What is the Real Measure of Vitamin B12 Deficiency?

By Associate Professor Chris Barnes
Published March 2026

The MBS changes introduced in 2025 have prompted necessary reflection on how vitamin B12 deficiency is best assessed in clinical practice. Our laboratory previously published an article outlining the potential advantages of Active B12 testing and its role in detecting early deficiency. With the MBS review in June 2025, it is timely to revisit that position, acknowledge the limitations of holoTC and consider alternative strategies that prioritise the detection of functional B12 deficiency. Functional vitamin B12 deficiency refers to impaired intracellular cobalamin activity despite normal or borderline serum B12 or holotranscobalamin levels (Active B12), leading to accumulation of the metabolic intermediates methylmalonic acid (MMA) and homocysteine. In the context of the 2025 MBS changes, this biochemical phenotype may be appropriately detected through reflex testing.

Revisiting the complexity of B12 deficiency

Vitamin B12 deficiency remains challenging because no single test can accurately diagnose or exclude deficiency in all clinical settings. Total serum B12 lacks sensitivity, and clinically meaningful (functional) deficiency can occur even when levels fall within the laboratory reference range.¹This is particularly relevant in older adults, where cognitive decline, neuropathy, anaemia, and frailty may develop subtly and be mistaken for ageing.²

Risk factors for the development of vitamin B12 deficiency are common and often cumulative, including:

Long-term proton pump inhibitor or H2-blocker therapy
Metformin use
Pernicious anaemia or autoimmune gastritis
Older age, gastric atrophy and reduced intrinsic factor production³

Active B12: useful but far from perfect

Although Active B12 reflects the biologically available fraction of cobalamin, several practical and biological limitations should be acknowledged.

Both serum vitamin B12 and holo-transcobalamin testing are characterised by a wide diagnostic “grey zone” that frequently necessitates confirmatory investigations and are significantly influenced by binding-protein biology.
Active B12 measures cobalamin bound to transcobalamin II, the carrier protein responsible for cellular delivery, rather than serving as a direct marker of intracellular B12 activity.
Transcobalamin II concentrations and receptor function are influenced by inflammation, liver and renal disease, ageing and genetic variation, such that holo-transcobalamin may appear normal or elevated despite impaired cellular utilisation, with documented cases demonstrating markedly elevated methylmalonic acid and homocysteine in the presence of normal serum and Active B12 levels.

A functional, clinically aligned pathway: reflex HCY testing

As methylmalonic acid is technically available under the MBS but requires specialised mass-spectrometry platforms and is not routinely accessible in many laboratories, and in response to the recent MBS changes aimed at improving identification of clinically significant (functional) vitamin B12 deficiency, our laboratory will implement reflex homocysteine (HCY) testing for all serum vitamin B12 results below 350 pmol/L, reframing this interval as “borderline” rather than “low-normal.”

This approach ensures that individuals at risk of early or functional deficiency, particularly older patients, receive additional biochemical clarification without the need for a separate test request or delays associated with referral MMA testing.

However, meaningful interpretation of HCY is essential:

Mild elevations (16–20 μmol/L) are often non-specific and may reflect renal function, folate or B6 deficiency, or pre-analytical factors
HCY >20 μmol/L correlates more strongly with clinically significant B12 deficiency and aligns with historic Active B12–based detection thresholds ^1,6

Reports will now include the updated interpretive comment:

“HCY is above the laboratory reference range, but values below 20 μmol/L are often non-specific and may not indicate clinically significant B12 deficiency. Levels >20 μmol/L are more consistent with vitamin B12 deficiency.”

Where does this leave GPs?

The inevitable question is: what is the real measure of B12 deficiency?

In practice, there is no single biochemical marker that answers this confidently. Clinical context, risk factors, and functional testing provide the most reliable pathway forward.

Our updated approach aims to:

Avoid missed deficiency in high-risk groups
Reduce over-calling of non-specific biochemical abnormalities
Provide clearer, more clinically aligned interpretive comments
Support GPs in making confident, evidence-based decisions in assessing patients at risk of vitamin B12 deficiency

How to Order Vitamin B12 Testing

For the initial assessment of a patient with no history: one Total B12 is claimable every 11 months (66838), and one Homocysteine is claimable every 11 months (66839).
For patients requiring follow-up treatment: Active B12 can be performed as a follow up of mild elevations via 66842.*
For patients with known clinical history: Total B12 is claimable via MBS item 66842.*
Eligible patients will not incur an out-of-pocket cost.

*Clinical Notes Requirement:

Patients may incur an out-of-pocket cost for Active B12 testing, or referred tests, if they do not meet the MBS criteria. Please note the reason the patient meets the MBS eligbility criteria in the Clinical Notes (see below for criteria).

MBS item 66842: Quantification of one or more of total vitamin B12, holotranscobalamin, methylmalonic acid or homocysteine for a patient:

(a) who: (i) is still experiencing symptoms of vitamin B12 deficiency 3 to 6 months after a service described in item 66838 or 66839 was rendered for the patient; or (ii) obtained inconclusive results from a service described in item 66839; or

(b) to whom one or more of the following applies: (i) the patient has a diet low in vitamin B12; (ii) the patient has a family history of vitamin B12 deficiency or an autoimmune condition; (iii) the patient has previously had abdominal or pelvic radiotherapy; (iv) the patient has previously had surgery involving the gastrointestinal tract; (v) the patient uses, or has a recent history of using, recreational nitrous oxide; (vi) the patient requires monitoring of vitamin B12 treatment; (vii) the patient uses vitamin B12‑antagonistic medicines; (viii) the patient has one or more clinical conditions with a recognised risk of vitamin B12 deficiency

References

Hunt A, Harrington D, Robinson S. Vitamin B12 deficiency. BMJ. 2014.
Wolters M et al. Cobalamin in the elderly. Prev Med. 2004.
O’Leary F, Samman S. Vitamin B12 in health and disease. Nutrients. 2010.
Russell-Jones G. ASEAN J Psychiatry. 2022.
Russell-Jones G. Paradoxical Vitamin B12 Deficiency. 2023.
Stabler SP. Vitamin B12 deficiency. N Engl J Med. 2013.

About the Author

Associate Professor Chris Barnes

MBBS FRACP FRCPA

Associate Professor Chris Barnes

MBBS FRACP FRCPA

Location: Clayton (VIC)

Speciality: Haematology

Areas of Interest:

paediatric haematology
non–malignant haematological conditions including
thrombosis and bleeding disorders

About

Associate Professor Chris Barnes joined Clinical Labs in 2016 and is the National Director of Clinical Pathology. Prior to this, he worked as a consultant haematopathologist with Healthscope Pathology from 2009. Chris is a dual-trained clinical and laboratory haematologist, having trained at the Royal Children’s Hospital and the Royal Melbourne Hospital. He also undertook training at The Hospital for Sick Children in Toronto, Canada. He holds a part-time public hospital appointment at the Royal Children’s Hospital, where he serves as the Director of the Henry Ekert Haemophilia Treatment Centre. Chris has extensive clinical research interests and is the principal investigator on eight separate clinical trials based at the Murdoch Children’s Research Institute. He has held numerous leadership positions, including Chair of the Medical Staff Association at the Royal Children’s Hospital and Chair of the Australian Haemophilia Directors Association. He is currently the Chair of the Australian Bleeding Disorders Registry with the National Blood Authority and a Director of Melbourne Haematology and Melbourne Paediatric Specialists. Chris’ focus within Clinical Labs is the national supervision and management of clinical pathology disciplines, including haematology, biochemistry, microbiology and immunopathology.