Immunohistochemistry for Aspergillus sp. with a Anti-Aspergillus Polyclonal Antibody: Comparison with In Situ Hybridization
Laurel Glaser, Amy Ziober, Li Ping Wang, Kathleen T Montone. Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
Background: Aspergillus infections are often detected on histopathologic review, however, it is well established that accurate species identification, particularly of filamentous fungal infections, can be problematic in formalin-fixed tissue specimens. While fungal culture is often considered the “gold standard” for identification, cultures may be negative, or in turn, may have been performed if a fungal pathogen was not suspected. In situ hybridization (ISH) for fungal rRNA is a means for determining the presence of Aspergillus sp. in tissue sections. More recently, commercially available antibodies targeting Aspergillus antigens have been developed. In this study a series of tissue specimens culture positive for Aspergillus sp. were studied by IHC and compared to a previously established ISH method for Aspergillus sp.
Design: Twenty-five formalin-fixed, paraffin embedded tissue specimens from non-invasive and invasive Aspergillus sp. culture positive cases were studied. These included specimens from a variety of tissue sites including lung, skin, sinonasal tract, gastrointestinal tract and, brain. Immunohistochemistry (IHC) was performed using a polyclonal rabbit anti-aspergillus antibody at a 1:1500 dilution. The immunogen was soluble extract from A. fumigatus, A. flavus, A. niger and A. terreus. ISH was performed with a previously established method using a locked nucleic acid probe targeting Aspergillus sp. 18S rRNA sequences. Slides from both the IHC and the IHC procedures were visualized using light microscopy.
Results: The IHC procedure resulted in a positive reaction in all 25 cases of known Aspergillus infection. The majority of organisms stained with the antibody and there was minimal background staining of the host tissue. ISH showed a positive reaction in 22/25 cases. In comparison, the IHC showed staining in more organisms than that seen by ISH which also show more background staining in the host tissue. Interestingly, both ISH and IHC staining for Aspergillus was weakest in fungal balls in comparison to invasive fungal infections. IHC and ISH on tissue specimens from other filamentous invasive fungal infections including Zygomycetes and Candida were negative.
Conclusions: Both IHC and ISH for Aspergillus can accurately identify Aspergillus sp. in tissue sections; however, IHC shows less background and stains more organisms. The fewer organisms seen by ISH may be explained by limited staining in non-viable organisms, the spatial localization of the fungal rRNA, method sensitivity or unknown factors.
Wednesday, March 21, 2012 1:00 PM
Poster Session VI # 251, Wednesday Afternoon