Optimizing diagnostic thresholds of total vitamin B12 (B12) for identifying cobalamin deficiency in adults with macrocytic anemia

Objectives: Identifying individuals with vitamin B12 (B12) deficiency is challenging due to poor harmonization across total B12 assays. To establish clinically meaningful thresh olds for the Roche assay, we characterized B12 concentra tions associated with deficiency by comparing individuals with macrocytic anemia and other anemia subtypes or no anemia. Methods: We retrospectively analysed 10 years of labora tory data from adults tested for total B12 and folate (Roche Cobas), homocysteine (Abbott Architect), and haemato logical parameters (Sysmex XE/XN). Individuals receiving vitamin supplementation or with isolated folate deficiency were excluded. Anemia subtypes (normocytic, microcytic, macrocytic) were classified using red blood cell count, hemoglobin concentration, and mean corpuscular volume relative to reference intervals. Results: Among 5,147 subjects (median age 65 years; 25th–75th percentile: 49–77), 36.8 % had anemia. Total B12 concentrations decreased by 2.3 ng/L for each 1μmol/L in crease in homocysteine and by 6.8ng/L per decade of age increase (p<0.0001). Macrocytic anemia (9.4 % of subjects), was associated with a mean reduction of 18.6ng/L in B12 levels compared with no anemia and microcytic anemia. Meanhomocysteineconcentrationsroseprogressively,from 15.9 to 21.5 μmol/L and then to 34.9μmol/L, as total B12 con centrations fell in the intervals: 342–447 ng/L, 341–258 ng/L, and 257– <50ng/L, respectively. Conclusions: Among individuals investigated for anemia, macrocytosis,ahallmarkofB12depletion,supportsthattotal B12 concentrations ≤257ng/L measured using the Roche assaylikelyreflectseveredeficiency.Levelsbetween258and 341 ng/L may indicate early depletion and warrant confir mation through elevated homocysteine concentrations.