combine the antibody targets and clinical relevance of the individual patterns, i.e., AC-4 and AC-5. Using the competent level AC-4/5 term may also be appropriate in mixed patterns caused by more than one autoantibody specificity in the sample.
Ideally, one should provide the most complete definition of the pattern (expert level), because these provide more specific information on antigenic associations and clinical relevance. For example, as indicated in the "antigen association section" of ICAP website, the nuclear coarse speckled pattern (AC-5) is associated mainly with anti-Sm and anti-U1-RNP antibodies, whereas the nuclear fine speckled pattern (AC-4) has a broader target antigen association. In the ICAP Note #2, AC-4 can be further divided into fine speckled with myriad discrete tiny dots (AC-4a), or simple fine speckled (AC-4b). These are defined patterns and have respective antibody targets, immunological associations, and clinical relevance. The nuclear dense fine speckled pattern (AC-2), on the other hand, should be distinguished from the nuclear speckled patterns AC-4 and AC-5 based on the positive staining of the metaphase plate. With exception of the centromeric pattern (AC-3), the clinical relevance of all patterns is only indicated if the antibody target has been identified.