Needle-Based Sampling Strategy for Traceable Fresh-Frozen Tissue Collection from Radical Prostatectomy Specimens

We aimed to develop and evaluate a needle-based strategy for collecting spatially traceable fresh-frozen tissue from radical prostatectomy specimens and linking the sampled cores to corresponding formalin-fixed, paraffin-embedded (FFPE) histology while preserving routine pathological assessment.

A total of 148 patients who underwent radical prostatectomy between August 2019 and December 2025 were enrolled in the study. Immediately after resection, cores were collected from four predefined sites using an 11- or 14-gauge biopsy needle. Each tract was marked with colored ink, and touch-imprint cytology was performed before embedding in the optimal cutting temperature compound and snap-freezing. Syringe needles were left in the tracts during formalin fixation to guide gross sectioning and localization in FFPE sections.

Of the 592 cores, 584 (98.6%) were localized in the FFPE sections. There was no sampling-related interference with routine grossing or diagnostic section interpretation, assessment of surgical margin status, pathological T stage, extraprostatic extension, seminal vesicle invasion, or tumor extent. Positive cytology had a positive predictive value of 92.7%, while negative cytology had a negative predictive value of 70.1%. In the nine selected cores, RNA integrity numbers ranged from 7.1 to 8.4, and DV200 (the percentage of RNA fragments longer than 200 nucleotides) ranged from 87% to 96%. All samples yielded RNA of sufficient quality and quantity for RNA-sequencing library preparation.

The strategy developed in this study enabled the collection of spatially traceable fresh-frozen prostate tissue while preserving routine pathological assessments in the present cohort. The resulting samples were suitable for exploratory gene expression profiling linked to the corresponding FFPE histology.