B-084 The Hook Effect in Immunoglobulin A (IgA) Measurements: A Diagnostic Challenge – A Case Series

Abstract Background The high-dose hook effect, or post-zone effect, occurs in immunoassays when extremely high analyte concentrations saturate antibody binding sites, leading to falsely low results. Turbidimetric and nephelometric assays measure analyte levels by forming antigen-antibody complexes that scatter light. Under optimal conditions, larger complexes form, increasing turbidity and allowing accurate quantification. However, when analyte levels are excessive, antibodies become saturated, preventing proper complex formation and leading to underestimation of concentrations. To mitigate this effect, laboratories use sample dilutions, multi-point calibrations, and automated flagging of non-linear assay responses. This phenomenon is particularly relevant in high-concentration analytes such as IgA, IgG, IgM, free light chains, and C-reactive protein (CRP). Failure to recognize the hook effect can lead to misinterpretation of results and potential mismanagement of patients. Objective: This case series highlights instances where the hook effect led to inaccurate IgA measurements, prompting changes in our laboratory workflow to enhance result accuracy and patient care. Methods We identified two recent cases with significant discrepancies between M-protein and IgA concentrations. At the time, our workflow involved integrating the interpretation of serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE), but without consistently completing quantitative IgA and free light chain measurements beforehand. Additionally, we employed delta checks, which were instrumental in identifying discrepancies, particularly in older patients. Following these cases, we revised our protocol to withhold quantitative values for any new patient until a pathologist reviews the SPEP findings. This allowed direct comparison between M-protein and immunoglobulin levels before finalizing reports. We adopted a more extensive dilution strategy for measuring free light chains and immunoglobulins in new patients to minimize antigen excess interference. Results Both cases demonstrated significant underestimation of IgA concentrations before dilution: Case 1: A newly diagnosed IgA multiple myeloma patient had an M-protein level of 7.2 g/dL, yet initial IgA testing yielded only 372 mg/dL. After a 1:99 dilution, the IgA concentration measured 5,246 mg/dL, which was confirmed by the reference laboratory (5,320 mg/dL). Recognizing the interference, we flagged this patient for mandatory dilution in future tests. Case 2: Two months later, another patient presented with an M-protein level of 4.9 g/dL and an initial IgA of 497 mg/dL. Due to concerns about the discordance, further dilution testing revealed an IgA level of 5,137 mg/dL, confirmed by the reference lab (5,270 mg/dL). Since implementing this workflow in 2020, a retrospective review suggests that over five years, up to 30 cases may have been affected by the hook effect before our intervention. Conclusion These cases highlight the importance of recognizing and mitigating the hook effect in IgA quantification. Failure to account for this issue can lead to underestimation of immunoglobulin levels, misdiagnoses, and inappropriate clinical decisions. Routine serum dilutions for samples, along with delayed result finalization until thorough review, are critical steps to ensure accuracy for any new patients. Laboratory professionals must remain vigilant for assay interferences and communicate effectively with clinicians to resolve discrepancies and improve patient management.