Variability in the recognition of distinctive immunofluorescence patterns in different brands of HEp-2 cell slides

Abstract

Introduction: Indirect immunofluorescence on HEp-2 cells is considered the gold standard for the detection of autoantibodies against cellular antigens. However, the culture conditions, cell fixation and permeabilization processes interfere directly in the preservation and spatial distribution of antigens. Therefore, one can assume that certain peculiarities in the processing of cellular substrate may affect the recognition of indirect immunofluorescence patterns associated with several autoantibodies. Objective: To evaluate a panel of serum samples representing nuclear, nucleolar, cytoplasmic, mitotic apparatus, and chromosome plate patterns on HEp-2 cell substrates from different suppliers. Materials and methods: Seven blinded observers, independent from the three selected reference centers, evaluated 17 samples yielding different nuclear, nucleolar, cytoplasmic and mitotic apparatus patterns on HEp-2 cell slides from eight different brands. The slides were coded to maintain confidentiality of both brands and participating centers. Results: The 17 HEp-2 cell patterns were identified on most substrates. Nonetheless, some slides showed deficit in the expression of several patterns: nuclear coarse speckled/U1-ribonucleoprotein associated with antibodies against RNP (U1RNP), centromeric protein F (CENP-F), proliferating cell nuclear antigen (PCNA), cytoplasmic fine speckled associated with anti-Jo-1 antibodies (histidyl synthetase), nuclear mitotic apparatus protein 1 (NuMA-1) and nuclear mitotic apparatus protein 2 (NuMA-2). Conclusion: Despite the overall good quality of the assessed HEp-2 substrates, there was considerable inconsistency in results among different commercial substrates. The variations may be due to the evaluated batches, hence generalizations cannot be made as to the respective brands. It is recommended that each new batch or new brand be tested with a panel of reference sera representing the various patterns.

V Brazilian consensus guidelines for detection of anti-cell autoantibodies on hep-2 cells

“…Therefore, it is suggested that laboratories consider the possibility of using more than one brand for samples generating unexpected results, considering the limitation of some substrates in the expression of certain patterns [26]. Another recommendation is that, for each new lot or new brand, representative samples of different patterns should be tested, preferentially contemplating different cell compartments.…”

“…It is the laboratory responsibility to address occasional problems with substrates that do not adequately express certain patterns, such as the fine speckled nuclear pattern associated with the presence of anti-SS-A/Ro antibodies (AC-4), the coarse speckled nuclear pattern associated with antibodies against RNP/Sm (AC-5), the pleomorphic nuclear pattern suggestive of antibodies anti-PCNA (AC-13), the compound pattern suggestive of the presence of anti-CENP-F antibodies (AC-14), the cytoplasmic fine speckled pattern suggestive of anti-Jo-1 (AC-20) antibodies, the cytoplasmic rods and rings pattern (AC-23), the anti-NuMA-1-like pattern (AC-26) and the anti-NuMA-2 pattern (AC-25) [10,26].…”

Interkit Reproducibility of the Indirect Immunofluorescence Assay on HEp-2 Cells Depends on the Immunofluorescence Reactivity Intensity and Pattern

“…Dellavance et al. compared eight HEp-2-IFA kits using 17 samples with well-defined IF patterns, including nuclear patterns (homogeneous/AC-1, dense fine speckled/AC-2, centromere/AC-3, coarse speckled/AC-5, multiple nuclear dots/AC-6, PCNA-like/AC-13, CENP-F-like/AC-14, nuclear matrix-like coarse speckled, quasi -homogeneous, and fine speckled with rare nuclear dots-AC-4/AC-7), nucleolar patterns (homogeneous/C-8, clumpy/AC-9, and punctate/AC-10), cytoplasmic patterns (fine speckled/AC-20 and dense fine speckled/AC-19), and mitotic apparatus patterns (NuMA-like/AC-26 and mitotic fuse/AC-25 ( 30 ) The samples were processed and analyzed blindly in three independent expert laboratories. The results show that some patterns (AC-1, AC-2, AC-3, AC-7, AC-8, AC-9, AC-10, AC-19, and nuclear quasi -homogeneous) were rather robust in that they were appropriately identified with all kits and in at least two of the three participating laboratories.…”

Response to ‘Titre-specific positive predictive value of anti-nuclear antibody patterns’ by Vulsteke et al

“…First, the genetically modified HEp-2000 substrate was used, whereas this substrate is currently not included in the ICAP consensus for reasons summarised in a recent ICAP correspondence 3. As stated in this correspondence, the ICAP decision to exclude the HEp-2000 substrate might be reconsidered, although a previous study observed that some patterns are not readily recognised in this cell line 4. Importantly, the SS-A/Ro60 pattern observed on the transfected HEp-2 cell line will reduce the PPV of the fine speckled pattern (AC-4) with anti-SS-A/Ro60 antibody-associated diseases.…”

“…3 As stated in this correspondence, the ICAP decision to exclude the HEp-2000 substrate might be reconsidered, although a previous study observed that some patterns are not readily recognised in this cell line. 4 Importantly, the SS-A/Ro60 pattern observed on the transfected HEp-2 cell line will reduce the PPV of the fine speckled pattern (AC-4) with anti-SS-A/Ro60 antibody-associated diseases. Indeed, the SS-A/Ro60 pattern was frequently observed in systemic lupus erythematosus (SLE; n=22), Sjögren's syndrome (SjS; n=27) and cutaneous lupus erythematosus (n=9).…”