The application and interpretation of laboratory biomarkers for the evaluation of vitamin B12 status

Abstract

Vitamin B₁₂ (cobalamin; B₁₂) is an essential micronutrient, but deficiency is common. The prompt diagnosis and treatment of B₁₂ deficiency protects against megaloblastic anaemia, neuropathy and neuropsychiatric changes. Biomarkers of B₁₂ status include the measurement of serum B₁₂ (also known as total B₁₂ or serum cobalamin), holotranscobalamin (holoTC or 'active B12'), methylmalonic acid (MMA) and total plasma homocysteine (Hcy). There is no 'gold standard' test for deficiency and the sensitivity and specificity of each biomarker for the evaluation of B₁₂ status is affected by analytical and biological factors that may confer a high degree of diagnostic uncertainty. Limited access to technical and clinical expertise can lead to an over-reliance on the serum B₁₂ test, which is readily available and highly automated. In some cases, the sequential use of different B₁₂ status biomarkers or the calculation of a composite B₁₂ status score, derived from a panel of B₁₂ biomarkers and adjusted for folate status and age, can be used to detect deficient states that may otherwise be overlooked when using a single biomarker approach. This review summarizes the utility of B₁₂-related biomarkers and describes approaches to their application and interpretation.

Keywords: NICE; Vitamin B12; active B12; homocysteine; methylmalonic acid; serum B12.

Figure 1.

Vitamin B₁₂ absorption and intracellular processing via two enzymatic pathways. In the absence of vitamin B₁₂, 5-MTHF becomes metabolically trapped in this form producing a pseudo folate-deficient state (methyl-trap) and cannot be utilized for regeneration of THF. Cbl, cobalamin; CBS, cystathionine beta-synthase; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; DHFR, dihydrofolate reductase; HC, haptocorrin; holoTC, holotranscobalamin; HO-Cbl, hydroxocobalamin; IF, intrinsic factor; MS, methionine synthase; Me-Cbl, methylcobalamin; MTHFR, methylene tetrahydrofolate reductase; MMA, methylmalonic acid; MCM, methylmalonyl-CoA mutase; 5-MTHF, 5-methyltetrahydrofolate; SAH, S-Adenosyl homocysteine; SAM, S-Adenosyl methionine; THF, tetrahydrofolate; TS, thymidylate synthase; TC, transcobalamin. Reproduced from reference 3 with permission.

Figure 2.

Data from the UK NEQAS Haematinics Scheme showing performance calculated over a rolling window of 6 months (18 External Quality Assurance specimens circulated) by seven analytical methods used for the analysis of vitamin B12 in serum. Methods clockwise from top left [UK NEQAS method abbreviation]: Abbott Architect [AB13]; Abbott Alinity [AB20]; Roche Cobas/Modular [BO5]; Beckman DxI [SF5]; Siemens Atellica [SM20]; Siemens Centaur [CO10]; Ortho Vitros [AM12]. The B score is the average bias of all Specimen % biases [(result – target)/target]×100% during the rolling 6-month window. The C score is the SD of the B score and shows consistency of bias over the same rolling time period. The grey box indicates the 5th to 95th centiles for each method. The unfilled box indicates the overall 5th to 95th centiles irrespective of method. The dotted box indicates limits of acceptable performance defined as ±20% B score and 20% C score. All analyses were performed during October 2023. With permission from Birmingham Quality, University Hospitals Birmingham NHS Foundation Trust.

Figure 3.

Data from the UK NEQAS Haematinics Scheme showing variation in result interpretation following analysis of distributed aliquots of a single specimen of serum B₁₂. All analyses were performed during October 2023 with laboratories applying their local reference range. Interpretation ranges from low B₁₂ status to high status. With permission from Birmingham Quality, University Hospitals Birmingham NHS Foundation Trust.

Figure 4.

Data from the UK NEQAS Haematinics scheme showing longitudinal bias over a window of 5 years by seven analytical methods used for the analysis of vitamin B12 in serum. Analyses were performed from 2019 to October 2023. With permission from Birmingham Quality, University Hospitals Birmingham NHS Foundation Trust.