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Published on: September 16, 2019
Enhanced diagnostic immunofluorescence using biopsies transported in saline
Robert M Vodegel1, Marcelus C J M de Jong, Hillegonda J Meijer
1Centre for Blistering Diseases, Department of Dermatology, Groningen University Hospital, Groningen, The Netherlands. R.M.VODEGEL@FREELER.NL
Insights
Transporting skin biopsies in normal saline for 24 hours improves direct immunofluorescence (DIF) accuracy for autoimmune skin diseases by reducing background noise. This method is a practical alternative to freezing or Michel's fixative for DIF diagnosis.
Area of Science:
- Dermatology
- Immunology
- Pathology
Background:
- Direct immunofluorescence microscopy (DIF) is crucial for diagnosing autoimmune and immune complex-mediated skin diseases.
- Preserving tissue-bound immunoreactants is key, with fresh-freezing or Michel's fixative as standard methods.
- High dermal background fluorescence can lead to false negatives in DIF, particularly with weak IgG staining.
Purpose of the Study:
- To compare normal saline with liquid nitrogen and Michel's fixative for transporting skin biopsies for DIF analysis.
- To evaluate the impact of different transport media on diagnostic accuracy and background fluorescence.
Main Methods:
- Twenty-five patients with autoimmune skin diseases provided four matched biopsies each.
- Biopsies were transported in normal saline (24h, 48h), liquid nitrogen, or Michel's fixative (48h).
- Direct immunofluorescence microscopy was used to assess immunoreactant staining and background fluorescence.
Main Results:
- Saline transport significantly reduced background fluorescence (p < 0.01) and enhanced specific IgG and IgA staining.
- Conclusive or tentative DIF diagnoses were achieved in 92% of cases after 24h saline, compared to 68% for liquid nitrogen and 62% for Michel's fixative.
- Higher diagnostic yields were observed with saline at both 24 and 48 hours compared to conventional methods.
Conclusions:
- Transporting skin biopsies in normal saline for 24 hours is an effective and practical method for routine DIF diagnosis.
- Saline incubation reduces IgG background fluorescence in the dermis and epidermis, leading to improved diagnostic accuracy.
- This approach offers an attractive alternative for DIF analysis in autoimmune and immune complex-mediated dermatoses.
Background:
The demonstration of tissue-bound immunoreactants by direct immunofluorescence microscopy (DIF) is a valuable parameter in the diagnosis of various autoimmune and immunecomplex-mediated skin diseases. For preservation of tissue-bound immunoreactants, biopsies are usually fresh-frozen in liquid nitrogen or transported in Michel's fixative. But even optimally preserved tissue specimens are no guarantee for the correct diagnosis by DIF, especially when weak to moderate IgG fluorescence of the epidermal basement membrane zone is involved. In such cases false negative results are easily obtained due to the relatively high dermal "background" fluorescence produced by polyclonal anti-human IgG fluorescein conjugates.
Methods:
In the present study we have compared the use of normal saline (0.9% NaCl) with liquid nitrogen and Michel's fixative as transport medium for skin biopsies. From 25 patients with an autoimmune skin disease (pemphigus, pemphigoid, lupus erythematosus and vasculitis) four matched skin biopsies were obtained and transported in either saline for 24 and 48 hours, liquid nitrogen, or Michel's fixative for 48 hours.
Results:
Direct IF microscopy showed significant reduction of background fluorescence (p < 0.01) and relatively enhanced desired specific (IgG, IgA) staining in biopsies transported in saline. A conclusive or tentative IF diagnosis was reached in 92% after 24 h saline, 83% after 48 h saline, 68% after freezing in liquid nitrogen, and 62% after 48 h Michel's medium (n = 25).
Conclusions:
We conclude that transporting biopsies without freezing in normal saline for 24 hours is an adequate and attractive method for routine IF diagnosis in autoimmune and immune complex-mediated dermatoses. The superior results with saline incubation are explained by washing away of IgG background in dermis and epidermis.
