Failure of the holotranscobalamin assay in vitamin B12-deficient patients

Background: It has been demonstrated that vitamin B12 determinations fail, especially in patients with pernicious anemia with high titers of intrinsic factor antibody. Consistent with this finding, we observed a case of falsely normal holotranscobalamin (HoloTC) results in a patient with pernicious anemia and severe vitamin B12 deficiency. We aimed to investigate whether such a situation can also be seen in other individuals. Methods: Within the frameworks of the SENIORLAB study and routine samples from a mixed patient population referred to a laboratory for investigation of B12 status, we searched for study participants displaying a normal HoloTC level (>= 50 pmol/L) together with a decreased total vitamin B12 level (<125 pmol/L). Thereafter, we determined whether samples with discrepant biochemical markers (i.e. low vitamin B12, normal HoloTC) also had increased functional markers of vitamin B12 deficiency (methyl malonic acid [MMA], homocysteine [Hcy]) and/or a low value of Fedosov's combined indicator of vitamin B12 status (<-0.5). Results: The prevalence of a normal HoloTC level and low total vitamin B12 level among the group of healthy seniors (n = 1451) was 0.21% (95% confidence interval [CI], CI, 0.08-0.6%). Among the 106,635 routine samples with concurrent HoloTC and total vitamin B12 determination, 176 (i.e. 0.17%, 95% CI, 0.14-0.19%) had discrepant biochemical markers. Among them, 24 who were identified as having discrepant biochemical markers and a diagnosis of vitamin B12 deficiency could be confirmed with functional markers. Conclusions: Initial and isolated screening for vitamin B12 deficiency using a HoloTC cut-off of >= 50 pmol/L in a small subset of patients may reveal false-negative (normal) results, meaning that patients with vitamin B12 deficiency may remain undetected.