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.

It is necessary that your microscope settings and your HEp-2 slides be assessed for the ability to display the AC-29 pattern as described by ICAP.  Some of the elements of the AC-29 patterns may not be readily identifiable. While the staining of the nucleoplasm and metaphase plate is very evident, the staining of the nucleolar organizer region (NOR), the nucleolus, and the cytoplasm requires explanation. For the NOR staining, it is important that different focal levels are analyzed by slowly adjusting the micrometer knob on the microscope up and down. The cytoplasmic staining may not be readily visible at lower serum dilutions and may be only apparent at higher dilutions. The staining of the nucleolus can be more inconsistent and varies considerably according to the HEp-2 slide brand/lot and other inter-manufacturer variables.

Using the reference standard for anti-Topo I (CAT#: IS2135. ANA #09) from the Autoantibody Standardization Committee (ASC), one should be able to identify all five subcellular regions. Otherwise, troubleshooting should consider adjustments to your microscope settings, the HEp-2 slides and/or reagents used.

The specificity of the definition of AC-29 pattern for anti-Topo I is more robust if all five elements are identified. If only the nucleolar staining is not observed, it is still acceptable to classify the sample as AC-29. The absence of any of the other four elements is incompatible with the AC-29 pattern provided that the lab can routinely observe the 5 elements using the ASC or other anti-Topo I internal standards.

Finally, AC-29 is likely a problem for automated IFA microscopic systems with a lower numerical aperture and shallow depth of field because AC-29 pattern recognition requires examination at different focal planes.