FAQ - Frequently Asked Questions

Traditional assays for anti-PCNA are double immunodiffusion and HEp-2 IFA. Positive anti-PCNA antibodies as defined by these assays are probably high titer and are associated with SLE. The “new” assays for anti-PCNA using ELISA, Line/Dot blot, CLIA, FEIA, ALBIA, and other solid-phase methods typically detect lower titer (or lower avidity) positives (e.g., higher sensitivity than IFA and immunodiffusion) and this may explain why studies using these methods no longer show a specific association of anti-PCNA with SLE (Vermeersch et al, 2009). More studies are needed on this topic.

A second consideration with anti-PCNA antibodies is that their reactivity has been reported to be highly dependent on the oxidation status of PCNA in the HEp-2 substrate (Tsai et al, 1992). The initial fascinating observation was that if the HEp-2 slide is taken out from the nitrogen-sealed packet at room temperature for 15-30 minutes before starting the first incubation of the diluted serum in the typical IFA protocol, the anti-PCNA staining was negative or substantially reduced while other ANA were not apparently affected. This loss of reactivity to PCNA was apparently due to air oxidation. The report showed that the free thiol group(s) on PCNA is important for anti-PCNA reactivity of samples from SLE patients. Thus, back to the question, the “negative (result) in HEp-2 IFA” would have to be considered more carefully by processing the slide immediately after opening the sealed packet, by using at least one additional HEp-2 slide brand, and by confirming the expected HEp-2 IFA AC-13 pattern with an available reference anti-PCNA serum.

Please note that there have not been any known recent reports that reproduced the data on the oxidation status-dependency of anti-PCNA staining as reported by Tsai et al. ~30 years ago. This is important because HEp-2 substrate processing, stabilization and fixation may have changed since then. In summary, anti-PCNA reactivity in a solid phase assay accompanied by the typical AC-13 pattern may be more specific for SLE compared to a positive reaction in solid phase assays alone.

References
Vermeersch P, De Beeck KO, Lauwerys BR, Van den Bergh K, Develter M, Marien G, Houssiau FA, Bossuyt X (2009) Antinuclear antibodies directed against proliferating cell nuclear antigen are not specifically associated with systemic lupus erythematosus. Ann Rheum Dis 68:1791-1793. DOI: 10.1136/ard.2008.104190

Tsai WM, Roos G, Hugli TE, Tan EM (1992) Influence of free thiol group(s) on autoantibody-defined epitope of proliferating cell nuclear antigen. J Immunol 149:2227-2233.

Mahler M, Miyachi K, Peebles C, Fritzler MJ. The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA). Autoimmun Rev. 2012 Aug;11(10):771-5.

Mahler M, Silverman ED, Fritzler MJ. Novel diagnostic and clinical aspects of anti-PCNA antibodies detected by novel detection methods. Lupus. 2010 Nov;19(13):1527-33.

Recognizing mitotic cells and the metaphase plate is an important step in learning to read and interpret HEp-2 IFA. The metaphase plate is like a special place in a cell where all the chromosomes line up perfectly along the middle, making sure everything is ready for the cell to divide, a phenomenon that occurs during the metaphase stage of the cell cycle. Therefore, the metaphase plate primarily contains the autoantigens that belong to chromosomes. The first step is to identify the cells in mitosis. If you use a conjugate that contains DAPI or Hoechst for DNA counterstain, you should be able to recognize metaphase cells using the appropriate filter, as shown in Figure 1.

Alternatively, you can train yourself in the recognition of cells in metaphase using well-characterized serum samples. For example, select a sample with anti-double-stranded DNA antibodies that produce a clear-cut homogeneous nuclear pattern (AC-1). You will see that the majority of cells will exhibit homogeneous staining within the nucleus during the interphase. Cells in metaphase, however, are less common and you might see only one in every 40X view. These cells look different because they have a specific intense staining of the metaphase plate in absence of staining of the rest of the cell's components. This staining phenomenon of the homogeneous pattern arises due to the integral presence of DNA within the chromosomes. In contrast, a sample with anti-U1RNP antibodies should yield the coarse speckled nuclear pattern (AC-5) in the interphase cells, whereas the metaphase cells present a dark transversal band (not stained) at the cell’s "equator". This is the negative metaphase plate (not stained), because U1RNP is not a chromosomal component. Figure 2 clearly illustrates positive and negative metaphase plates.

It is not uncommon that ANA (or HEp-2 IFA) positivity can change over time. This may be especially true when the first test showed weak reactivity, as it was in this case. Such fluctuations between positive and negative are common in cases with weak reactivity and can simply be the result of the variability of the IFA technique itself. The serum levels of anti-SS-A/Ro antibodies can also fluctuate over time (1).

It should be noted that anti-SS-A/Ro often includes two distinct autoantibodies that recognize 60kD (Ro60) and 52kD (Ro52) antigens. To ensure reactivity is detected in a sensitive manner, ideally solid phase specific assays for Ro52 and Ro60 should be included, next to HEp-2 IFA to complete the analysis.

The other issue relevant to the result for anti-SS-A/Ro that was positive last year is its clinical significance. Anti-SS-A/Ro antibodies occur in diseases, such as lupus and Sjögren´s syndrome. The prevalence of anti-Ro52 autoantibodies is similar to that of anti-Ro60 in every connective tissue disease cohort except for myositis, where anti-Ro52 is common but not anti-Ro60. Occasionally, anti-SS-A/Ro may also occur in people with no apparent disease.

It is very important to keep in mind that the autoantibodies, by themselves, are not sufficient for the diagnosis of any disease. It is important that the physician match the results of the autoantibody tests with the entire clinical picture of the patient to come to an appropriate diagnosis.

1.Faria AC, Barcellos KSA, Andrade LEC: Longitudinal oscillation of antibodies to extractable nuclear antigens in systemic lupus erythematosus. J Rheumatol 32: 1267-72, 2005.

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.

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: In the literature, there is general acceptance that accurate assessment of IFA patterns on HEp-2 cells requires a minimum total magnification of 400x (40x objective and 10x ocular) when analyzing the slides directly through the microscope. However, a similar statement has not been made by the ICAP Executive Committee.

Many semi-automated IFA readers use 200x magnification, which is sufficient to allow interpretation of many AC patterns. However, digital images taken on semi-automated instruments (or from traditional microscopes) are different from viewing live images through microscope eyepieces. For images submitted to ICAP, it is recommended that the minimum magnification should be 400x.

To fully appreciate all the 5 features of AC-29 (topo I-like) and the nuclear tiny speckles of AC-4a, 400x magnification is recommended. A 400x magnification is also needed most of the times to differentiate the nucleolar patterns (AC-8,9,10) and the nuclear envelope patterns (AC-11,12).

Answer: We refer to simple patterns when we observe single and isolated clear-cut HEp-2 IFA patterns, e.g. AC-23 or AC-3. The terms multiple, mixed, and composite IFA patterns refer to close but distinct concepts that go beyond the simple patterns.
1. The term "multiple patterns" applies to situations where two or more individual and clearly discernable patterns, e.g. AC-1 + AC-23 or AC-4 + AC-22, are observed in an individual serum sample (i.e., each of the patterns is clearly identified).
2. The term "mixed patterns" applies to observations where a serum sample produces more than one pattern in the same cell compartment and it is not possible to clearly identify each of the components. As an example, AC-4 + AC-5 can commonly be seen together but we are unable to distinguish each of the components (AC-4 and AC-5); in those cases, the HEp-2 IFA staining pattern is regarded as a mixed nuclear-speckled pattern.
3. Finally, the term "composite patterns" is applied to cases in which a single antibody causes staining of two or more cell compartments and the composite image is so characteristic that it always evokes the presence of that autoantibody. For example, the AC-26 pattern is an interphase nuclear staining pattern accompanied by mitotic spindle fiber staining and is typically associated with anti-NuMA antibodies. Similarly, the AC-14 pattern, typically associated with anti-CENP-F antibodies, is characterized by nuclear fine speckled staining with variable intensity in interphase cells, a delicate centromere staining only in mitotic cells, staining of the inter-cellular bridge, and nuclear envelope staining exclusively in prophase cells.

Answer: The competent-level designation is ideally aimed at laboratories starting to train on reading HEp-2 IFA patterns and they can focus on covering all the competent-level patterns first. In other words, the competent-level covers all patterns considered essential to be reported by a laboratory testing for HEp-2 IFA. However, ICAP is not implying that labs should stop there with a “don’t cross this line” thinking.

Thus, if a lab can recognize AC-18 (cytoplasmic discrete dots), they can just report it as AC-18. How should we consider a lab being competent with AC-18? It would be ideal that they establish an internal standard reference for AC-18 (based on external reference material as provided by the IUIS autoantibody standardization committee https://asc.dental.ufl.edu/reference-sera/) and check their HEp-2 substrate whenever they have new batches. This ensures that they should be able to recognize AC-18.

If a lab does not recognize AC-18 by the above policy/reference, they can report just as cytoplasmic.

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.

Answer: In most cases, a sample with anti-dsDNA antibodies is associated with a homogeneous nuclear pattern (AC-1). In rare occasions, no nuclear staining is observed and that happens because the DNA epitopes recognized by the antibodies in that sample are present in the highly compacted Crithidia luciliae kinetoplast DNA, but not in commercially-prepared HEp-2 cells that are commonly used. In those cases, it may be helpful to do the HEp-2 IFA test with another HEp-2 kit, which may or may not be positive. In your case, it is clear that you have confirmed the negative result in different commercial kits. In some cases, the anti-dsDNA antibodies target DNA epitopes that are hidden by interaction with histones and other proteins (e.g., high mobility group proteins) in the nucleus. In such cases, extraction of histones and other nuclear proteins with 0.1N HCl prior to the IF reaction may allow the anti-dsDNA antibodies to produce the expected AC-1 pattern. Finally, it must be certified that the immunofluorescence reaction that you observed in the Crithidia assay corresponds to the kinetoplast and not to the basal body.

With all things being equal, same slide manufacturer with same slide lots, same conjugate, and same serum dilution to ensure there are no technical issues, HEp-2 IFA patterns can change over time. This may include a change from no detectable pattern (AC-0) to any AC pattern and vice-versa. Many factors such as disease progression or overlap syndrome, new interventions (drugs, biologicals), new exposures (infectious disease, cancer, etc.) can also affect such changes. It is important to know if the end-point titer also changed (increased or decreased by at least 2 dilutions).
What is the immunological basis for changes in HEp-2 IFA status along the course of SLE? Autoantibody levels do fluctuate along the disease course. Some of them (i.e., native DNA and nucleosome, for example) may fluctuate in relation to disease activity, while others (i.e., anti-Ro60 and anti-Sm) appear to fluctuate independently of disease activity.
Can HEp-2 IFA temporal changes be related to disease activity? Most people would say “no”, but the answer is not so simple. A recent study from the Andrade lab evaluated 269 SLE patients regarding their HEp-2 IFA titer and pattern in a transverse (active x intermediary x inactive disease activity) and prospective (change in disease activity status in the same patient along 1-year follow-up) (1). The report showed that patterns and titer change can change and there was some association with disease activity status. High titer and the AC-1 pattern were associated with active disease, whereas low titer and the AC-4 pattern were associated with disease remission. Interestingly, the diagnostic performance (ROC curve AUC) of HEp-2 titer for the definition of disease activity was in the range of traditional disease activity biomarkers (anti-dsDNA, anti-nucleosome) and much greater than C3 and C4. However, the HEp-2 IFA is not recommended as a test for monitoring SLE disease activity. Similar conclusions may apply to other ANA-related rheumatic diseases.
Thus, coming back to the index case, the change from AC-5 to AC-1 may indicate the appearance of anti-nucleosome and/or anti-dsDNA antibodies. It is recommended to be aware of these autoantibodies and possible disease flare.
1. Prado MS, Dellavance A, Rodrigues SH, Marvulle V, Andrade LEC (2020) Changes in the result of antinuclear antibody immunofluorescence assay on HEp-2 cells reflect disease activity status in systemic lupus erythematosus. Clin Chem Lab Med 58:1271-1281. DOI: 10.1515/cclm-2019-0638

The short answer to your question is that when mitotic patterns (AC-24 to AC-28) are detected, they should be reported as “ANA positive” (see reference below).

The long answer involves some considerations. We know that there is quite a difference in how mitotic (or even cytoplasmic) patterns are reported depending on the region of the world. Some countries report positive mitotic patterns as ANA+ and others as ANA-. Even within a country, some laboratories may report positive mitotic patterns as ANA+ and others as ANA-. It is important to harmonize your reported test results with the local ‘culture’ and practices where you reside. Nevertheless, we recommend following the ICAP suggestions at www.anapatterns.org.

The most important part is to ensure that your “customers” understand your results. ICAP recommends that you avoid using terms like “ANA positive” or “ANA negative.” In fact, the term ANA is outdated, and we prefer to refer to the actual assay, which is an indirect immunofluorescence assay using HEp-2 slides or HEp-2 IFA, as a transition to a new description for this test: Anti-Cell Antibodies (see reference below).

We have been trying to disseminate the concept that there is more in the HEp-2 IFA test than just "positive" or "negative." We know that clinicians (and laboratories) are familiar with this dichotomous concept, but we also learned that clinicians learn fast how to use HEp-2 IFA patterns for better clinical decisions.

Reference
von Mühlen et al. (2021) How to report the Antinuclear Antibodies (Anti-Cell Antibodies) test on HEp-2 cells: guidelines from the ICAP initiative. Immunol. Res., in press.

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.

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.

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.


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.

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.

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.

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.

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.

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. 

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.
   

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.