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.
Date: January 28, 2019