Abstract
After developing two 5‐antigen multiplex (Mx) immunofluorescence assays using qualitative tissue controls in formalin‐fixed, paraffin‐embedded (FFPE) prostate cancer (CaP) tissue sections, we proposed to optimize control evaluation by quantification of signal‐derived features. Three CaP cell lines (LNCaP, PC‐3, DU‐145) were used as substrates for a nuclear antigen in each Mx [androgen receptor (AR) and Ki‐67]. Cells suspended in agar were FFPE as arrays (CLA) containing all three cell lines. Mx results are recorded by a computer‐driven microscope system with a Nuance(tm) camera (CRI, Woburn MA) and filters to capture serial images 10 nm apart (wave length range: 520 ‐ 720 nm) including emission spectra for all fluors used. Grey scale images identifying antigen distribution by its spectral profile are created by unmixing fluor signals in each image series. High throughput image analysis scripts and algorithms previously developed to interrogate tissue sections were adapted to measure intensity/area features of AR or Ki‐67 in CLA nuclei (classified as "DAPI objects"), expressed as (1) mean intensity of all cells, and (2) mean and (3) percentage of positive cells above a threshold derived from negative control sections. Results from a test run are thus validated by control metrics within established antigen/cell line ranges, an objective approach surpassing in accuracy and efficiency subjective visual criteria.
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