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.

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.

With all things being equal, same slide manufacturer with same slide lots, same conjugate, and same serum dilution to ensure there are no technical issues, HEp-2 IFA patterns can change over time. This may include a change from no detectable pattern (AC-0) to any AC pattern and vice-versa. Many factors such as disease progression or overlap syndrome, new interventions (drugs, biologicals), new exposures (infectious disease, cancer, etc.) can also affect such changes. It is important to know if the end-point titer also changed (increased or decreased by at least 2 dilutions).
What is the immunological basis for changes in HEp-2 IFA status along the course of SLE? Autoantibody levels do fluctuate along the disease course. Some of them (i.e., native DNA and nucleosome, for example) may fluctuate in relation to disease activity, while others (i.e., anti-Ro60 and anti-Sm) appear to fluctuate independently of disease activity.
Can HEp-2 IFA temporal changes be related to disease activity? Most people would say “no”, but the answer is not so simple. A recent study from the Andrade lab evaluated 269 SLE patients regarding their HEp-2 IFA titer and pattern in a transverse (active x intermediary x inactive disease activity) and prospective (change in disease activity status in the same patient along 1-year follow-up) (1). The report showed that patterns and titer change can change and there was some association with disease activity status. High titer and the AC-1 pattern were associated with active disease, whereas low titer and the AC-4 pattern were associated with disease remission. Interestingly, the diagnostic performance (ROC curve AUC) of HEp-2 titer for the definition of disease activity was in the range of traditional disease activity biomarkers (anti-dsDNA, anti-nucleosome) and much greater than C3 and C4. However, the HEp-2 IFA is not recommended as a test for monitoring SLE disease activity. Similar conclusions may apply to other ANA-related rheumatic diseases.
Thus, coming back to the index case, the change from AC-5 to AC-1 may indicate the appearance of anti-nucleosome and/or anti-dsDNA antibodies. It is recommended to be aware of these autoantibodies and possible disease flare.
1. Prado MS, Dellavance A, Rodrigues SH, Marvulle V, Andrade LEC (2020) Changes in the result of antinuclear antibody immunofluorescence assay on HEp-2 cells reflect disease activity status in systemic lupus erythematosus. Clin Chem Lab Med 58:1271-1281. DOI: 10.1515/cclm-2019-0638