Inflammation within the spinal subarachnoid space leads to arachnoid hypercellularity. Multiplex immunohistochemistry (MP-IHC) enables the quantification of immune cells to assess arachnoid inflammation, but manual counting is time-consuming, impractical for large datasets, and prone to operator bias. Although automated colocalization methods exist, many clinicians prefer manual counting due to challenges with diverse cell morphologies and imperfect colocalization. Object-based colocalization analysis (OBCA) tools address these issues, improving accuracy and efficiency. We evaluated semi-automated and automated OBCA techniques for quantifying colocalized immune cells in human arachnoid tissue sections. Both methods demonstrated sufficient reliability across morphologies (P <
0.0001). While automated counts differed significantly from manual counts, their strong correlation (R