Review Article

Annals of Clinical Biochemistry

2025, Vol. 62(1) 22–33
© The Author(s) 2024

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DOI: 10.1177/00045632241292432
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The application and interpretation of laboratory

biomarkers for the evaluation of vitamin B12 status

Dominic J Harrington1,2, Emma Stevenson3  and Agata Sobczyńska-Malefora1,4

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
Vitamin B12, active B12, homocysteine, methylmalonic acid, serum B12, NICE

Accepted: 30th September 2024

Introduction – biochemical relevance and methylmalonic acid (MMA), a product of the hydrolysis of
risk factors for deficiency excessive methylmalonyl-CoA, which cannot be converted to
succinyl-CoA.1
Vitamin B12 (cobalamin; B12) is an essential micronutrient. In In the United Kingdom (UK), the prevalence of B12 defi-
humans, microgram quantities of B12 (2–3 µg/d) are required ciency is estimated to be up to 20% in some cohorts.2 Risk
to produce methylcobalamin and adenosylcobalamin, which factors for developing deficiency include prolonged omission of
are used as cofactors in two reactions (Figure 1). One of the
reactions takes place in the cytoplasm, where methionine
synthase catalyses the transfer of the methyl groups from 50 -
1
The Nutristasis Unit, Synnovis, St Thomas’ Hospital, London, UK
2
School of Biosciences and Medicine, University of Surrey, Guildford, UK
methyltetrahydrofolate to homocysteine (Hcy) via the cofactor 3
Department of Clinical Biochemistry, Pathology, Gloucestershire Royal
methylcobalamin to form methionine and tetrahydrofolate. Hospital, Gloucester, UK
Serum concentrations of Hcy increase in response to a sub- 4
Faculty of Life Sciences and Medicine, King’s College London, London, UK
optimal supply of methylcobalamin. The other B12-dependent
reaction takes place in the mitochondria, where the conversion Corresponding author:
Dominic J Harrington, The Nutristasis Unit, 4th Floor North Wing, St.
of methylmalonyl-CoA to succinyl-CoA by methylmalonyl- Thomas’ Hospital, Westminster Bridge Road, Synnovis, London SE1
CoA mutase requires the cofactor adenosylcobalamin. A 7EH, UK.
deficiency of adenosylcobalamin leads to an accumulation of Email: [email protected]
Harrington et al. 23

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.

B12 containing food from the diet, such as meat, fish and dairy, However, macrocytosis is a non-specific pathological indicator
for example, in veganism and vegetarianism; autoimmune of advanced B12 deficiency and some deficient patients express
gastritis, for example, pernicious anaemia; intestinal diseases; no haematological abnormality.4 Patients should have their B12
infections or surgical interventions; the increased requirement status evaluated if they have at least one risk factor for de-
for B12 during pregnancy and in neonatal life; pharmaceutical ficiency and at least one symptom (Table 1). Clinical judge-
interactions and nitrous oxide (N2O) abuse. In older people, ment should be used when deciding whether to test patients
there is a pronounced increase in the prevalence of B12 defi- who have no risk factors but at least one sign or symptom.5
ciency, mainly caused by gastritis and inadequate dietary intake.2 The best characterized biomarkers of B12 status are
The signs and symptoms of B12 deficiency vary con- serum B12 (also known as total B12 or serum cobalamin),
siderably from person-to-person. Classical features that may holotranscobalamin (also known as holoTC or ‘active
present include anaemia, gastrointestinal and neurological B12’), MMA and Hcy.6 The utility of B12 status biomarkers
symptoms, and those relating to psychological and psy- has not, however, been adequately studied outside of adult
chiatric disturbances.3 populations and there is a need to evaluate their use in
children, during each trimester of pregnancy and in some
ethnic groups. Audit has shown that a diagnosis of B12
Current clinical practice deficiency may take several years.7 Delays in diagnosis and
B12 deficiency is most commonly diagnosed and treated in treatment of B12 deficiency have a negative impact on
primary care with patients presenting with non-specific quality of life and increase the likelihood of permanent
complaints, such as tiredness. Clinicians may be guided to- neurologic damage.5 Despite studies consistently demon-
wards a diagnosis of B12 deficiency by the incidental detection strating that no single biomarker of B12 status exhibits the
of macrocytosis when routine blood tests are performed. performance characteristics necessary to definitively define
24 Annals of Clinical Biochemistry 62(1)

Table 1. An example assessment algorithm for vitamin B12 status evaluation. Clinical decision limits for test results are based on those
used in the National Institute for Health and Care Excellence (NICE) guidance on vitamin B12 deficiency in over 16s: Diagnosis and
management (NG239).5 Laboratories should evaluate the applicability of clinical decision limits locally.

status in all patients, the majority of diagnostic laboratories Hcy and MMA are not widely utilized as first-line bio-
rely solely on serum B12. markers. Hcy is less suitable than serum B12 and holoTC be-
To overcome the performance limitations of individual cause of stringent pre-analytical requirements in which serum/
B12 status biomarkers, laboratories should implement first- plasma must be separated from whole blood, ideally within
line testing strategies that maximize diagnostic sensitivity 60 min of sample collection, and kept on ice prior to separation
and, where initial test results are indeterminate, second-line to prevent falsely elevated results. Such prompt sample sepa-
testing strategies that maximize diagnostic specificity.6,8,9 ration is often not available in primary care and Hcy increases by
These strategies are discussed below. 1 µmol/L in 3 hours in unseparated specimens kept at room
temperature.10 Alternatively, samples can be collected into tubes
containing stabilizing preservatives.11 In contrast, MMA sam-
ples are pre-analytically stable but high-throughput automated
First-line B12 status testing
assays for routine laboratories are not yet widely available.
The high demand in healthcare for B12 status investigations One notable caveat is that neither serum B12 nor holoTC
limits the selection of first-line biomarkers to serum B12 and have diagnostic utility for the evaluation of B12 status in
holoTC, since both are pre-analytically relatively stable and can people in whom N2O abuse is suspected (refer to Hcy
be performed on highly automated clinical chemistry analysers. section below).
Harrington et al. 25

Serum B12 However, results were generated using a radioassay

(Quantaphase, Bio-Rad Laboratories, Richmond, CA) and a
Since the 1960s, the concentration of B12 in serum has been microbiologic assay using Lactobacillus leichmannii rather
the most commonly used biomarker for the assessment of than assays used in clinical laboratories today. Patients were
B12 status.6 Contemporary serum B12 methods are readily considered to be B12 deficient if they had characteristic
available on highly automated clinical chemistry platforms features that responded to B12 treatment and the authors
by competitive protein-binding assays using intrinsic factor recognized this as a limitation of their study.18
as the binding protein with chemiluminescence or fluo- Results from serum B12 assays are interpreted against a
rescence detection systems.12 reference range or a clinical decision limit for deficiency.
A low serum B12 concentration is indicative of defi- Serum B12 results are known to be subject to inter-assay
ciency but results within and above the reference interval variation (Figure 2) and, therefore, manufacturer-dependent
should be interpreted with an understanding of the limita- validated cut-offs should be used.
tions of the assay (see also Assay Limitations section be- Setting a clinical decision limit is a compromise between
low). All serum B12 tests detect the total concentration of sensitivity and specificity (Table 2). Consequently, it is good
B12, which comprises cobalamin bound to the ß-globulin practice to define an indeterminate range between frank
protein transcobalamin, forming holoTC, and B12 bound to deficiency and sufficiency. NICE NG239 suggests an in-
the ß-globulin haptocorrin, forming holohaptocorrin (hol- determinate range of 180 to 350 ng/L (133 to 258 pmol/L)5;
oHC). Only the B12 bound to transcobalamin is transported however, this will not be appropriate for all serum
into cells (holoTC), whereas there is no evidence that B12 B12 assays and laboratories should evaluate its applicability
from holoHC contributes to B12 status.13 Further, the half- locally. Second-line testing should be considered for serum
life of holoTC in blood is rapid compared with holoHC, so B12 results that fall within the indeterminate range.5,26
the majority of B12 measured by serum B12 assays is
holoHC, even though newly absorbed B12 mainly binds to
transcobalamin. Therefore, serum B12 concentrations may Limitations of approach
not reflect metabolic B12 status and the test is considered a Age, ethnicity and pregnancy are known to influence serum
late indicator of deficiency when compared with holoTC, B12 concentrations, yet cohort-matched reference ranges are
Hcy and MMA. rarely available to interpret clinical results. Children of all
ethnicities have much higher B12 concentrations than adults
and age-specific reference ranges are warranted.14,27 The
Result interpretation
reasons for much higher B12 concentrations in children have
Endogenous serum B12 concentrations in adults residing in not been elucidated.14 The application of unified reference
south east London, UK, are ∼182 to ∼692 ng/L (∼134 to ranges in Black patients may lead to an overestimation of
∼511 pmol/L) for Asian and White patients and ∼225 to B12 status because people with Black family backgrounds
∼1091 ng/L (∼166 to ∼805 pmol/L) for Black patients (data have significantly higher serum B12 concentrations when
generated using the Abbott Architect serum B12 assay).14 compared with people with Asian and White family
However, variation may be seen across the UK population backgrounds.14 In pregnancy, serum B12 are subject to an
and with different serum B12 assay manufacturers. For ∼50% fall that is related to haemodilution and a decrease in
example, in the predominantly White patient population of the synthesis of haptocorrin.28
Gloucestershire, UK, endogenous serum B12 concentrations
in adults are ∼145 to ∼424 ng/L (∼107 to ∼313 pmol/L)
when generated using the Beckman DXI platform (un-
Assay limitations
published data, author ES). Other published work shows The diagnosis of B12 deficiency is partly laboratory- and
lower reference limits for the Roche and Siemens assays that manufacturer-dependent (Figure 3). This unwarranted
align well with the authors’ published data using the Abbott variation is driven by a lack of harmonization and stan-
Architect assay. A similarly negative bias was also reported dardization by manufacturers of serum B12 assays.6 Serum
when prospectively generating a reference interval using the B12 assays are often calibrated independently by manu-
Beckman DXI platform compared to other assays.15 facturers with traceability to an internally manufactured
Results from serum B12 assays are interpreted against a standard material rather than to an international standard.6
reference range with the clinical decision point for defi- Longitudinal variation in the performance of manufacturer-
ciency commonly set in the region of 200 ng/L (148 pmol/ specific assays is revealed through external quality as-
L).16 This approach is partly informed by a study in which it sessment (EQA) performance (Figure 4). Importantly, assay
was estimated that 90 to 95% of patients with B12 deficiency bias and longitudinal variation do not align with the ac-
had concentrations <200 ng/L (<148 pmol/L), 5 to 10% had companying reference ranges. For example, assays pro-
concentrations 200 to 300 ng/L (148 to 221 pmol/L) and less vided by Abbott Diagnostics have higher reference range
than 1% had concentrations >300 ng/L (>221 pmol/L).17 upper limits, yet it is assays on the Roche platforms that are
26 Annals of Clinical Biochemistry 62(1)

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.

slightly positively biased when results are compared using antibody interference.29 High haptocorrin concentrations
EQA. The Beckman systems consistently give lower results are another cause.30
than other manufacturers. In people with signs and symptoms of B12 deficiency
False-normal serum B12 concentrations have been de- who are shown to be B12-replete, folate status should be
scribed in patients with high-titre intrinsic factor antibodies investigated. Coexisting iron deficiency or thalassemia trait
and may also occur because of the presence of heterophile may mask macrocytic changes seen on full blood counts.
Harrington et al. 27

Table 2. Summary of studies on the diagnostic performance of serum B12, Holotanscobalamin (Active B12), methylmalonic acid,
homocysteine and combined indicator of vitamin B12 status (4cB12).

Index test of deficiency Sensitivity Specificity

Biomarker Study cohort (decision point) AUC (95%CI) (decision point) (decision point) Reference

Serum 204 controls & MMA >271 nmol/L 0.836 45% (211 ng/L) 98% (211 ng/L) Herrman
B12 vegetarians 87% (359 ng/L) 56% (359 ng/L) et al.
200319
937 individuals with MMA >750 nmol/L 0.85 (0.77 – - - Hyas &
↑MMA (not 0.94) Nexo
treated) 20059
1279 neurology MMA >397 nmol/L 0.72 (0.65 – - - Schrempf
patients (>47 µg/L) 0.78) et al.
201120
1359 clinical samples MMA >300 nmol/L & 0.632 72% (308 ng/L) 41% (308 ng/L) Herrmann &
HoloTC >22 pmol/L Obeid
201321
5196 clinical samples MMA >260 nmol/L ROC (SE) 73% (157 ng/L) 74% (157 ng/L) Bolann et al.
with ≥50% reduction after 0.810 (0.034) 200022
B12 replacement
360 clinical samples MMA >450 nmol/L 0.63 (0.56 – 53% (197 ng/L) 81% (197 ng/L) Heil et al.
0.70) MMA 64% (244 ng/L) 64% (244 ng/L) 201223
320 nmol/L
0.70 (0.61 –
0.79) MMA
450 nmol/L
0.73 (0.60 –
0.87) MMA
770 nmol/L
224 clinical samples MMA >376 nmol/L - 40% (230 ng/L) 98% (230 ng/L) Hølleland
serum et al.
B12 <300 pmol/L 199924
HoloTC 204 controls & MMA >271 nmol/L 0.879 87% (35 pmol/L) 75% (35 pmol/L) Herrman
vegetarians et al.
200319
937 individuals with MMA >750 nmol/L 0.90 (0.83 – - - Hyas and
↑MMA (not 0.97) Nexo
treated) 20059
1279 neurology MMA >397 nmol/L 0.66 (0.51 – - - Schrempf
patients (>47 µg/L) 0.82) et al.
201120
1359 clinical samples MMA >300 nmol/L 0.714 72% (35 pmol/L) 54% (35 pmol/L) Herrmann &
Obeid
201321
360 clinical samples MMA >450 nmol/L 0.70 (0.64 – 83% (32 pmol/L) 60% (32 pmol/L) Heil et al.
0.77) (MMA 64% (21 pmol/L) 88% (21 pmol/L) 201223
320 nmol/L)
0.78 (0.69 –
0.87) (MMA
450 nmol/L)
0.92 (0.85 –
0.98) (MMA
770 nmol/L)
Hcy 5196 clinical samples MMA >260 nmol/L 0.768 (SE 73% 68% Bolann et al
with ≥50% reduction after 0.037) (15.0 mmol/L) (15.0 mmol/L) 200022
B12 replacement 90% 38%
(11.3 mmol/L) (11.3 mmol/L)

(continued)
28 Annals of Clinical Biochemistry 62(1)

Table 2. (continued)

Index test of deficiency Sensitivity Specificity

Biomarker Study cohort (decision point) AUC (95%CI) (decision point) (decision point) Reference

4cB12 887,871 clinical Subclinical deficiency: Subclinical Subclinical Campos

samples 4cB12 ≤0.5 and >1.5 deficiency: deficiency: et al
Serum B12 Serum B12 202025
Serum B12 86.1% (310 ng/L) 77.7% (310 ng/L)
(0.9)
HoloTC HoloTC
HoloTC (0.92) 85.7% (<45 pmol/ 81.2%
L) (<45 pmol/L)
Hcy Hcy
Hcy (0.78) 67.7% (>15 umol/ 76.7%
L) (>15 umol/L)
MMA MMA
MMA (0.91) 81.8% (>245 nmol/ 83.4%
L) (>245 nmol/L)
Possible or probably Possible or Possible or
deficiency: probable probable
4cB12 ≤1.5 deficiency: deficiency:
Serum B12 Serum B12
94.8% (226 ng/L) 92.3% (226 ng/L)
HoloTC HoloTC
93.1% (27 pmol/L) 96% 27 (pmol/L)
Hcy Hcy
87.9% 80.9%
(>16.4 µmol/L) (>16.4 µmol/
L)
MMA MMA
94.8% (>466 nmol/ 96.4%
L) (>466 nmol/L)
Abbreviations: area under the curve (AUC); reciever operating characteristic (ROC); standard error (SE); methylmalonic acid (MMA); holotranscobalamin
(holoTC); homocysteine (Hcy); combined B12 status indicator consisting of four biomarkers serum B12, holoTC, Hcy and MMA (4cB12).

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.
Harrington et al. 29

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.

Significance of high results Holotranscobalamin (holoTC, ‘active B12’)

High concentrations of serum B12 are frequently seen by clinical The measurement of holoTC in serum provides an estimate
laboratories, but seldom investigated or issued with a supporting of the abundance of B12 available for receptor-mediated
clinical comment.31 High concentrations often reflect B12 transportation into cells. HoloTC assays are available on
treatment; however, in some instances, they may be a conse- automated clinical chemistry platforms, including those
quence of underlying liver disease or haematological malig- from Abbott (since 2006), Siemens Healthineers (2016),
nancy.32 It has also been demonstrated that immune complexes Roche Diagnostics (2017) and Beckman Coulter (2020), at
between serum immunoglobulins and B12 binding protein a cost that is higher to perform per test when compared with
(macro-B12) develop in some individuals that cause assay in- serum B12 assays. If B12 is absent from a diet, this will be
terference, potentially masking a B12 deficient state.33 Methods reflected in the holoTC concentration, which will decrease
to remove these immune complexes have been described.34 rapidly in response to the lack of newly absorbed B12.35
30 Annals of Clinical Biochemistry 62(1)

Result interpretation methylmalonic aciduria and small-bowel overgrowth with

bacteria producing high amounts of propionic acid, the
A holoTC result of <25 pmol/L indicates deficiency. Re- precursor of MMA.39,40 The single nucleotide polymor-
ceiver operating characteristic curves show HoloTC to be a phism rs291466 in 3-hydroxyisobutyryl-CoA hydrolase
moderately superior indicator of B12 status when compared (HIBCH) also influences variation in MMA concentration
with serum B12 measurement (Table 2). However, in much via the valine degradation pathway.41
the same way as serum B12, any assigned cut-off point for
the interpretation of holoTC assays is a compromise be-
tween assay sensitivity and specificity. As an alternative to a
single clinical decision point, results in the range of 25 to Homocysteine
70 pmol/L may be considered as indeterminate and second- The formation of methylcobalamin, to remethylate Hcy to
line testing should be considered to clarify B12 status.5,6,8 methionine, is dependent on adequate supply of 50 -
Concentrations >70 pmol/L indicate sufficiency.5 methyltetrahydrofolate, vitamin B12 and vitamin B2. The
Population-specific reference ranges have not been de- catabolic route of Hcy disposal requires vitamin B6. The
termined. In pregnancy, holoTC is considered the preferred diagnostic utility of Hcy for the evaluation of B12 status
B12 status biomarker because, unlike serum B12 assays, it is therefore rests on the patient being replete for folate, vitamin
not impacted by the decreased synthesis of haptocorrin.28,36 B6 and vitamin B2.42,43 Assays for the measurement of Hcy
Commercially available assays for holoTC use calibra- are widely available and range from the highly automated
tors traceable to a common primary reference calibrator held immuno- and enzymatic-assays to those that are mass
by Axis-Shield Diagnostics Ltd, Dundee, Scotland, which spectrometry-based. Besides rapid separation of plasma/
confers superior harmonisation when compared with serum serum from red cells for homocysteine analysis, other
B12 assays. factors which may be taken into consideration are post-
prandial and orthostatic variations. Samples should ideally
Second-line testing be collected after an overnight fast and venepuncture should
not be performed after venous stasis or following the subject
Second-line testing should be considered when results from resting in a supine position.
first-line testing are indeterminate (see serum B12 and When interpreting Hcy results, it is recommended that
holoTC Result Interpretation sections).5,26 The best de- age- and sex-specific reference ranges are applied, although
scribed biomarkers that are considered suitable for second- this is seldom done in practice. Children and pregnant
line testing of B12 status are MMA and Hcy. women have lower Hcy, while higher concentrations seen in
the elderly are likely due to an increased prevalence of
intestinal malabsorption, reduced enzymatic activity and
Methylmalonic acid reduced kidney function. Adult males have higher Hcy
Although no ‘gold standard’ biomarker for the determi- concentrations than females.
nation of B12 status has been identified, MMA is most Plasma Hcy should be the first-choice test to assess B12
commonly used as the index test for B12 deficiency. Some status in patients exposed to N2O.3 The gas is commonly
diagnostic laboratories cite a serum MMA used for sedation and pain relief, but long-term abuse with
concentration >280 nmol/L as indicative of suboptimal B12 large doses causes severe B12 deficiency, that can present
status in patients <65 years with normal renal function.8 with multiple neurological symptoms and consequences, for
Interpretation is more challenging in the elderly and those example, inability to walk, incontinence, disruption to the
with impaired renal function where MMA is likely to be reproductive system, depression, demyelinating poly-
elevated independently of B12 status.37 A large decrease in neuropathy and, most significantly, subacute combined
MMA concentration after treatment with B12 is considered degeneration of the spinal cord. Nitrous oxide oxidizes the
confirmatory of a previously B12-deficient state. An MMA active cobalt of cobalamin, which eventually leads to dis-
concentration >750 nmol/L is accepted as indicative of sociation of cobalamin from methionine synthetase (MS)
‘definite’ B12 deficiency.38 and inactivation of apo-MS. As a result, Hcy cannot be
Unlike Hcy, MMA concentrations are not influenced by remethylated to methionine. Consequently, Hcy is the first
folate, vitamin B6 or vitamin B2 status. Contrasting with the biomarker to respond (increase) in patients who abuse N2O.
other three biomarkers of B12 status, automated MMA This is followed by a slow decrease in serum B12 and
immunoassays are not available. An automated LC-MS/MS holoTC, as well as slight elevations in MMA. Serum B12 is
based assay for MMA analysis is available, however, and often normal at presentation, which may lead to an incorrect
capable of processing several hundred samples daily.12 differential diagnosis. Although there is no specific marker
In addition to impaired renal function, other B12-inde- of N2O abuse according to levels of consumption, it has
pendent causes of increased serum MMA concentration are been suggested that MMA may have utility as a marker of
states of dehydration, impaired thyroid function, inherited clinical gravity.44
Harrington et al. 31

Composite biomarker B12 MMA measurement should be considered in people who

status evaluation have symptoms or signs of B12 deficiency and an inde-
terminate test result (see Result Interpretation sections).5
A composite score, cB12, which combines the biochemical The cut-offs for serum B12 should be applied to adult pa-
measurements of serum B12, holoTC, MMA and Hcy – tients of White and Asian origin. For children, people of
while also accounting for low folate status and corrected for Black ethnicity, and in pregnancy, appropriate reference
age – can be used to evaluate B12 status.45 The composite intervals should be used.14
score provides an index that relates biomarkers of the in- B12 replacement can commence if an initial B12 test result
dividual to the reference combination at the stipulated age. indicates deficiency, or if the initial test is indeterminate in
This reference combination has been derived from a large patients with signs or symptoms of B12 deficiency and an
database following mathematical modelling. The formula is MMA result is elevated. B12 replacement should not be
expressed as: cB12 = log10 [holoTC × B12/MMA × Hcy]  delayed if the total B12 result is indeterminate and the patient
[3.79/1 (age/230)2.6]. Depending on the cB12 value ob- has a condition or symptom that may deteriorate rapidly and
tained, B12 status is classified as: elevated, adequate, low, have a major effect on quality of life (e.g. neurological or
possible B12 deficiency and probable B12 deficiency. Less haematological conditions like ataxia or anaemia), a condi-
comprehensive formulas that utilize two or three biomarkers tion or suspected condition that is an irreversible cause of B12
have also been derived (2cB12 or 3cB12) and have been deficiency (e.g. autoimmune gastritis), had surgery that can be
shown to outperform commonly used one-biomarker test an irreversible cause of B12 deficiency (e.g. gastrectomy,
approaches.46 terminal ileal resection or some types of bariatric surgery) or
There have been no large-scale genetic epidemiological are pregnant or breastfeeding.5
studies aimed at describing the basis of variability in For people with an indeterminate result from first-line
cB12 among adults and the composite score is not yet testing and no symptoms or signs of B12 deficiency, no
widely used. further testing is required unless signs or symptoms of
deficiency develop.
If results from first-line testing for serum B12 or holoTC
Suggested approaches for the evaluation of indicate sufficiency, including during pregnancy or
breastfeeding, then B12 deficiency is unlikely. If, however,
B12 status symptoms or signs are present 3–6 months later, then the
Suggested approaches for the evaluation of B12 status are initial test should be repeated.5
shown in Table 1. Although the diagnosis of B12 deficiency
is a global challenge, the availability of clinical and tech-
nological expertise, as well as resources, vary considerably,
Establishing the cause of deficiency
and this will influence the approach taken by different A detailed review of approaches to establishing the cause of a
healthcare providers. B12 deficient state is beyond the scope of this article. A brief
Blood samples for diagnostic tests should be collected summary is provided in Table 1. Establishing the cause of
before starting B12 replacement. B12 replacement treatment deficiency is important, as in some cases the cause will be found
should not, however, be delayed while waiting for the test to be reversible and indicate short term treatment is required. In
results of people with megaloblastic anaemia or subacute other cases, the cause will be found to be irreversible, leading to
combined degeneration of the spinal cord. obligatory lifelong replacement. B12 deficiency is most com-
Either serum B12 or holoTC assays should be used as the monly corrected with oral or intramuscular doses of the vitamin.
initial test for suspected B12 deficiency, unless the test needs No toxicity is associated with the treatment.
to be performed during pregnancy or infancy, or if N2O use
is the suspected cause of deficiency. HoloTC should be used
to assess the B12 status of pregnant women and infants,
Summary
whereas Hcy should be used in N2O exposure. No single laboratory biomarker is suitable for the assess-
The interpretation of first-line B12 tests can be compli- ment of B12 status in all patients. Using biomarkers in
cated if the person is already receiving supplements since combination leads to a reduction in the number of B12-
elevated results may reflect recent exposure to B12 rather deficient people incorrectly classified as replete and B12-
than metabolic B12 status. replete people incorrectly classified as deficient. In people
Deficiency can be confirmed in people with a serum B12 with a condition or symptom that may progress rapidly,
concentration below the assay manufacturer-dependent val- treatment should not be delayed while waiting for results of
idated cut-off or a holoTC concentration <25 pmol/L.5 After a laboratory test. Treatment should continue until the cause
exposure to N2O, elevated Hcy concentration should be taken of B12 deficiency has been identified and B12 deficiency
as indicative of deficiency; Hcy concentrations >50 µmol/L reversed, or lifelong treatment should continue if the cause
are often seen in such patients. is identified as irreversible.
32 Annals of Clinical Biochemistry 62(1)

Acknowledgements management (GID-NG10176). Project information. Vitamin

B12 deficiency in over 16s: diagnosis and management.
The authors thank Renata Gorska of the Nutristasis Unit for her
Guidance. London: NICE.
artistic skills in drawing Figure 1. We are also grateful to Rachel
6. Harrington DJ. Laboratory assessment of vitamin B12 status.
Marrington and Finlay MacKenzie from UK NEQAS Haematinics
J Clin Pathol 2017; 70: 168–173.
for providing Figures 2, 3 and 4. The UK NEQAS Haematinics
7. Hooper M, Hudson P, Porter F, et al. Patient journeys: di-
service is provided by Birmingham Quality, which is part of the
agnosis and treatment of pernicious anaemia. Br J Nurs 2014;
University Hospitals Birmingham NHS Foundation Trust. This
23: 376–381.
article was prepared at the invitation of the Clinical Sciences
8. Sobczyńska-Malefora A, Gorska R, Pelisser M, et al. An audit
Reviews Committee of the Association for Clinical Biochemistry
and Laboratory Medicine. of holotranscobalamin (‘Active’ B12) and methylmalonic acid
assays for the assessment of vitamin B12 status: application in
a mixed patient population. Clin Biochem 2014; 47: 82–86.
Declaration of conflicting interests
9. Hvas AM and Nexo E. Holotranscobalamin – a first choice
The author(s) declared no potential conflicts of interest with re- assay for diagnosing early vitamin B12 deficiency? J Intern
spect to the research, authorship and/or publication of this article. Med 2005; 257: 289–298.
10. Bailey LB, Stover PJ, McNulty H, et al. Biomarkers of nu-
Funding trition for development-folate review. J Nutr 2015; 145:
The author(s) received no financial support for the research, au- 1636S–1680S.
thorship, and/or publication of this article. 11. Sobczynska-Malefora A. Methods for assessment of folate
(vitamin B9). In: DJ Harrington (ed) Laboratory assessment
Ethical approval of vitamin status. 1st ed. Cambridge, MA: Academic Press,
2018, pp. 219–264.
Not required.
12. Harrington DJ. Methods for assessment of vitamin B12. In:
DJ Harrington (ed) Laboratory assessment of vitamin status.
Guarantor
1st ed. Cambridge, MA: Academic Press, 2018, pp. 265–300.
Dr Katharine Bates. 13. Nexo E, Christensen A, Hvas A, et al. Quantification of
holotranscobalamin, a marker of vitamin B12 deficiency. Clin
Contributorship Chem 2002; 48: 561–562.
DJH was the primary author. ES and AM-S contributed to the 14. Sobczyńska-Malefora A, Katayev A, Steed D, et al. Age- and
manuscript equally. In particular ES contributed to the text on ethnicity-related reference intervals for serum vitamin B12.
limitations on applying standardized cut-offs of result interpre- Clin Biochem 2023; 111: 66–71.
tation and the comparison of different platforms. In particular AM- 15. Jassam N, Bancroft T, Luvai A, et al. Vitamin B12 reference
S contributed to performance characteristics of 1st and 2nd line intervals on Beckman, Roche and Siemens analytical plat-
testing. forms. Ann Clin Biochem 2023; 60: 417–422.
16. Devalia V, Hamilton MS, Molloy AM, et al. Guidelines for
ORCID iD the diagnosis and treatment of cobalamin and folate disorders.
Br J Haematol 2014; 166: 496–513.
Emma Stevenson  https://orcid.org/0000-0002-9509-8826
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