Enhanced diagnostic immunofluorescence using biopsies transported in saline

Abstract

BackgroundThe 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.MethodsIn 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.ResultsDirect 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).ConclusionsWe 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.

Orange Palpebral Spots

“…Melanin or iron deposits are ruled out by the characteristics of the deposit. The negative first 4 fluorescence results were attributed to the negative effect of Michel’s fixative used for storage and transport of the samples [16]. It was positive in the fifth case, frozen-stored.…”

Management of bullous pemphigoid: the European Dermatology Forum consensus in collaboration with the European Academy of Dermatology and Venereology

“…DIF studies typically demonstrate linear deposits of IgG and/or C3 along the dermoepidermal junction; occasionally IgA and IgE are also found with a similar pattern …”

British Association of Dermatologists’ guidelines for the management of bullous pemphigoid 2012

“…A biopsy for direct IF (DIF) is taken from uninvolved skin about 1 cm away from a fresh blister and is immediately snap‐frozen in liquid nitrogen or transported in either Michel’s transport medium or normal (0·9%) saline. If using saline, the biopsy must be processed within 24–48 h; 19 with Michel’s medium prompt handling is to be preferred, but a longer delay of up to 2 weeks may still yield results 20 . Indirect IF (IIF) is performed on serum, and if this is not obtainable, on blister fluid.…”