Holotranscobalamin-based algorithm for the identification of macro-vitamin B12 in analytical hypercobalaminemia

Interference by vitamin B12 (B12) macrocomplexes is common but underappreciated in clinical practice. When unexpected hypercobalaminemia is detected, analytical interference must be considered. Holotranscobalamin (HoloTC) reflects the biologically active B12 fraction, but the impact of B12 macrocomplexes (M-B12) on HoloTC immunoassay performance remains unclear. We collected 528 adult serum samples. Total B12 and HoloTC were measured by commercial immunoassays. M-B12 presence was defined as B12 recovery after polyethylene glycol precipitation (PEG) <50%. Samples were classified as HyperB12 (hypercobalaminemia) or NormoB12. M-B12 was detected in 16.0% (66/411) of HyperB12 samples but in none of NormoB12. In three M-B12 cases, B12 fell below the lower reference limit after PEG. B12 recovery was higher in NormoB12 than in NoM-HyperB12 ( p < 0.001). HoloTC was measured in a subgroup of samples (NormoB12 n = 16; NoM-HyperB12 n = 41; M-HyperB12 n = 40). HoloTC was higher in M-HyperB12 (median 300 pmol/L) than in NoM-HyperB12 (median 120 pmol/L), while NormoB12 did not differ from NoM-HyperB12. After PEG precipitation, HoloTC recovery was lower than B12 recovery in HyperB12 samples (NoM-HyperB12: 29.8% vs 73.1%; M-HyperB12: 11.0% vs 27.5%), whereas the difference was not significant in NormoB12 (62.9% vs 83.5%). B12 and HoloTC concentrations showed correlation before ( r = 0.45, p = 0.004) and after PEG ( r = 0.52, p < 0.001) precipitation only in M-B12 group. HoloTC identified M-B12 presence (AUC 0.870) with 75.0% sensitivity and 95.1% specificity at 201 pmol/L. HoloTC concentrations are influenced by the presence of B12 macrocomplexes. The HoloTC assay may serve as a biomarker to screen for M-B12 in patients with analytical HyperB12. • Macro-B12 found in 16% of hypercobalaminemia; absent in normo-B12. • HoloTC polyethylene glycol recovery was lower than B12 recovery in HyperB12. • HoloTC detected macro-B12 (AUC 0.870); cutoff 201 pmol/L (Se 75.0%, Sp 95.1%). • HoloTC-first algorithm may reduce manual PEG precipitation in hypercobalaminemia.