STABILITY AND ACCURACY OF ACRIDINE ORANGE FLOW CYTOMETRY FOR SPERM DNA FRAGMENTATION IN INFERTILITY: A 2,000+ EXAM ROLLING‑AVERAGE STUDY

Objective: To quantify sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) by acridine orange flow cytometry (WHO Laboratory Manual 6th Ed.) using a proprietary algorithm in men undergoing infertility work up, and to evaluate assay stability and reproducibility using a 100 sample rolling average analysis. Design: Retrospective study of 2,174 consecutive acridine orange flow cytometry exams performed from June 1, 2024 to April 30, 2025 at a PALC certified private reference laboratory in Brazil. Materials and Methods: Semen samples were collected at client laboratories, frozen, and shipped to a private reference laboratory. Samples were subjected to acid detergent denaturing and stained with acridine orange. 6,000 sperm were analyzed in a Beckman Coulter DxFlex flow cytometer. All tests were run in duplicates. Raw fluorescence data were processed through a proprietary algorithm developed by one of the authors (RPB) to compute DNA Fragmentation Index (DFI) and High DNA Stainability (HDS). Rolling means and SDs across 100 sample windows yielded coefficients of variation (CV), benchmarked to an “excellent” CV < 10% and an “acceptable” CV < 30%. DFI was categorized as low (<15%), moderate (15–25%), high (25–40%), and very high (>40%). Results: Across all windows, the rolling mean DFI was 25.4% (range: 20.4–31.9%), with CVs spanning 5.0–7.9%. Rolling mean HDS was 6.9% (range: 5.2–8.5%), with CVs of 5.1–9.4%, consistently below the 30% acceptable threshold, or the 10% excellent threshold. DFI category distribution revealed 28.7% low, 29.5% moderate, 22.3% high, and 19.5% very high DFI—indicating that nearly 70% of patients exhibited moderate to severe DNA damage. Conclusions: Acridine orange flow cytometry, coupled with our proprietary algorithm, provides a rapid, precise, and highly reproducible assessment of sperm DNA integrity. All CVs remained well below the accepted 30% limit, underscoring assay robustness. The high prevalence of moderate to very high DFI in this cohort highlights the assay’s critical role in guiding infertility diagnosis and personalized treatment strategies. Support: all exams were run in a private clinical laboratory in Brazil.