Can direct immunofluorescence testing still be accurate if performed on biopsy specimens after brief inadvertent immersion in formalin?


      Direct immunofluorescence is useful in the diagnosis of autoimmune, vesiculobullous, and connective tissue diseases. Michel medium is typically indicated for transport, but clinicians may inadvertently place samples into formalin.


      We set out to determine the amount of time that specimens can remain in 10% buffered formalin and still retain their diagnostic properties.


      Biopsy samples were examined from cases with established diagnoses of bullous pemphigoid (n = 12), dermatitis herpetiformis (n = 6), and pemphigus vulgaris (n = 6) and exposed to formalin for time points ranging from 2 minutes to 4 hours.


      We found that immunoreactants were detectable in the majority of samples when subjected to 2 minutes of formalin exposure. Dermatitis herpetiformis and pemphigoid samples retained immunogenicity for 10 minutes, whereas pemphigus showed reduced immunogenicity for all samples studied. A nonimmunologic nuclear fluorochroming pattern was noted in some of the specimens after formalin immersion.


      Sample size, only examining 3 disease processes, and samples already having been in Michel medium were the major limitations in the study.


      In direct immunofluorescence studies, formalin exposure to biopsy specimens causes two types of artifactual changes: (1) the shortest exposure (2 minutes) causes complete loss of diagnostic markers of pemphigus; and (2) prolonged exposure changes tissue to a form that allows fluorescein-labeled antibodies to give fluorochroming reactions of nuclei (which can be mistaken for in vivo antinuclear antibody reactions of lupus erythematosus). After time intervals of 10 minutes to 2 hours, direct immunofluorescence studies of proven cases of bullous pemphigoid and dermatitis herpetiformis retained variable levels of specific reactivity.

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        • Vassileva S.
        Immunofluorescence in dermatology.
        Int J Dermatol. 1993; 32: 153-161
        • Morrison L.H.
        Direct immunofluorescence microscopy in the diagnosis of autoimmune bullous dermatoses.
        Clin Dermatol. 2001; 19: 607-613
        • Michel B.
        • Milner Y.
        • David K.
        Preservation of tissue-fixed immunoglobulins in skin biopsies of patients with lupus erythematosus and bullous diseases–preliminary report.
        J Invest Dermatol. 1972; 59: 449-452
        • Vodegel R.M.
        • de Jong M.C.
        • Meijer H.J.
        • Weytingh M.B.
        • Pas H.H.
        • Jonkman M.F.
        Enhanced diagnostic immunofluorescence using biopsies transported in saline.
        BMC Dermatol. 2004; 4: 10
        • Jonsson R.
        • Kristensson-Aas A.
        • Kutti J.
        Assessment of a tissue transport-medium in preservation of tissue-fixed immunoglobulins and complement demonstrated by direct immunofluorescence: a pilot study with skin from SLE patients.
        J Clin Pathol. 1978; 31: 823-826
        • Mera S.L.
        • Young E.W.
        • Bradfield J.W.
        Direct immunofluorescence of skin using formalin-fixed paraffin-embedded sections.
        J Clin Pathol. 1980; 33: 365-369
        • Firth N.A.
        • Rich A.M.
        • Radden B.G.
        • Reade P.C.
        Direct immunofluorescence of oral mucosal biopsies: a comparison of fresh-frozen tissue and formalin-fixed, paraffin-embedded tissue.
        J Oral Pathol Med. 1992; 21: 358-363
        • Beutner E.H.
        • Sepulveda M.R.
        • Barnett E.V.
        Quantitative studies of immunofluorescent staining: relationships of characteristics of unabsorbed antihuman IgG conjugates to their specific and non-specific staining properties in an indirect test for antinuclear factors.
        Bull World Health Organ. 1968; 39: 587-606
        • Beutner E.H.
        Field trials by ten laboratories of six commercial conjugates prepared from antisera to human IgG.
        Ann N Y Acad Sci. 1971; 177: 361-404