Frequently asked questions answer frequently asked questions

This is not an uncommon phenomenon and there are several possible explanations for this apparent discrepancy.

It is recognized that each immunoassay platform (IFA, WB, ELISA, immunoprecipitation (IP), etc.) is dependent upon a given set of epitopes of relevant autoantigens (1). Broadly, autoantibodies recognize linear and/or discontinuous (native or conformational) epitopes. In the context of discontinuous epitopes, it is important to appreciate that many target autoantigens are part of macromolecular complexes where unique epitopes might be represented as quaternary structures. Immunoassays where the epitope structures are best preserved include IP of cell lysates and HEp-2 IFA. However, there is an important caveat for HEp-2 IFA because the cells have been fixed in methanol and/or acetone (or other undisclosed ‘trade secret’ fixatives) that likely alter some epitopes as well as well as exposing others. As to the question of native epitope preservation, WB presents the largest challenge because the protocols of boiling proteins under deliberate denaturing conditions most likely alters epitopes significantly. It is true that some may renature/refold after electro-transfer of proteins to nitrocellulose membrane, but many most likely do not. When “antibodies by blot” are “not related to the pattern observed in HEp-2 cells by IFA,” one explanation is that antibodies to epitopes recognized in IFA are not identical to or represent only a proportion of those (or even different ones) recognized in a WB. One example for this is anti-fibrillarin; IFA typically shows an AC-9 pattern and yet negative in immunoblot because a high percentage of anti-fibrillarin antibodies do not recognize denatured fibrillarin in immunoblot.

It is common that a given serum sample (especially in SLE) has more than one autoantibody specificity. Hence, another explanation is that when two (or more) completely different antibody specificities co-exist in the same sera, confounding results can be observed, especially on the HEp-2 IFA. In that case, the HEp-2 IFA pattern may reflect the combination of IFA reactivity of all present autoantibodies and the resultant pattern may be different from the one expected if there were only one autoantibody specificity in the serum.

1.  Mahler M, Bluthner M, Pollard KM. Advances in B-cell epitope analysis of autoantigens in connective tissue diseases. Clin Immunol. 2003 May;107:65-79.

Date: August 14, 2020

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 vitrotranslation 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.

Date: June 18, 2020

This is an interesting question and reflects a challenge frequently encountered by ANA laboratories. At the present time, the “pseudo-DFS” nomenclature has not been endorsed by ICAP or included in the ICAP algorithm. Nevertheless, there is published evidence that not all sera having a nuclear “dense” fine speckled pattern with similarly stained mitotic chromatin contain detectable anti-DFS70 autoantibodies. This situation may reflect a HEp-2 IFA pattern that is distinct from the anti-DFS70 positive AC-2 pattern even though it stains the interphase nuclei and mitotic chromatin with a speckled texture. However, the speckled texture is slightly different from the genuine AC-2 pattern (1, 2). The anti-DFS70-positive AC-2 pattern has a speckled texture that is heterogeneous within each nucleus, with some denser speckled areas adjacent to sparser areas; and areas with larger and brighter speckles intercalated with areas with smaller and dimer speckles. The same applies to the staining of mitotic chromatin.  Some investigators have suggested the term “pseudo-DFS” to designate sera that do not have these precise AC-2 staining characteristics (1, 2), The Autoantibody Standardizing Committee offers free of charge reference sera with several HEp-2 IFA patterns and antibody specificities (http://www.autoab.org/), including a reference material for AC-2 pattern (with reactivity to DFS70) (3). This can help in the exact recognition of the AC-2 pattern. Note that the specific autoantibody target(s) and the clinical relevance for the pseudo-DFS pattern but anti-DFS70 negative remains to be published.

1.  Mahler M, Andrade LE, Casiano CA, Malyavantham K, Fritzler MJ. Anti-DFS70 antibodies: an update on our current understanding and their clinical usefulness. Expert Rev Clin Immunol. 2019;15:241-250.
2.  Infantino M, Bizzaro N, Grossi V, Manfredi M. The long-awaited 'pseudo-DFS pattern'. Expert Rev Clin Immunol. 2019 15(5):445.
3.  Dellavance A, Baldo DC, Zheng B, Mora RA, Fritzler MJ, Hiepe F, Ronnelid J, Satoh M, Garcia-De La Torre I, Wener MH, Chan EKL, Andrade LEC. Establishment of an international autoantibody reference standard for human anti-DFS70 antibodies: proof-of-concept study for a novel Megapool strategy by pooling individual specific sera. Clin Chem Lab Med. 2019;57:1754-63.


Date: November 26, 2019

Some laboratories report the fluorescence intensity as numerical (0, 1, 2, 3, 4) or symbols (-, +, ++, +++, ++++) representing a semi-quantitative approximation of the fluorescence intensity. This may be a rough approximation of the actual titer. For economic and practical reasons, many laboratories dilute only up to a given dilution (e.g., 1/640) and report the titer as ≥1/640 when the sample is still bright at 1/640. In your case, this seems the most appropriate approach rather than ‘guesstimating’ the end-point titer. This approach is recommended because one cannot really establish a tight correlation between the numerical or symbolic systems and actual titration. For example, some samples look very bright at 1/80, but become negative very early in the titration process whereas some samples that do not show very bright at 1/80 may become even brighter as the sample is titrated to 1/320 and 1/640. This has been referred to as ‘low dilution antibody systems’ in the former and ‘high dilution systems’ in the latter. For example, anti-centromere (AC-3) is typically a high dilution system. Also note that one may not be able to discern the true ANA pattern without titering the sample out carefully. In any case, it is not recommended that one states a given titer (e.g., 1/1280) if the real titration process is not performed. Clearly the most scientifically sound approach is to do the actual titrations and report as the titer just below the one at which the reaction becomes negative.

Date: June 22, 2019

Increase in UV light intensity can be accomplished with a simple setting change with a modern UV light source and it is clearly important to take note on the effect on ANA positivity in the HEp-2 indirect immunofluorescence assay (HEp-2 IFA). With older microscopes, an increase in intensity often comes with the replacement of new UV bulb. Either increase in light source intensity can affect interpretation of HEp-2 IFA positivity as illustrated in this question. The recommended magnification for HEp-2 IFA is x400 (x40 objective lens) and lower magnification will cause low-intensity positive samples to be interpreted as negative.

Each laboratory should have a working set of positive and negative human serum sample controls for this specific purpose to ensure that the light source setting is appropriate. A brief discussion on the selection of negative HEp-2 IFA samples is relevant here. HEp-2 IFA cutoff should be determined within the local population using the specific reagents and microscope setting. In a practicing clinical laboratory, it should be clear that there are HEp-2 IFA-negative sera that range from almost completely negative to borderline positive. The latter may be most appropriate to help as reference controls for light intensity setting. Another point to be considered is the fluorescent conjugate. Considering the variability in the microscope settings worldwide it is unrealistic to assume that the ready-to-use conjugate provided with the HEp-2 slide kits will fit all customers. Therefore, it is wise to titrate the conjugate against known negative and low-intensity positive samples in a checkerboard fashion.

Date: June 8, 2019

The nuclear envelope patterns (AC-11 and AC-12) present a distinctive staining of the peripheral border of the nucleus in interphase cells unaccompanied by staining of mitotic cells where the nuclear envelope has broken down. In addition, there may be a faint staining across the interphase nucleus, representing the overlying nuclear envelope. Other than the autoantigen targets represented by AC-11 and AC-12, we are not aware of any other autoantigens that are exclusively located at the periphery of the HEp-2 cell nucleus. Occasionally, some samples yield a nuclear homogeneous staining (AC-1) with a slight enhancement in the vicinity of the nuclear envelope. In our experience, this finding is not consistently observed when we test the sample in other HEp-2 slide brands or with a different lot of the same brand. This observation indicates that this particular fluorescence distribution may be an artifact from cell culture and fixation conditions. One other possibility is the artifact caused by accidental drying of the cell substrate during the incubation steps of the IFA procedure.

Date: April 5, 2019

Many typical HEp-2 kits come with a fluorescein isothiocyanate (FITC) conjugate such as goat anti-human IgG. For the double immunofluorescence IFA procedure, you need a fluorescence microscope fitted with exciter and barrier filters that are suited to multi-color imaging. In addition, a monoclonal or polyclonal antibody directed against a protein that is specific to the subcellular compartment you are interested in, will be required. This animal antibody will be the tracer for that subcellular domain. If we take the nucleolus as an example, then you need an antibody against a nucleolar protein (e.g. fibrillarin, nucleolar phosphoprotein B23, etc.). Let's say you can obtain a commercially available mouse monoclonal anti-fibrillarin IgG antibody. Most commonly, you will need a red fluorochrome (e.g. rhodamine or Alexa586) conjugated goat anti-mouse IgG to differentiate this from the human anti-nucleolar antibody stained with FITC. It is important that the fluorochrome against mouse IgG is different from FITC and the light signal does not overlap in excitation/emission/barrier filter spectrum used for the FITC conjugate. For the procedure, in the same HEp-2 well, you will incubate the human antibody and the mouse monoclonal anti-fibrillarin antibody. Both should be at a dilution that yield a good fluorescence signal. It is advised that you first optimize the reaction with the monoclonal antibody.

For the double IFA protocol, there are three options for the primary antibody incubation step:

1) Human antibody ==> wash (do not allow the well to dry)=> apply the mouse monoclonal ==> wash ==> add a mixture of the two conjugate secondary antibodies at appropriate dilutions ==> wash ==> mounting media (e.g. buffered glycerin) and coverslip
2) Mouse monoclonal ==> wash ==> apply human antibody ==> wash ==> mix of the two conjugate secondary antibodies at appropriate dilutions ==> wash ==> mounting media and coverslip
3) Mix of both the mouse monoclonal and human antibody, taking into account that dilution for both is now effectively reduced by one-half ==> wash ==> mix of the two conjugate secondary antibodies at appropriate dilutions ==> wash ==> mounting media and coverslip.

In some protocols, it is often helpful to counterstain with DAPI (blue stain). Which of the above 3 options to use may or may not be important. One may work better than the others depending on the specific antibodies you will use. If one method doesn't work well, try the others.

You will then be able to see the same field in the microscope under the filters for each of the fluorophores. That will tell you if your human primary antibody stains the same subcellular domain as the monoclonal antibody. It is obviously an advantage to take high resolution photographs of the same field of view and then overlap the images using an image processing program such as Adobe PhotoShop as needed.

Date: March 23, 2019

This is an issue that the ICAP committee has discussed extensively but there has been no consensus to date. In many countries, sera showing cytoplasmic staining alone are considered ANA test positive, while in other countries, such sera are considered ANA-negative but cytoplasmic positive. In other jurisdictions, cytoplasmic staining is not reported at all because it is not strictly definable as nuclear staining. There is wide agreement that, sera with cytoplasmic staining alone should not be ignored (1). Other discussions on the topic has suggested renaming he ANA test as anti-cellular antibodies (1), which is consistent with the nomenclature that ICAP has chosen for the various IFA patterns (i.e. AC = anti-cellular). How you report this remains somewhat a local issue. However, whether it is identified as ANA positive or negative has implications for some disease classification criteria. ICAP is working towards a consensus recommendation in the coming year.

N. Agmon-Levin, J. Damoiseaux, C. Kallenberg, U. Sack, T. Witte, M. Herold, X. Bossuyt, L. Musset, R. Cervera, A. Plaza- Lopez, C. Dias, Sousa M. Jose, A. Radice, C. Eriksson, O. Hultgren, M. Viander, M. Khamashta, S. Regenass, L. E. Coelho Andrade, A. Wiik, A. Tincani, J. Ronnelid, D. B. Bloch, M. J. Fritzler, E. K. Chan, I. Garcia-de la Torre, K. N. Konstantinov, R. Lahita, M. Wilson, O. Vainio, N. Fabien, R. A. Sinico, P. Meroni, and Y. Shoenfeld. International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. Ann.Rheum.Dis. 73:17-23, 2014.

Date: March 14, 2019

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. 

Date: March 7, 2019

If the indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) is positive at 1/80, it is recommended that the serum be titered to a dilution of at least 1/640 and thereafter the report should indicate if the titer is

• >1/80 – <1/640
  

• =1/640
  

• >1/640

A dilution of 1/640 is suggested as the minimum requirement. Some laboratories do not dilute beyond 1/640 for logistic and budgetary considerations, while others who use some automated ANA IFA technologies will obtain digitally calculated end point titer results.

Additional notes:

Although it may be true that for some antibodies, higher titers are more indicative of an autoantibody-associated rheumatic diseases (AARD), there are caveats when considering only the HEp-2 IFA method:

1. It is not true for the full spectrum of HEp-2 IFA (ANA) patterns described by ICAP. The most obvious example is the nuclear dense fine speckled pattern (AC-2 pattern) that has been shown to occur at high titer in the general population (1), but uncontrolled experience suggest the same is true for Golgi, centrosome, and some other cytoplasmic patterns.

2. A point in time ANA is not really that revealing on its own. What is more important to know is whether titers are decreasing, increasing or staying the same over time.

Reference:

1. Mariz HA, Sato EI, Barbosa SH, Rodrigues SH, Dellavance A, Andrade LE. Pattern on the antinuclear antibody-HEp-2 test is a critical parameter for discriminating antinuclear antibody-positive healthy individuals and patients with autoimmune rheumatic diseases. Arthritis Rheum. 2011;63:191-200.
   

Date: January 29, 2019

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