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"Normal colonic crypt epithelium adjacent to the tumour, lymphoid cells and stromal cells served as internal positive controls. In addition, on slide positive controls are a routine practice in our IHC laboratory. Technically, failed slides were excluded as the IHC process is automated and there are established standard operating procedures and quality assurance measures where every slide is vetted by a pathologist medical director and finally by the reporting pathologist. All cases in the study were internal to the institution and were fixed with 10% neutral‐buffered formalin and fixed for 24–48 h prior to sectioning as per departmental prescribed operating procedures. This ensured local uniform pre‐analytical standardisation (in the absence of universally accepted pre‐analytical guidelines for MMR IHC testing) for fixation and grossing protocols. Hence, the immunohistochemical findings were based on quality control‐verified bona fide stained cases for the MMR proteins.
Heterogeneous staining was defined according to the criteria established by Joost and colleagues as tumours showing intra‐glandular heterogeneity (strongly immunoreactive cells admixed with negative cells) and/or zonal loss (confluent areas of staining loss involving multiple adjacent glands) 14. IHC was repeated twice on each case using the same blocks. In all cases labelled as showing MMR heterogeneity according to the patterns described above, there was a distinct loss of nuclear staining in tumour cells, while normal stroma and lymphocytes showed strong nuclear staining in the same areas, thus excluding artefact and/or staining failure. An arbitrary cut‐off value of approximately 10% of the tumour showing either retention or loss of MMR proteins was used. This facilitated microdissection of the differently stained areas."
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