Interference by macro-B12: multicenter evaluation of polyethylene glycol precipitation and establishment of reference intervals using four different immunoassay methods

Abstract Objectives Macro-B12 interference is an underestimated cause of hypercobalaminemia that may lead to misleading clinical interpretations. The aims of this study were to evaluate the diagnostic performance of polyethylene glycol (PEG) precipitation for the detection of macro-B12 using four different immunoassay methods, establish reference intervals for post-PEG vitamin B12 concentrations and recovery values, and develop a screening algorithm applicable to routine clinical laboratory practice. Methods This was a prospective, multicenter observational study conducted across eight hospital laboratories. A total of 126 healthy volunteers were included for reference interval establishment, and 290 patients with elevated, non-supplemented vitamin B12 concentrations were enrolled. Vitamin B12 was measured using the Atellica (Siemens), DxI-800 (Beckman Coulter), Alinity (Abbott), and Cobas (Roche) immunoassay platforms. The presence of macro-B12 was confirmed by size-exclusion chromatography. Results Macro-B12 was identified in 58 patients (20 %), with IgG-associated immunocomplexes being the predominant form. Patients with macro-B12 exhibited significantly higher vitamin B12 concentrations than those without macro-B12. Recovery values demonstrated excellent diagnostic performance, with sensitivities ranging from 96.6 to 100 % and specificities from 96.1 to 99.6 %, outperforming post-PEG vitamin B12 concentrations. Agreement among the four immunoassay methods was very good. Liver diseases and active solid malignancies were negatively associated with the presence of macro-B12. Conclusions Calculation of recovery following PEG precipitation is a simple, reproducible, and highly effective tool for the detection of macro-B12. Implementation of the proposed screening algorithms may improve the interpretation of hypercobalaminemia and help avoid unnecessary diagnostic procedures.