Discrepancies in hepatitis B surface antigen testing: A systematic review of false-positive results and underlying mechanisms with implications for laboratory hematology

INTRODUCTION

The global burden of hepatitis B virus (HBV) infection affects over 254 million people, with hematological implications extending to transfusion-transmitted infections and chronic liver disease in patients with blood disorders.^[1] In laboratory hematology, Hepatitis B surface antigen (HBsAg) detection through immunoassays is pivotal for blood donor deferral, prenatal screening, and monitoring in hematological malignancies. Despite advancements in assay sensitivity – now detecting HBsAg at concentrations as low as 0.05 IU/mL – false-positive results remain a persistent issue, complicating clinical decision-making and eroding trust in diagnostic pathways.^[2]

False positives in HBsAg testing, defined as reactive results not confirmed by neutralization or HBV DNA assays, arise from analytical interferences rather than true viremia. Reported rates vary from 0.1% in high-prevalence regions to 9% in low-endemic areas, influenced by assay type, population demographics, and underlying comorbidities.^[3,4] In hematology, these discrepancies are amplified by patient factors such as immunosuppression, frequent transfusions, and concurrent autoimmune conditions, which heighten the risk of antibody-mediated artifacts.^[5]

Historically, early enzyme-linked immunosorbent assays (ELISAs) suffered from cross-reactivity with rheumatoid factor (RF) and heterophilic antibodies (HAs), leading to overdiagnosis in rheumatological cohorts.^[6] Modern Chemiluminescent immunoassays (CLIAs), like the Elecsys HBsAg II, boast specificities exceeding 99.5%, yet isolated reports document persistent false positives post-vaccination or in paraneoplastic syndromes.^[7,8] The novelty of this review lies in its hematology-centric lens: While prior meta-analyses focused on general virology,^[9] we integrate discrepancies in blood banking and oncological hematology, where false positives can delay chemotherapy or mandate unwarranted antiviral prophylaxis.

Potential mechanisms include:

1. Immunological interferences: HAs, RF, and human anti-mouse antibodies bind non-specifically to assay capture antibodies, mimicking HBsAg reactivity.^[10]

2. Iatrogenic factors: Transient antigenemia from HBV vaccines or granulocyte-colony stimulating factor (G-CSF), common in myelosuppressive therapies.^[11]

3. Assay artifacts: Sample-related issues such as lipemia, hemolysis, or gel separator dislodgment in serum tubes.^[12]

4. Genetic variants: Escape mutations in the major hydrophilic region (MHR) of HBsAg, though more linked to false negatives, can indirectly cause confirmatory discrepancies.^[13]

This review aims to systematically appraise these mechanisms, quantify their prevalence in hematological populations, and propose mitigation strategies. By refining diagnostic protocols, we seek to bolster the fidelity of HBsAg testing in Journal of Hematology and Allied Sciences (JHAS)-aligned domains of clinical and laboratory hematology.

METHODS

This systematic review adhered to Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2020 guidelines for transparency and reproducibility.

RESULTS

DISCUSSION

This review illuminates the multifaceted nature of HBsAg false positives, with immunological interferences emerging as the predominant culprit in hematological settings – a finding underexplored in prior virology-focused syntheses.^[9,1] The 3.5% rate in blood donors underscores a public health imperative: erroneous deferrals exacerbate donor shortages, particularly in low-prevalence regions where positive predictive value plummets below 50%.^[4] Novelty herein lies in quantifying hematology-specific risks; for instance, G-CSF’s 10% interference rate in myeloid disorders necessitates pre-therapy HBsAg retesting, absent from current ASH guidelines.

Mechanistic insights reveal assay vulnerabilities: Sandwich formats amplify HA effects, as IgG/IgM bridges evade blocking agents in 25% of cases.^[10] Vaccination transients, while self-limiting, pose ethical dilemmas in mandatory screenings – e.g., deferring healthcare workers post-booster.^[17] Analytical artifacts, though less common, highlight pre-analytical standardization needs; adopting lithium-heparin tubes reduced discrepancies by 40% in one cohort.^[7]

Comparative analysis with HBcrAg assays shows analogous issues (9% false positives), suggesting multiplex panels for resolution.^[19] Limitations include publication bias toward Asian studies (high endemicity masks low-prevalence artifacts) and heterogeneity precluding meta-analysis. Strengths: Comprehensive grey literature inclusion and QUADAS-2 rigor ensure robustness.

Implications for laboratory hematology: Implement a tiered algorithm – initial CLIA screening, HA/RF reflex testing, and HBV DNA confirmation – potentially halving false positives.^[3] In transfusion medicine, integrating genotype-specific assays for MHR variants could safeguard supplies. Future directions: Prospective trials of HA-neutralizing reagents (e.g., serum diluents) and artificial intelligence - driven S/CO pattern recognition in CLIAs.

This synthesis not only refines our understanding but also equips hematologists with evidence-based tools to navigate HBsAg pitfalls [Table 1].

CONCLUSION

False-positive HBsAg results, driven chiefly by HAs and iatrogenic factors, afflict 1–9% of tests, with amplified risks in hematology. This review’s novel integration of mechanisms and populations advocates confirmatory algorithms to enhance diagnostic precision, averting clinical harms. As JHAS advances laboratory hematology, adopting these insights promises safer blood practices and equitable care.