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.

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.