Answer: To appreciate the characteristic myriad tiny dots of the AC-4a nuclear speckled pattern, one should carefully adjust the fine adjustment knob to notice the tiny dots that may be present at slightly different focal planes. The characteristics of AC-4a may not be evident at the screening 1/80 dilution and become clearer as the sample is further diluted. In general, the 400x magnification is appropriate for the identification of the AC-4a pattern characterized by innumerable tiny pinprick-like dots slightly variable in size. In addition, we noticed that AC-4a is more evident with certain HEp-2 slide brands (e.g. Bion-MBL, Aesku, Medipan, BIO-RAD, and Inova) than with others, and this may depend on distinct details of the cell culture and fixation methods applied by different manufacturers. Therefore, one needs to identify how the AC-4a pattern shows up in the particular HEp-2 slide brand in use in the laboratory. The availability of the IUIS/ASC reference serum IS2105 will also help laboratories to identify this AC-4a pattern efficiently. In summary, the best tip for identifying AC4a is to have enough time to devote to reading using a good microscope and not just relying on an automatic reading system.

References

Rober N, Dellavance A, Ingenito F, Reimer ML, Carballo OG, Conrad K, Chan EKL, Andrade LEC (2021) Strong association of the myriad discrete speckled nuclear pattern with anti-SS-A/Ro60 antibodies: consensus experience of four international expert centers. Front Immunol 12:730102. DOI: 10.3389/fimmu.2021.730102

Dellavance A, Alvarenga RR, Rodrigues SH, Barbosa SH, Camilo AC, Shiguedomi HS, Rodrigues SS, Silva CG, Andrade LE (2013) Autoantibodies to 60kDa SS-A/Ro yield a specific nuclear myriad discrete fine speckled immunofluorescence pattern. J Immunol Methods 390:35-40. DOI: 10.1016/j.jim.2013.01.006

Answer: This is a phenomenon that not many people are aware of, but it may occur in some practice. Some samples at low dilution yield an apparently negative HEp-2 IFA result and as we dilute the sample there is a progressive appearance of a nice and defined reactivity. This does not mean we should titer every sample with a negative result at 1/80, but it may be worthwhile to give it a try for those that produce a bright fuzzy staining (no defined pattern) at 1/80. Failure to be aware of this potential problem may account for false-negative results in some cases.

Anti-Ro52 antibodies (also known as anti-TRIM21) characteristically do not produce a distinctive staining pattern on commercially prepared HEp-2 cells using the indirect immunofluorescence assay (HEp-2 IFA). They also do not show reactivity in double immunodiffusion or classical radioimmunoprecipitation assays. One explanation put forth is that these autoantibodies recognize predominantly denatured epitopes in the middle leucine zipper domain, which are poorly available in these assays. Consistent with this hypothesis is that anti-Ro52 are readily detected in immunoblot, ELISA and chemiluminescent immunoassay (CLIA) and bead-based immunoassays. However, it has also been reported that antibodies to "native" Ro52 were detected in ~50% of anti-Ro52 positive sera using an N-terminal Ring finger domain in an in vitro translation and immunoprecipitation assay (1). Whether the native epitope is blocked for antibody access in other immunoassays is unclear.

If your anti-Ro52 patient sample presents any reactivity in HEp-2 IFA, it probably contains additional autoantibodies against antigens other than anti-Ro52. In other words, samples with exclusive anti-Ro52 reactivity tend to be negative in HEp-2 IFA.

1.         Buyon JP, Slade SG, Reveille JD, Hamel JC, Chan EKL. Autoantibody responses to the "native" 52-kDa SS-A/Ro protein in neonatal lupus syndromes, systemic lupus erythematosus, and Sjogren's syndrome. J Immunol. 1994 Apr 1;152:3675-84.

There may be some significant variations in HEp-2 immunofluorescence assay (IFA) staining depending on the manufacturer of HEp-2 cell slides employed. Some autoantigens and epitopes may not be available in some cell preparations. To ensure that the HEp-2 slide in use can detect anti-Ro60/SS-A and anti-La/SS-B staining, appropriate reference materials, such as those from www.AutoAb.org, should be used to verify the HEp-2 slide and other reagents employed in the assay. Experts in the field also advise to have low titer positive controls to test each new batch of HEp-2 slides for appropriate sensitivity. If well-defined standards for anti-Ro60/SSA and anti-La/SS-B do not show nuclear staining, this is a clear indication that the IFA has not been optimized for those autoantigens. The Ro60 autoantigen, in particular, seems to be labile and can be extracted by mild solvents and even prolonged exposure to some buffers.

 Anti-La/SS-B - in general, high titer positive anti-La/SS-B sera as determined by solid phase assay (SPA) are expected to be positive in HEp-2 IFA. It is unlikely that a high titer anti-La/SS-B serum would be negative in HEp-2 cell staining. A caveat is that monospecific anti-La/SS-B sera are very rare and hence any IFA staining may be related to a second antibody such as anti-Ro60/SS-A.

 Anti-Ro60/SS-A - there are reports that anti-Ro60/SS-A sera positive by SPA may be negative by HEp-2 IFA. Anti-Ro60/SS-A normally gives AC-4 nuclear speckled staining, but in certain commercially available HEp-2 slides, anti-Ro60/SS-A may be negative. One manufacturer provides HEp-2 cell slides that overexpress the Ro60/SS-A antigen, a substrate that reportedly has higher sensitivity and specificity to detect this antibody than conventional HEp-2 substrates. In contrast, the Ro52 (TRIM21) autoantibody is not regularly recognized in the HEp-2 IFA test. Thus, a monospecific anti-Ro52/TRIM21 serum may have a completely negative HEp-2 IFA.