Detection of Tissue Eosinophils and Evidence of Extensive Degranulation in Archived Clinical Biopsies Using a Novel Anti-Eosinophil Peroxidase Monoclonal Antibody

RATIONALE: Allergen-mediated inflammatory responses are associated with a massive expansion in the number of accumulating tissue eosinophils that often also display evidence of activation/degranulation. Unfortunately, because functionally sensitive eosinophil-specific antibodies useful in immunohistochemistry (IHC) platforms are rare, high throughput assays to assess archived clinical biopsies have been out of reach.METHODS: We have developed a novel anti-eosinophil peroxidase (EPO) mouse monoclonal antibody by sensitizing EPO knockout mice. This antibody is capable of reliably detecting both tissue eosinophils and evidence of released EPO using the most common archived clinical samples - formalin-fixed paraffin-embedded tissues.RESULTS: A standardized anti-EPO based IHC assay was used to examine gastrointestinal biopsies from patients who displayed physical symptoms suggestive of allergic inflammation (i.e., peripheral eosinophilia, atopy, diarrhea/flushing with certain trigger foods) but whose pathology reports were otherwise inconclusive. This new IHC assay confirmed that in many of these cases eosinophils were present and their numbers were not particularly elevated. However, and in contrast to the “near-normal” numbers of intact eosinophils in these cases, the EPO-based IHC assay revealed evidence for extensive and often regionally-specific eosinophil degranulation. Specifically, this assay was capable of detecting and visualizing the presence of both enucleated cytoplasmic fragments and large numbers of individual free eosinophil secondary granules.CONCLUSIONS: The availability of this new anti-EPO monoclonal antibody-based assay provides the pathology community with an invaluable resource for high throughput IHC screens of biopsies to aide in the assessments of allergic patients. RATIONALE: Allergen-mediated inflammatory responses are associated with a massive expansion in the number of accumulating tissue eosinophils that often also display evidence of activation/degranulation. Unfortunately, because functionally sensitive eosinophil-specific antibodies useful in immunohistochemistry (IHC) platforms are rare, high throughput assays to assess archived clinical biopsies have been out of reach. METHODS: We have developed a novel anti-eosinophil peroxidase (EPO) mouse monoclonal antibody by sensitizing EPO knockout mice. This antibody is capable of reliably detecting both tissue eosinophils and evidence of released EPO using the most common archived clinical samples - formalin-fixed paraffin-embedded tissues. RESULTS: A standardized anti-EPO based IHC assay was used to examine gastrointestinal biopsies from patients who displayed physical symptoms suggestive of allergic inflammation (i.e., peripheral eosinophilia, atopy, diarrhea/flushing with certain trigger foods) but whose pathology reports were otherwise inconclusive. This new IHC assay confirmed that in many of these cases eosinophils were present and their numbers were not particularly elevated. However, and in contrast to the “near-normal” numbers of intact eosinophils in these cases, the EPO-based IHC assay revealed evidence for extensive and often regionally-specific eosinophil degranulation. Specifically, this assay was capable of detecting and visualizing the presence of both enucleated cytoplasmic fragments and large numbers of individual free eosinophil secondary granules. CONCLUSIONS: The availability of this new anti-EPO monoclonal antibody-based assay provides the pathology community with an invaluable resource for high throughput IHC screens of biopsies to aide in the assessments of allergic patients.