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 thresholds for the Roche assay, we characterized B12 concentrations associated with deficiency by comparing individuals with macrocytic anemia and other anemia subtypes or no anemia. Methods We retrospectively analysed 10 years of laboratory data from adults tested for total B12 and folate (Roche Cobas), homocysteine (Abbott Architect), and haematological 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 1 mu mol/L increase in homocysteine and by 6.8 ng/L per decade of age increase (p<0.0001). Macrocytic anemia (9.4 % of subjects), was associated with a mean reduction of 18.6 ng/L in B12 levels compared with no anemia and microcytic anemia. Mean homocysteine concentrations rose progressively, from 15.9 to 21.5 mu mol/L and then to 34.9 mu mol/L, as total B12 concentrations fell in the intervals: 342-447 ng/L, 341-258 ng/L, and 257 - <50 ng/L, respectively. Conclusions Among individuals investigated for anemia, macrocytosis, a hallmark of B12 depletion, supports that total B12 concentrations <= 257 ng/L measured using the Roche assay likely reflect severe deficiency. Levels between 258 and 341 ng/L may indicate early depletion and warrant confirmation through elevated homocysteine concentrations.