These images are provided as examples of what are considered HEp-2 IFA-negative as viewed with an immunofluorescence microscope. Since a negative HEp-2 IFA can be represented by different non-specific and ill-defined fluorescence images, it should be clear that these AC-0 examples [1] should not be regarded as the entire array of possibilities, but rather representative examples only. The guiding features that link these images are the absence of clear-cut staining of any subcellular structure(s) and the dim and ill-defined nature of the fluorescent signal. This definition is both subjective and semi-quantitative.

In viewing these AC-0 images, it is important to note that the viewing screen default brightness and contrast settings may vary from computer (device) to computer (device) and adjustments may be necessary for appropriate interpretation. For example, for computers with two screens, the brightness and contrast between both screens is sometimes not synchronized, which can be attributed to image variations on a single computer. These AC-0 images were selected to show a marginal definition of cells, including cells in metaphase. If one cannot see the cells, the screen brightness is likely set too dark. On the other hand, if these images show too much green staining, please consider that the screen brightness may be set too bright. In summary, viewers of these images should make appropriate adjustments to their viewing screen brightness settings.

Note that with the Computer-Aided Diagnosis microscope systems becoming common in many clinical laboratories, the setting for AC-0 needs to be considered accordingly as it is often not reflecting the equivalent in the traditional microscope.

As to how HEp-2 IFA-positive vs -negative cut-off is determined, there is consensus that cut-offs should be determined experimentally and locally using apparently healthy and age-relevant population controls. The cut-off is highly dependent on the HEp-2 substrate used by individual laboratories, including factors specific to different HEp-2 slide manufacturers, lot-to-lot variations, fluorochrome-conjugated secondary antibody reagents, microscope optical system and light power, camera and viewing monitor settings, serum screening dilutions, and other variables.

The absolute screening dilution used for HEp-2 IFA is not universally agreed upon [2]. It is recommended that a 1:160 dilution should be used by laboratories testing samples with low pre-test probability (i.e., general population screening) and 1:80 dilution for samples with high pre-test probability (i.e., patients with a strong clinical suspicion of autoimmune disease). To maintain transparency and comparability, it is also strongly recommended that the final HEp-2 IFA report includes the specific screening dilution used [3].

Also note that the AC-0 designation infers no clinically relevant patterns are observed. AC-0 does not necessarily mean the absence of autoantibodies and/or autoimmune disease because sometimes the autoantibodies in a particular sample do not recognize epitopes of the target autoantigen as expressed in the HEp-2 cell. This is particularly relevant for antibodies against TRIM21/Ro52 [4], TROVE2/Ro60 [4], tRNA synthetases (i.e., Jo-1, PL-7, PL-12, etc), and ribosomal P. Also note that AC-0 should not be confused with AC-XX (unclassified AC- pattern).