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• W1608146575 abstract "The method that widely used for ANA screening is the immunofluorescent assay (IFA) using human epithelial cell lines (HEp-2) cells as substrate antigen. However, most of the IFA-ANA tests are commercial products. The practical data for HEp-2 cells preparation has not been reported and fixative reagents for in-house preparation remain unclear. The proposes of this study were to determine the optimal condition in the preparation of HEp-2 cells culture for detecting antinuclear antibody by IFA and to compare fixative reagents between methanol-acetone and 1% paraformaldehyde. HEp-2 cells were cultured on 12-wells glass slide and harvested at different time points (12, 24, 36 and 42 hours), followed by fixing with two different fixatives: methanol-acetone or 1% paraformaldehyde (using methanol-acetone as permeabilizer). Next, the ANA-IFA was performed with known clinical samples to evaluate the five common ANA patterns and compared with commercial kit. The results showed that the optimal time of cells harvesting was at 42 hours. The spreading of cells on slide was 90-100% confluences and amount of cells after staining were 2-5 mitotic cells observing under phase contrast microscope at 400X magnification. For ANA pattern results, both fixatives represented the correct ANA positive patterns for homogeneous, speckle, nucleolar and centromere pattern. However, 1% paraformaldehyde fixed cells demonstrated higher background than the methanol-acetone fixed cells. In one case of nuclear membrane pattern, the staining pattern could not be interpreted due to masking of cytoplasm staining. This problem was solved with alternative fixation protocol: 1% paraformaldehyde and permebilyzed with Triton-X100. Our results suggest that methanol-acetone was the effective fixative for ANA screening using IFA. This protocol would be useful for in-house ANA test development." @default.
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• W1608146575 date "2010-04-01" @default.
• W1608146575 modified "2023-09-24" @default.
• W1608146575 title "Preparation of HEp-2 Cells for Antinuclear Antibody Detection Using Immunofluorescent Technique" @default.
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