Enhanced diagnostic immunofluorescence using biopsies transported in saline.
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.
Vodegel
Robert M
RM
Centre for Blistering Diseases, Department of Dermatology, Groningen University Hospital, Groningen, The Netherlands. R.M.VODEGEL@FREELER.NL
de Jong
Marcelus C J M
MC
Meijer
Hillegonda J
HJ
Weytingh
Marijn B
MB
Pas
Hendri H
HH
Jonkman
Marcel F
MF
D001327:Autoimmune Diseases / Q000378:metabolism / Q000473:pathology*; D001706:Biopsy / Q000379:methods; D015925:Cryopreservation; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D010391:Pemphigoid, Bullous / Q000473:pathology; D012680:Sensitivity and Specificity; D012867:Skin / Q000378:metabolism / Q000473:pathology; D012871:Skin Diseases / Q000378:metabolism / Q000473:pathology*; D012965:Sodium Chloride; D013048:Specimen Handling / Q000379:methods*; D014021:Tissue Preservation / Q000379:methods
D007074:Immunoglobulin G; D012965:Sodium Chloride