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Cellular Imaging: A Case Study on Indirect Immunofluorescence

The antinuclear antibody (ANA), which refers to a unique group of autoantibodies targeting at the nuclear contents of the cell, has significant relation to specific AD, such as systemic lupus erythematosus (SLE), systemic sclerosis and rheumatoid arthritis. The identification of ANAs plays an important role in the clinical medicine and clinical immunology.

Indirect immunofluorescence (IIF) is the original approach for ANA test described by Coons and Kaplan more than 50 years ago [2]. Over time, other methods of detecting ANA have been developed such as solid phase immunoassays, e.g. including enzyme-linked immunosorbent assay (ELISA), and the multiplex platform [3, 10, 23], which is much easier and cheaper compared to IIF. However, the new tests usually show lower sensitivity [9, 11, 12]. In addition, different commercial ELISA kits produce different results [22] and they cannot avoid the problem of false positive results. Recently, the American College of Rheumatology issued a position statement stressing that the IIF method based on HEp-2 cells for ANA assay remains the gold standard [13]. HEp-2 cells, originally considered to originate from a human larynx carcinoma, are now known to have been established from a HeLa cell contamination [1]. They have many advantages, such as: they are a more sensitive substrate that allows identification of many patterns; human origin ensures better specificity than animal tissues; the nuclei are much larger, so complex nuclear details can be detected; the cell monolayer ensures that all nuclei are visible; cell division rates are higher so that antigens produced only in cell division are easily located, e.g. centromere and mitotic spindle patterns; there is no obscuring of the intercellular matrix; antigen distribution is uniform [1].

The technique for performing the IIF test in the Clinical Immunology Laboratory of Tan Tock Seng Hospital, Singapore is shown in Fig.1.3 [14]. Briefly, the process of IIF is as follows: First, diluted patient’s sample is incubated with the slides containing fixed HEp-2 cells. After a period, unbound antibodies are washed off and a fluorescein-conjugated anti-human immunoglobulin will be applied. Then, washing after the second incubation, any unbound secondary immunoglobulin will be removed. The ANAs are finally revealed as fluorescent cells under the fluorescence microscope. Both fluorescence intensity and positive staining patterns for each slide image are identified by highly qualified and skillful physicians.

IIF procedure

Fig. 1.3 IIF procedure

In practice, the intensity of fluorescence cells is reported on a scale of values from 0 to 4+ as follows:

  • • 4+: bright green fluorescence;
  • • 3+: less bright green fluorescence;
  • • 2+: diminished fluorescence yet clearly observable;
  • • 1+: very subdued fluorescence;
  • • 0: negative.

It is worth noting that negative intensity or level 0 indicates that the patient is normal. Staining patterns are recorded for samples with positive intensity (i.e., from 1+ to 4+), since different patterns are related to specific diseases. There are more than thirty kinds of staining patterns in the world, however, the most frequent staining patterns of HEp-2 cells in clinical practice are as follows [1, 5]:

  • Centromere: characterized by 40-60 discrete speckles diffused over all the nuclei in the interphase and characteristically found in the condensed nuclear chromatin during mitosis as a bar of closely related speckles;
  • Nucleolar: characterized by large coarse speckles (less than six) within the nucleoli of interphase cells;
  • Homogeneous: characterized by a uniform distributed fluorescence of the entire interphase nuclei and fluorescence of the nuclear chromatin during mitotic;
  • Speckled: there are two sub-categories:
    • - Fine Speckled: characterized by a fine granular nuclear staining of nuclei in the interphase, which is distributed uniformly;
    • - Coarse Speckled: characterized by dense, intermediate sized particles in interphase nuclei together with large speckles;
  • Cytoplasmic: characterized by staining of the cytoplasm exclusive of the nucleus.

The staining patterns mentioned above are shown in Fig.1.4. As a matter of fact, according to different needs of the lab, the categorization of staining patterns can be changed slightly. In some cases, mixed staining patterns reveal when patient’s serum contains more than one autoantibody specificity. However, mixed patterns are

Typical HEp-2 cells with different staining patterns atypical and occur rarely in clinical diagnosis

Fig. 1.4 Typical HEp-2 cells with different staining patterns atypical and occur rarely in clinical diagnosis. In most situations, same pattern is shown in the same slide then it can be classified into common class. In academic community, only the images with the same staining pattern are used to study.

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