Monday, April 24, 2017

Lab Series #16: Anti Nuclear antibodies

Occasionally in this blog, I write about immunology topics. But I have rarely discussed diagnostic immunology in this blog. Of course, I have written a post long back on how immunology-based methods are used for diagnostics but not really anything about diagnosing an immunological disorder. Autoimmune diseases are a huge topic to discuss and write about. Ask a rheumatologist, there are hundreds of different types of them. To talk about everything is a huge task (I will have to start writing a new blog for that!!). Instead, I want to focus on a common test, casually called as ANA- Global and Profiling tests. I specifically chose this topic since there are some particular confusions that many don’t clearly understand about.

Photo 1: LE cell. Source
In 1948, the observation of LE cells in a patient with SLE was a key realization that autoimmune reactions are a significant clinical condition. This discovery lead to more discoveries on characterization of antibodies directed against nuclear antigens what is now designated as “ANA or anti-nuclear antibodies”. An LE cell is a neutrophil or macrophage that has phagocytized the denatured nuclear material of another cell. For a long time, LE cell was used as a diagnostic methodology. The principle is very simple. Make a buffy coat preparation from patient blood sample and break some cells such that the nucleus is exposed. Make a smear of the preparation. If ANA is present, this will trigger phagocytosis of nuclear material and can be seen as an LE cell. A smear is considered positive when 10 or more characteristic LE cells are seen in a smear preparation (See Photo 1). The test is laborious and have some demerits. In modern day practice, we no more do this test. Instead, we screen for ANA by a screening test using indirect Immunofluorescence. The screening test is called ANA global and identifying the antibody is called as ANA profiling.

George Friou was the first who applied the immunofluorescence technique for the detection of ANA. In the later years, different immunofluorescence patterns of ANA were observed and lead to the realisation that there are several different possible ANA. By the 1970’s the indirect immunofluorescence test was established using tissue sections as substrate. By 1980’s HEp-2 (Human epithelial cell line) was established as a substrate for ANA screening. Currently, Immunofluorescence testing method is considered as a gold standard for diagnosis. 

So, what makes a Hep-2 cell the standard? HEp-2 cells are laboratory maintained cells (derived from human laryngeal carcinoma) and hence it brings in the uniformity of testing. But a more important reason is these cells are neoplastic in nature and have a large nucleus which gives an advantage in viewing the results. The methodology of ANA- IF testing is very simple. The exact details differ between manufacturers kits though the underlying idea is uniformly the same. Patient plasma/serum sample is overlaid on the HEp-2 cell line. If auto antibodies are present it binds to the antigens in the cell whcih is detected using a secondary antibody. A negative Immunofluorescence (IF) result is a confirmation of negative for autoimmune antibodies.

Interpretation of the findings is based on 3 different parameters.

  • Fluorescence pattern
  • Substrate tissue
  • Sample dilution (titer)
Fluorescence pattern:

There is a huge number of possible patterns recorded in literature that can be seen in the IF testing. Though technically speaking the test is specific for nuclear antibodies, the substrate in use is a complete cell and hence if there are autoantibodies to antigens other than the nuclear antigens you are bound to see those patterns too. There are 3 basic possible patterns that can be seen in an ANA-IF global testing. See Fig 1, for some basic idea of classification.

Fig 1: Common immunofluorescence patterns.
HEp-2 cells, as already stated are neoplastic cells. Most of the cells have a higher nuclear DNA content than you would see in a normal cell and rate of cell division is higher. Hence you will see in any given HEp-2 cell line coated with a slide, a number of cells that are in interphase and a good number of cell in metaphase. This is advantageous cause there are certain patterns better visualised in interphase cells and certain patterns in metaphase cells.

A good number of people interpret the results just by looking into a couple of fields. Though it is time-saving, it is sometimes beneficial in checking more than a few fields under a microscope. Another common mistake is to interpret the positivity of test by fluorescence intensity alone. Washing step after labelling is a crucial step and sometimes despite good and skilled hands, there tends to be a low background fluorescence. This is often referred to as background noise. Sometimes, this fluorescence can be stronger and relying too much on fluorescence maybe misleading to falsely identify a negative as low positive. The best method is to follow a rule - If you see the same pattern all over the slide (in any part of the preparation you observe) and if you can clearly differentiate nucleus and cytoplasm by fluorescence, with a specific pattern only then it is a positive.
Fig 2: Most common nuclear patterns observed under fluorescence microscopy. Source

Substrate tissue:

As already discussed above, substrate tissue determines what antigens you are looking at. ANA kits though are ideally made to detect only ANA, the occurrence of other auto-antibodies has prompted to include more cell lines in a single assay (Example: Euroimmun biochip). Certain antibodies are neagtive on mouse kidney or rat liver but are positive on HEp-2 cell line. For most purposes, ANA is considered classically on HEp-2 cells. 

ANA titer:

The dilution of the sample at which the fluorescence is positive is an indicator of the quantity of the auto antibody. There is a lack of consensus on what should be the minimum titer for a sample to be declared as positive. This is because a good number of normal population is positive for ANA at lower titers. For example, at a dilution of 1:40 roughly 20% of the population is positive. This percentage changes depending on location, Age, Sex etc. By increasing the titer to 1:160 this positive rate comes down to about 3%. A good number of kits now recommend 1:160 as an optimal titer. It should be noted that there is a still a small amount of false positive rate.

Table 1: ANA Profiling tests for detection of specific antibodies.
A global assay (Generic assay) is a screening test. It cannot predict the autoantibody. For example, a homogenous pattern indicates antibody against ds- DNA, histone or nucleosome. So when an ANA global is positive, a profile is ordered.

In the earlier days, antigen preparation was not advanced. Only a handful of the antigens could be purified in the laboratory. These antigens that could be extracted are called as ENA (Extractable Nuclear antigen). Some of the common known ENA's include dS-DNA, Histone, Centromere, JO-1, LA(SS-B), RNP, RO (SS-A), SCL, Sm etc. Today more and more antigens are purified or available in a pure form to test. There are several methods available in current practice for profiling the ANA. See Table 1. Fig 3, shows an example multiparametric immunoblot from Euroimmun for differentiation and confirmation of the 23 ANA in a single test run.

Fig 3: ANA 23 profile. Source

The ICAP (international consensus on antinuclear antibody pattern) has renamed the nomenclature for reporting ANA. Instead of name, each pattern is assigned an AC code. For example, the homogenous pattern will be called as AC-1 (Read as Anti-cell pattern-1). Also, they have categorised the patterns into competent level reporting and expert level reporting based on clinical relevance and easiness of identification. The requirement is that the patterns under competent level should be reported and expert level reporting is optional.

Fig 4: Nomenclature and Classification Tree. Source

Kumar Y, Bhatia A, Minz R. Antinuclear antibodies and their detection methods in diagnosis of connective tissue diseases: a journey revisited. Diagnostic Pathology. 2009;4(1):1 DOI: 10.1186/1746-1596-4-1

Chan Ek et al. Report of the First International Consensus on Standardized Nomenclature of AntinuclearAntibody HEp-2 Cell Patterns 2014-2015. Front Immunol. 2015 Aug 20;6:412. doi: 10.3389/fimmu.2015.00412.

The official website for the International Consensus on Antinuclear Antibody (ANA) Pattern (ICAP).