Abstract Background Vitamin B12 is an essential water-soluble vitamin required for critical biological processes such as DNA synthesis, erythrocyte maturation, and maintenance of nervous system integrity. Deficiency of vitamin B12 can lead to serious clinical outcomes, including megaloblastic anemia and potentially irreversible neurological damage. Conversely, hypercobalaminemia may be associated with severe disorders, including solid neoplasms and hematological malignancies, or may result from inappropriate supplementation or immunoglobulin–B12 macrocomplexes. Current guidelines recommend total serum vitamin B12 and holotranscobalamin (holoTC) as first-line biomarkers for assessing cobalamin status. Content Despite guideline recommendations, total serum vitamin B12 remains the most widely used test in routine clinical practice. However, holoTC represents the biologically active fraction of vitamin B12 available for receptor-mediated cellular uptake and may therefore provide a more reliable assessment of cobalamin status, particularly in specific clinical settings. Compared with total vitamin B12 measurement, holoTC is evaluated using a more limited number of analytical methods, and most commercially available assays are traceable to the WHO reference standard, improving interassay harmonization. This review examines the role of vitamin B12 and holoTC, focusing on analytical challenges and clinical interpretation. Summary The available evidence suggests that holoTC may offer important advantages over total serum vitamin B12 in the evaluation of B12 status. Its biological relevance and improved analytical harmonization support its potential utility as a more reliable biomarker in selected clinical contexts.
