Background/Objectives: Grey zone serologic results in blood donor screening pose challenges for transfusion safety, donor management, and blood supply sustainability. In Saudi Arabia, standardized national protocols for managing grey zone outcomes remain lacking. This study aimed to evaluate the prevalence and follow-up outcomes of grey zone serologic results among blood donors at a Saudi hospital over a five-year period. Methods: Serological screening results of six transfusion-transmissible infections (TTIs) markers were extracted alongside nucleic acid testing (NAT) results for HBV, HCV, and HIV. The grey zone was defined as a signal-to-cutoff (S/CO) of 0.90–0.99. Repeat and follow-up results, including subsequent donations, were assessed for seroconversion. Results: A total of 48,241 donations from 38,524 donors were analyzed. Anti-HBc showed the highest reactivity (n = 2312; 4.8%), followed by HbsAg (n = 2292; 0.31%) and syphilis (n = 218; 0.5%). Grey zone results were rare, and most frequent in anti-HBc (n = 76; 0.16%), HCV (n = 39; 0.08%), and HBsAg (n = 28; 0.06%). Grey zone-to-reactive conversion upon subsequent donation was rare. Three donors who initially tested in the grey zone for anti-HBc later tested reactive in subsequent donations, but their HBV NAT remained negative. Conclusions: While grey zone outcomes were infrequent, a subset involving HBV markers showed low-level reactivity on repeat testing. For other TTIs markers, grey zone results likely reflected assay variability rather than true infection. We propose a six-month temporary deferral with follow-up serologic and NAT testing, allowing conditional re-entry for donors with consistently non-reactive results, supporting both transfusion safety and a more sustainable donor pool.

To assess the prevalence, temporal trends, and diagnostic concordance of transfusion-transmitted viruses (TTVs) using combined serological and nucleic acid testing (NAT) in a large donor population in Jeddah, Saudi Arabia. This retrospective study analysed all blood donations screened from 2019 to 2024 at King Fahad Armed Forces Hospital, using serological assays and NAT. Prevalence estimates were calculated with 95% confidence intervals. Temporal trends were evaluated using generalised estimating equations logistic regression. Multivariable logistic regression identified predictors of Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) positivity. Diagnostic agreement between serology and NAT was assessed using Cohen’s kappa statistic. Among 48,151 donors, seroprevalence was 0.4% for HBsAg, 0.3% for HCV Ab, 0.1% for HIV Ab, and 0.02% for HTLV Ab. HBc Ab was detected in 4.7%. NAT identified HBV and HCV in 0.2% and 0.1%, respectively. Logistic regression showed a significant annual decline in HBsAg and NAT-HBV. Older age and non-Saudi nationality were linked to higher HBsAg and HCV positivity. Concordance between serology and NAT was substantial for HIV but negligible for HBV and HCV. TTVs rates are low and declining, but HBV/HCV concordance underscores the need for continued dual testing to ensure transfusion safety.