Hepatitis Serology Reference Ranges: Kit-Specific Variability and Laboratory Requirements
Yes, hepatitis serology reference ranges and units differ substantially between assay kits, and each laboratory must establish and display its own specific cut-off values rather than relying on universal standards.
Why Reference Ranges Vary Between Kits
Each commercial kit expresses results in its own arbitrary units because no international reference calibrator exists for most hepatitis serologic assays, making direct comparisons between different manufacturers' kits impossible. 1 The cut-off values can differ by up to five-fold between different assay systems, even when testing identical samples 1.
For hepatitis B surface antigen (HBsAg) quantitation specifically, good correlation exists between major manufacturers (Abbott, Diasorin, Bio-Rad, Roche), but small systematic differences persist with mean differences ranging from -0.27 to 0.11 log₁₀ IU/mL 2. These differences, while statistically acceptable, underscore why each laboratory cannot simply adopt another facility's reference ranges.
Laboratory Responsibilities for Establishing Cut-Offs
Each laboratory—even those using commercial kits—must determine its own local cut-off value by testing at least 50 (preferably 100) healthy normal individuals. 1 The control group should predominantly include women, as these antibodies appear more frequently in females 1.
The cut-off must be calculated using the method of percentiles rather than adding standard deviations to the mean, because the distribution of antibody values is not Gaussian 1. Kit manufacturers should indicate unit values corresponding to the 95%, 97.5%, and 99% percentiles of the control group distribution, in addition to their recommended cut-off 1.
Standardization Efforts and Current Limitations
For hepatitis C antibody (anti-HCV) testing, the specificity of enzyme immunoassays exceeds 99%, yet among populations with infection prevalence below 10%, approximately 35% of screening-test-positive results are false positives (range: 15–60%) 1. This high false-positive rate in low-prevalence populations occurs despite excellent kit performance, emphasizing why laboratories must validate cut-offs against their specific patient demographics.
Serum HBV DNA levels should now be expressed universally in international units per milliliter (IU/mL) to ensure comparability between assays and clinical trials. 1 The World Health Organization has defined an international standard for HBV DNA nucleic acid amplification techniques, and several quantification assays have been normalized to this standard 1. In general, one IU equals approximately 5–6 copies, depending on the specific assay 1.
Practical Implications for Clinical Laboratories
Laboratories must display their specific cut-off values on test reports because:
- Arbitrary units are not interchangeable: Different kits measure the same analyte using different capture antibodies, detection systems, and calibration standards 1
- Sensitivity varies dramatically: Super-high-sensitive HBsAg assays can be 10–40 times more sensitive than conventional chemiluminescence kits, detecting 16 additional HBV-infected patients in six months that conventional kits missed 3
- Specificity differs by kit: Among nine HCV antibody test kits, false-positive frequencies ranged from 0.2% to 1.8%, meaning even high-specificity kits produce one false positive per 500 samples 3
Common Pitfalls to Avoid
Do not assume that a "positive" result from one laboratory is equivalent to a "positive" from another laboratory using a different kit. The optical density cut-off values differ widely—up to five-fold—between commercial assays 1. For the majority of commercial kits, manufacturers provide no indication in the package insert about how to calculate the cut-off value 1.
Do not rely solely on manufacturer-stated cut-offs without local validation. Even when using the same kit, patient population characteristics (age distribution, sex ratio, prevalence of occult infection) influence the optimal cut-off for a given laboratory 1.
For hepatitis B genotyping, use direct sequencing-based techniques as the gold standard because all mutations can be detected, which is particularly important for identifying resistance patterns 1. Reverse hybridization techniques and real-time PCR are acceptable alternatives if validated against sequencing 1.
Bottom Line
There is no "unique fixed unit" for hepatitis serology across all laboratories. Each facility must establish and document its own reference ranges based on the specific assay kit used, validated against an appropriate local control population. This requirement applies universally to qualitative antibody/antigen detection and quantitative viral load assays, ensuring accurate interpretation and appropriate clinical decision-making 1.