Over the past decades, major advances in diagnostic assays for infectious diseases have improved the safety of blood donations. Currently, most blood collection organizations (BCOs) employ highly sensitive laboratory screening tests to detect transfusion-transmissible infections (TTIs), thereby minimizing the risk of transfusing contaminated blood products and making TTIs a rare event [1–3]. Globally, routine TTI screening typically includes tests for human immunodeficiency virus (HIV) [4,5], hepatitis B virus (HBV) [6], hepatitis C virus (HCV), human T-lymphotropic virus (HTLV) [7], and syphilis [8,9]. Additional testing for other TTIs includes Chagas disease, West Nile virus, Babesia, Malaria, and dengue, which are performed according to local epidemiology and risk assessment [10–12].

A major consequence of universal testing with highly sensitive diagnostic assays is the occurrence of false-positive results, which can have several undesirable implications. These include the unnecessary discard of safe blood units, increased laboratory workload and costs related to confirmatory testing, and significant emotional distress for donors, who may face permanent deferral or become reluctant to return for future donations [13–15].

False-positive serologic results may arise through several mechanisms. First, antibody cross-reactivity can occur in the presence of autoimmune disorders, heterophile antibodies, recent vaccinations, or unrelated infections [16,17]. Second, early-stage infections may present with incomplete antibody responses, leading to a reactive screening test but a non-reactive confirmatory result, thus interpreted as false positive [18–20]. Third, test-related technical factors, such as lot-to-lot variations or reduced assay specificity, can contribute to false reactivity [21]. Lastly, false-positive results may occur by chance, without a clearly identifiable underlying cause.

Previous studies have explored factors such as sex, educational attainment, and race/ethnicity as potential predictors of false reactivity in blood donation screening tests [13–15]. Although findings vary across infections and populations, some studies suggest that female sex, lower educational attainment, or African-American race and Hispanic ethnicity are associated with higher odds of false-positive results [13–15].

Despite these findings, recent data addressing how demographic factors influence the frequency of false-positive results using modern, high-performance assays remain limited. A clearer understanding of these associations could improve donor counseling, aid interpretation of discordant results, and encourage donors with previous false-positive findings to continue donating blood.

This study investigated the overall and annual frequencies of positive serological screening and false-positive serological results for syphilis, HIV, HCV, and HBV over a 10-year period at a blood center in São Paulo, Brazil. It also evaluated demographic factors associated with both false-positive and true-positive serologic screening results.

Data from >120,000 blood donors were analyzed in this 10-year cross-sectional study conducted at a major blood center in São Paulo, Brazil, to assess annual trends in positive screening and false-positive results for syphilis, HIV, HCV, and HBV. Demographic factors associated with false-positive and true-positive results were also examined. Nearly all donations had nonreactive screening results (98.7%), with reactive results most frequent for syphilis (0.5%) and HBV (0.5%). Among positive screening tests, false positivity was most common for HIV (92.0%) and HCV (72.7%).

As for temporal trends, given the relatively small number of reactive samples, year-to-year variability likely reflects random variations rather than true epidemiologic shifts, when considering positive serology and false-positive results for syphilis, HBV, and HCV. For HIV, a marked decline in screening reactivity occurred in 2017 and remained stable until 2022, likely reflecting the replacement of the bioMérieux CLIA platform and Ortho Clinical Diagnostics with assays from Abbott Diagnostics that year. Decreases in false-positive HIV results in 2018, 2020, and 2022 further support an assay-related effect.

In the multivariable Poisson regression models, female sex was associated with a higher prevalence of false-positive results in the HIV model, consistent with previous studies [14]. This pattern may be attributable to higher frequencies of autoantibodies and nonspecific antibody reactivity among women [23]. Such cross-reactivity is a known source of false-positive serologic findings [16,17,24].

Older age was also associated with false-positive results in the overall, syphilis and HCV models though not consistently across infections. This finding aligns with previous research suggesting that older individuals may produce higher titers of nonspecific antibodies after immune stimulation, which can increase assay cross-reactivity [25].

Lower educational attainment was another consistent predictor of false-positive results across most analyses. This association may indirectly reflect socioeconomic status: individuals with lower education may be more exposed to infections or immunologic stimuli, potentially increasing antibody cross-reactivity [13,26]. Lower educational attainment was also associated with true positivity, which may reflect a lower access to healthcare and diagnostic testing [27]. Importantly, limited health literacy associated with lower educational attainment [28] may influence the donor’s ability to understand the meaning and implications of false-positive results, potentially increasing the risk of misinterpretation and emotional distress if results are not clearly explained.

First-time donors demonstrated the strongest and most consistent association with false-positive results. Similar findings have been reported previously [7,13,14]. This association may be partially influenced by selection bias, as donors with false-positive results may be discouraged from future donations. Repeat and sporadic donors who have previously tested negative may represent a self-selected population less likely to present false reactivity, resulting in a repeat donor population with inherently lower reactivity rates. First-time donor status was also consistently associated with true-positive results, likely driven by a similar structural mechanism: donors with prior true-positive results are permanently deferred from future donation. Lastly, another possible mechanism is associated with risk behaviors: studies show that repeat donors have fewer high-risk behaviors when compared to first-time donors [29]. Return rates among donors with false-positive results were not evaluated, which could provide further insight into donor retention and behavioral responses following reactive screening.

No significant associations were found between race/skin color or marital status and false-positive results in this study although other reports have described higher false reactivity among non-White donors [7,13,14]. Such discrepancies may reflect regional demographic differences or variations in assay performance.

Due to the small number of true-positive cases, analyses to assess factors associated with true-positive results were not conducted for HIV and HCV. In the overall, syphilis and HBV models, male sex and older age were associated with higher prevalence of true-positive results, consistent with known epidemiologic patterns of TTIs. Non-white race/skin color was associated with true positivity in the overall and syphilis models but not for HBV. Across all models, lower educational attainment, being single or divorced/separated and first-time donation were consistently related to true positivity.

These findings reinforce the importance of demographic and behavioral screening in donor selection, while highlighting the balance between high assay sensitivity and the occurrence of nonspecific reactivity in maintaining transfusion safety. However, the lack of consistent overlap between variables associated with false-positive and true-positive results suggests that the mechanisms underlying false reactivity differ from those driving true infection risk.

False-positive serologic results are commonly attributed to nonspecific antibody cross-reactivity, which may occur following vaccination, infection, allergic reactions, autoimmune processes or exposure to transplantation antigens [16,17,24]. Differences in assay design and antigen composition can also influence specificity and lead to spurious reactivity [30].

The observed association between female sex and HIV false positivity may reflect immunologic cross-reactivity due to the higher prevalence of autoantibodies in women compared to men [23]. Similarly, the association between lower education and false reactivity may indicate increased immune stimulation from exposure to infections in lower socioeconomic contexts [26]. Older individuals may have accumulated immunologic experiences or demonstrate more pronounced polyclonal antibody responses, contributing to the age-related trend [25].

Although false positivity can also result from incomplete antibody responses during early infection - where screening is reactive but confirmatory testing is negative [18–20] - the lack of consistent overlap between predictors of false and true positivity in the present study suggests that this mechanism was not the predominant cause.

This study has several limitations. Results from subsequent donations of participants who experienced prior false-positive outcomes were not compared. This omission limits the possibility to analyze the underlying mechanisms that drive these occurrences. On false-positive results, additional testing was not conducted to exclude incipient infections, neither was testing to explore cross-reactivity. Although nucleic acid testing (NAT) is routinely performed in the BCO, these data were not included, and some cases classified as false positive may represent early window-period infections. However, given the low prevalence of such window-period infections, this potential misclassification is unlikely to substantially affect the overall findings [31]. Moreover, the change in HIV screening assays in 2017 led to an immediate reduction in reactivity rates, which may limit the interpretation of time trends for this infection. No similar assay transitions occurred for the other infections. Additionally, the study did not collect behavioral or health-related variables, such as sexual activity, history of infections or chronic diseases, which could influence false-positive rates. Nonetheless, this large ten-year dataset provides a robust overview of serologic reactivity trends and associated factors among Brazilian blood donors.

The findings of this study demonstrate that certain demographic characteristics, particularly first-time donation and lower educational attainment, are consistently associated with a higher prevalence of false-positive serologic results in blood donor screening. Older age showed a weaker and less consistent association. The lack of overlap between predictors of false-positive and true-positive results supports the hypothesis that most false-positive findings arise from cross-reactive antibody mechanisms rather than incomplete antibody responses.

These results underscore the need for further studies investigating immune and technical mechanisms underlying false reactivity in blood donation screening. Future research should also explore the psychological and operational impacts of false-positive results and evaluate strategies to support donor retention following false reactivity notifications.

The authors received no specific funding for this work

The data that support the findings of this study are available from the corresponding author upon reasonable request.

L.K.S. performed the research, analyzed the data, wrote the first draft of the manuscript and reviewed and edited the manuscript. L.D.S. collected data, designed the study and reviewed and edited the manuscript. V.F.D. collected data, designed the study, supervised the research and reviewed and edited the manuscript. J.M.K. collected data, supervised the research and reviewed and edited the manuscript. C.B.B. designed the study, supervised the research and reviewed and edited the manuscript.