Digital Rectal Examination

The design of the Swedish arm of the ERSPC in Göteborg, Sweden, has been previously reported [22 (link)]. In brief, the study population consisted of all males living in Göteborg, Sweden, on 12 December 1994, and who were born between 1 January 1930 and 12 December 1944 (n = 32,298). Of these men, 9972 were randomly selected to undergo initial PSA testing between 1995 and 1996. All men were re-invited for PSA testing every second year up to 2005, unless they were diagnosed with prostate cancer, were aged over 70 or had died. Men with a level of total PSA in serum ≥3.0 ng/ml were invited to undergo clinical examination by an experienced urologist. This examination included digital rectal examination (DRE), and transrectal ultrasound guided laterally directed sextant biopsy of the prostate [23 (link)]. All biopsy specimens were evaluated by a single pathologist. Tumors were classified according to the 1997 International Union Against Cancer staging system [24 ] and graded according to the Gleason grading system [25 ].
Seven milliliters of blood was collected by venipuncture in Vacutainer^® tubes from every man who signed informed consent to undergo PSA testing. The blood was allowed to clot, and serum was separated from blood cells by centrifugation at 3000 g for 20 minutes, separated and frozen within 3 hours from collection, and kept frozen at -20°C until analysis. Free and total PSA were measured within 2 weeks from the blood draw by Dr Lilja's laboratory at the Wallenberg Research Laboratories, Department of Laboratory Medicine, Lund University, University Hospital UMAS in Malmö, Sweden, using the dual-label DELFIA Prostatus^® total/free PSA-Assay (Perkin-Elmer, Turku, Finland) as reported previously [22 (link)]. During 2005 and 2006, analyses of intact PSA and hK2 were performed at Dr Lilja's laboratory. Samples had been frozen at -20°C for up to 2 years, thawed and aliquoted once, and then stored frozen at -70°C until analysis. All analyses were conducted blind to biopsy result.
The performance of the Prostatus^® assay has been comprehensively documented previously [26 (link)]. The combination H117/H50 detects free PSA and PSA bound in complex to 1-anti-chymotrypsin (ACT) in an equimolar fashion [27 (link)], and also fully cross-reacts with hK2 [28 (link)]. The combination of Mabs (monoclonal antibody) H117/5A10 used to measure free PSA does not cross-react with hK2 [28 (link)], or with PSA-ACT (<0.2%) [27 (link)].
Immunodetection of hK2 is based on previously reported in-house research assays [28 (link),29 (link)] where important modifications provide enhanced low-end precision and linearity [30 (link)]. Mab (6H10) has 5% cross-reaction to PSA, but this cross-reaction to PSA is eliminated (0.005%) by the addition of PSA-blocking Mabs (2E9, 2H11, 10, 36). We have previously described the performance of this assay [31 (link)]. Our method for assaying intact PSA has been published [8 (link),17 (link),32 (link)]. Briefly, biotinylated 5A10 antibody is used to capture the free PSA; after incubation and a wash step, we add as detection antibody europium-labeled intact PSA antibody 5C3 that loses binding to PSA when PSA is internally cleaved at Lys145–Lys146. The analytical detection limit of the assay is 0.035 ng/ml.

Fecal samples were collected during a single visit to each farm. Individual disposable gloves were used for collection of each sample and samples were stored in individual 15 mL polypropylene sample tubes. Rectal samples were collected on 8 farms from the recto-anal junction with individual rectal palpation sleeves. Pasture samples were collected as fresh feces (approximately 30–50 g) via gloved hand from the top and center of a freshly voided fecal pat, where the individual animal was observed to defecate. Samples were transported on ice to the laboratory at the University of California, Davis where they were kept refrigerated at 4°C if culture could be performed within 48 h or stored at −80°C in tryptic soy broth with 25% glycerol.

This was a retrospective observational study approved by the local institutional review board. Initially, 1203 patients who underwent systematic transrectal ultrasound (TRUS)-guided and MRI-targeted prostate biopsies in institution 1 (First Hospital of Shanxi Medical University, Taiyuan, China) between January 2018 and June 2021 were included for model development. All patients underwent evaluation for elevated serum PSA levels (4 ng/mL) or abnormal results of a digital rectal examination (DRE) or TRUS. Exclusion criteria were previous prostate biopsy, prior treatment for PCa (prostatectomy, radiotherapy, focal therapy, hormonal therapy, etc.), and incomplete data. Finally, 847 patients were enrolled for evaluation (Supplementary Fig. 1).
A total of 317 patients in the external validation cohort underwent biopsies between May 2019 and January 2022 from institution 2 (Shanxi Bethune Hospital, Taiyuan, China). The biopsy indications and exclusion criteria were the same as those for the development queue. Of the 317 patients, 208 were enrolled in cohort 2 (Supplementary Fig. 2).