Ki‐67 assessment—agreeability between immunohistochemistry and flow cytometry in canine lymphoma

Recent literature suggests a combination of flow cytometric determination of Ki‐67 and immunophenotype as a reliable tool to classify canine lymphomas. Immunohistochemistry (IHC) on histological samples is the gold standard technique assessing Ki‐67 index. Agreement between IHC and FCM derived Ki‐67 indices has never been investigated. The aim of this study was to investigate the agreement between IHC and FCM in the assessment of Ki‐67 expression/index, in order to evaluate whether FCM may serve as a non‐invasive alternative method for the estimation of proliferative activity in canine lymphoma. Dogs with previously untreated canine lymphoma undergoing diagnostic lymphadenectomy were prospectively enrolled. Ki‐67 expression/index was assessed by FCM and IHC and expressed as percentage of positive cells. 39 dogs classified by histopathology matched the inclusion criteria. With both methods, Ki‐67 expression/index was higher in intermediate/high‐grade lymphomas. Spearman's coefficient of correlation was ρ = 0.57; (95% CI0.33‐0.75) suggesting a moderate correlation. A Bland‐Altman plot revealed a negative constant bias of −3.55 (95% CI: −10.52 to 3.42) with limits of agreement from −45.71 to 38.61. The study confirmed agreement albeit with wide confidence intervals between the values of Ki‐67 expression/index assessed with FCM and IHC. Discrepancies were observed in a subset of cases. Possible explanation could be that Ki‐67 index in IHC is determined in the most proliferative areas of the slide, which could introduce kind of sampling bias, whereas FCM evaluates many more cells in cell suspension. Further studies are warranted to investigate this phenomenon.