Validation of an automated chromogenic in situ hybridization protocol for detection of cytomegalovirus in formalin-fixed, paraffin-embedded renal graft biopsies.

Abstract

Introduction: Histopathological diagnosis of human cytomegalovirus (HCMV) infection in formalin-fixed, paraffin-embedded (FFPE) tissues stained with hematoxylin-eosin relies on the identification of characteristic cytopathic changes, including eosinophilic intranuclear and cytoplasmic viral inclusions. Chromogenic in situ hybridization (CISH) enables the localization of specific nucleic acid sequences in histological sections, increasing diagnostic sensitivity. Automated CISH platforms allow standardized and reproducible detection of viral RNA or DNA in FFPE tissues. This study aimed to validate an automated CISH protocol for the detection of HCMV in multiple tissue types, including renal allograft biopsies processed at the Anatomical Pathology Division of UERJ.

Methods: Two groups of FFPE samples were analyzed. The first group included ten samples from various tissues, including kidney, palate, esophagus, stomach, and colon; nine positive and one negative for HCMV by immunohistochemistry (IHC). The second group included twenty renal allograft biopsies; nineteen without previous diagnosis and one positive by IHC. For CISH, fluorescein-conjugated oligonucleotide probes targeting HCMV RNA expressed during the early replication phase were used, with hybridization and detection performed on an automated platform.

Results: In Group 1, all samples, including the case previously negative by IHC, were positive for HCMV by CISH. In Group 2, six of the twenty renal biopsies were positive, including the sample already identified as positive by IHC.

Conclusions: The automated CISH protocol demonstrated high sensitivity and reproducibility for HCMV detection, supporting its validation and use in the diagnosis of renal biopsies and other tissues, as well as its incorporation into the routine workflow of Anatomical Pathology laboratories.