Background: Immunohistochemistry is essential to the pathological diagnosis of formalin-fixed paraffin-embedded (FFPE) tissue biopsies for suspected lymphomas. At present, multiple pathologist-selected single-plex chromogenic stains are performed on separate tissue sections, potentially leading to tissue exhaustion, incomplete diagnosis, and re-biopsy in a proportion of cases. Comprehensive multiplex immunohistochemistry (mIHC) on a single tissue section, such as sequential immunofluorescence (seqIF™, PMID 37813886) performed on the COMET™ platform, could potentially overcome these shortcomings, but the diagnostic utility of mIHC in pathological tissue assessment has not yet been evaluated.

Methods: A 38-marker seqIF™panel comprising most markers required for lymphoma diagnosis was optimised on the automated staining and imaging COMETTM platform (Lunaphore, a Bio-Techne brand), including 37 antibodies against immune lineage proteins and 1 RNA probe against EBER. Each stain was evaluated compared to standard clinical chromogenic stains by semi-quantitative clinical assessment scoring (CAS). 15 retrospective lymphoma cases, comprising 3 Hodgkin lymphomas, 6 low-grade lymphomas, 2 high-grade lymphomas, 2 T-cell lymphomas, and 2 reactive lymphadenopathies were processed on the COMET™. Each case was evaluated by 5 hematopathologists to assess diagnostic utility and usability of the seqIF™ panel alongside a haematoxylin and eosin stain of the tissue. Diagnoses rendered on seqIF™ were correlated with the diagnoses previously rendered on single-plex-chromogenic stains.

Results: All 38 markers in the seqIF™ panel showed equal, or better, CAS scores compared to standard-of-care single-plex chromogenic markers. Diagnostic assessment of the 15 cases (75 interactions) showed 100% correlation with the diagnosis rendered using chromogenic single-plex stains. In 5/75 (6%) of diagnostic interactions, the comprehensive seqIF™ panel contained a marker crucial to diagnosis, which was not selected in the initial panel by the assessing pathologist. Usability of the seqIF™ panel was rated equivalent to chromogenic staining in 37/75 (49.3%) interactions, and better than chromogenic staining in 38/75 (50.8%) interactions. Assessment of co-expression was rated equal to chromogenic staining 71/74 (91%) of interactions.

Conclusions: This is the first investigation into the diagnostic utility of a comprehensive mIHC panel, such as seqIF™, for lymphoma, demonstrating that the technique is safe and effective for lymphoma diagnosis. Widespread adoption of comprehensive seqIF™ for diagnosis could improve turnaround times, facilitate interpretation, reduce diagnostic error and obviate the requirement for repeat biopsies due to tissue exhaustion.

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2025
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