A HIGH THROUGHPUT PROTOCOL FOR SPERM DNA FRAGMENTATION ANALYSIS USING THE SDF-INDEXTM ALGORITHM.

OBJECTIVE: To determine whether delayed post-staining acquisition can support higher-throughput in sperm DNA fragmentation testing in acridine orange flow cytometry testing with the SDF-Index™ algorithm. DESIGN: Prospective repeated-measures analytical validation study. MATERIALS AND METHODS: Semen samples were diluted, exposed to acid-detergent denaturation for 30 seconds, stained with acridine orange, maintained at 4-8 °C, and read by flow cytometry at 2.5 minutes and serially through 7 days. The SDF-Index™ algorithm was used to quantify DNA fragmentation (% DFI) and immaturity (% HDS). DFI categories were low <15%, average 15-<25%, high 25-40%, and very high >40%; HDS was normal <25% or elevated ≥25%. Stability was assessed using origin-traced change in percentage points (pp), Lin concordance correlation coefficient (CCC), tolerance retention, classification agreement, and paired tests with Holm adjustment. All samples were from a biorepository, and appropriate waiver was obtained from the IRB committee. RESULTS: Seventy tests from 26 specimens were used. Baseline DFI was 19.0±12.8%, with 39 low, 14 average, 11 high, and 6 very high results. Baseline HDS was 9.5±5.5%, and all HDS results were normal. DFI remained quantitatively stable through 60 minutes, with mean non-significant changes of +0.11 pp at 15 minutes, +0.28 pp at 30 minutes, and +1.04 pp at 60 minutes. Across these time points, CCC remained high (0.946-0.973), and 81%-91% of tests remained within ±5 pp of origin. DFI category discordance at 15, 30, and 60 minutes was 15.7%, 17.1%, and 21.4%, respectively; most discordant classifications originated within 5 pp of a category boundary, indicating threshold vulnerability rather than broad analytical failure. At 120 minutes, significant DFI change increased to +3.40 and CCC declined to 0.797, while DFI category discordance increased to 30.0%. HDS remained stable up to 240 minutes. CONCLUSIONS: DFI values generated by acridine orange flow cytometry and analyzed with the SDF-Index™ algorithm were sufficiently stable through 60 minutes after staining under cold storage. Later acquisition reduced quantitative and categorical interchangeability with the 2.5-minute origin. HDS remained normal-classification stable, although elevated-HDS specimens were not represented.