Indirect immunofluorescence assay (IFA) has been used for detection of autoantibodies against cellular antigens for more than 50 years. Originally using rodent tissue as substrate, the method was optimized by using the human immortal HEp-2 cell line derived from a larynx epidermal carcinoma. The HEp-2/IFA platform allows for optimal visualization of several cellular domains recognized by autoantibodies in the samples being tested. Serial dilution allows for the estimation of the concentration (titer) of the autoantibodies in the sample. Judicious analysis of the topographic distribution of the immunofluorescence (pattern) provides useful hints on the most plausible autoantigens being recognized, vis-à-vis the cognate autoantibodies. The importance of the HEp-2/IFA pattern has been recently emphasized by the International Consensus on ANA Patterns (ICAP), an initiative that established a comprehensive classification of the most relevant and prevalent HEp-2/IFA patterns (designated anti-cell (AC) patterns) and harmonized its nomenclature. The former designation “antinuclear antibody test” has been progressively replaced by the term “anti-cell antibody test,” due to the recognition that the HEp-2/IFA method in fact allows the detection of autoantibodies to several cellular domains, such as the cytoplasm and mitotic apparatus.
The performance of the HEp-2/IFA test is strongly influenced by several technical details, including cell culture conditions, cell fixation and permeabilization methods, choice and titration of fluorochrome-conjugated secondary antibody, use and choice of blocking solutions, washing buffers, and antifading mounting medium. The several steps of the procedure must be carefully performed in order to avoid the formation of false positive fluorescent artifacts. The quality control of the assay involves the use of serum standards for negative, low positive and strongly positive reaction in each run of the assay. In addition, every new lot or new brand of HEp-2 slides should be evaluated by using a panel of standard sera yielding the most relevant AC patterns. Special attention should be dedicated to the training of personnel for the analysis of the slides at the microscope. These should be able to identify possible artifacts, recognize all relevant AC patterns, and formulate possible reflex tests according to the observed AC patterns.
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