Homogeneous and regular fluorescence across all nucleoplasm. The nucleoli maybe stained or not stained depending on cell substrate. Mitotic cells (metaphase, anaphase, and telophase) have the chromatin mass intensely stained in a homogeneous hyaline fashion.

Speckled pattern distributed throughout the interphase nucleus with characteristic heterogeneity in the size, brightness and distribution of the speckles. Throughout the interphase nucleus, there are some denser and looser areas of speckles (very characteristic feature). The metaphase plate depicts strong speckled pattern with some coarse speckles standing out.

Discrete coarse speckles (40-80/cell) scattered in interphase cells and aligned at the chromatin mass on mitotic cells. e.g. anti-CENP-B                        

Fine tiny speckles across all nucleoplasm. The nucleoli may be stained or not stained. Mitotic cells (metaphase, anaphase, and telophase) have the chromatin mass not stained. e.g. anti-SS-A/Ro, anti-SS-B/La     

Coarse speckles across all nucleoplasm. The nucleoli may be stained or not stained. Mitotic cells (metaphase, anaphase, and telophase) have the chromatin mass not stained. e.g. anti-Sm, anti-U1 RNP                        

Countable discrete nuclear speckles (6 to 20 nuclear dots/cell). e.g. SP-100                        

Countable discrete speckles (1 to 6 nuclear dots/cell in most cells). These are known as Cajal bodies or coiled bodies. e.g. anti-p80-coilin                        

Diffuse fluorescence of the entire nucleolus, while the metaphase plate shows no staining. e.g. anti-PM-Scl, anti-Th/To.                        

 Irregular staining of the nucleoli and Cajal bodies with a peri-chromosomal staining at the metaphase plates. e.g. anti-fibrillarin.                        

Densely distributed but distinct grains seen in the nucleoli of interphase cells. In metaphase cells, up to 5 bright pairs of the nucleolar organizer regions (NOR) can be seen within the chromatin body. The cytoplasm of mitotic cells may be slightly positive. e.g. anti-NOR-90, anti-RNA polymerase I

Homogeneous staining of the nucleus with greater intensity at its outer rim and no staining at the metaphase and anaphase chromatin plates. There is a peculiar accentuation of the fluorescence at the points where adjacent cells touch each other. e.g. anti-lamin B.

Nuclear envelope reveals a punctate staining in interphase cells, with accentuation of fluorescence at the points where adjacent cells touch each other. No staining of the metaphase and anaphase chromatin plates. e.g. anti-gp210.

Pleomorphic speckled nucleoplasmic staining, with variability in size and brightness of the speckles. In interphase, some cells are negative (G1 phase), some are intensely stained (S-phase) and some present rare and scattered speckles with occasional nucleolar staining (late S and early G2 phases). Mitotic cells are not stained.

Nuclear speckled pattern with striking variability in intensity with the strongest staining in G2 phase and weakest/negative staining in G1. The centromeres are positive only in prometaphase and metaphase, revealing multiple aligned small and faint dots. Prometaphase cells frequently show a weak staining of the nuclear envelope. During anaphase and telophase, some sera demonstrate intense staining in the ring located at the midzone (i.e. mid-body, stem body) where the division of the daughter cells is taking place. The surrounding cytoplasm of the mitotic cells is diffusely stained.

The Topo I-like pattern can comprise staining of five subcellular regions:

This 5-element compound staining pattern has been observed in most commercial HEp-2 cell slides, but there may be some variations in the expression of each element according to the slide brand. The detection of all 5 elements may be a challenge especially when only using a single serum dilution (e.g. strong mitotic chromatin staining obscures NOR) or in many semi-automated systems when images are often selected on a single focal plane (e.g. NOR or cytoplasmic staining not in same focal plane as interphase nuclei).

Practical recommendation how to routinely screen for AC-29 with a HEp-2 slide that show this pattern using a traditional microscope setting: if the above 1) and 2) features are observed on routine samples suggestive of the AC-29, the next step should be to look for 3) positive NOR staining by searching different focal planes for NORs on mitotic chromatins. Next, the presence of the 4) cytoplasmic staining and lastly 5) the nucleolar staining should be evaluated. In some HEp-2 slides, the nucleolar staining is only visible near the edge of the well.

References

Dellavance A, Gallindo C, Soares MG, da Silva NP, Mortara RA, Andrade LE. Redefining the Scl-70 indirect immunofluorescence pattern: autoantibodies to DNA topoisomerase I yield a specific compound immunofluorescence pattern. Rheumatology (Oxford). 2009;48:632-7.

Andrade LEC, Klotz W, Herold M, Conrad K, Ronnelid J, Fritzler MJ, von Muhlen CA, Satoh M, Damoiseaux J, de Melo Cruvinel W, Chan EKL. International consensus on antinuclear antibody patterns: definition of the AC-29 pattern associated with antibodies to DNA topoisomerase I. Clin Chem Lab Med. 2018;56:1783-8.

INOVA HEp-2 images illustrating the AC-29 pattern with staining in all 5 compartments. Panel A is a merged image from the other 3 panels (A', A'', A''') representing different optical sections/focal planes, each illustrating the unique stained structures not obvious in the other focal planes.  In addition to the obvious nucleoplasmic and mitotic condensed chromatin staining, panel A' illustrates two bright NORs in focus (arrow) on condensed chromatin in the mitotic cell; panel A'' shows another NOR in focus (arrow) in the same cell; A''' shows characteristic cytoplasmic (arrowhead) and weak perinucleolar staining (short arrow).