False-normal vitamin B12 results in a patient with pernicious anaemia

P. Wainwright, S. Narayanan, P. Cook

PII: S0009-9120(15)00389-6
DOI: doi: 10.1016/j.clinbiochem.2015.08.009
Reference: CLB 9114

To appear in: Clinical Biochemistry

Received date: 19 May 2015

Revised date: 17 July 2015
Accepted date: 7 August 2015

Please cite this article as: Wainwright P, Narayanan S, Cook P, False-normal vitamin
B12 results in a patient with pernicious anaemia, Clinical Biochemistry (2015), doi:
10.1016/j.clinbiochem.2015.08.009

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 ACCEPTED MANUSCRIPT

False-normal vitamin B12 results in a patient with pernicious anaemia

P Wainwright specialist registrar in chemical pathology and metabolic medicine1, S

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Narayanan consultant in haematology2 and P Cook consultant in chemical pathology and

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metabolic medicine1

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1
Department of Clinical Biochemistry, University Hospital Southampton, Southampton, UK,

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SO16 6YD

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Department of Haematology, University Hospital Southampton, Southampton, UK, SO16

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6YD
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Address for correspondence:
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Dr Paul Cook, Consultant in Chemical Pathology and Metabolic Medicine, Department of

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Clinical Biochemistry, University Hospital Southampton, Tremona Road, Southampton, SO16

6YD
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Email: paul.cook@uhs.nhs.uk
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Tel no: 02381 206419

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Word count: 1326

Conflict of Interest: None to declare.

Funding: This research received no specific grant from any funding agency in the public,
commercial, or not-for-profit sectors

Contributors: PW and PC conceived the study and searched the literature. PW wrote the 1st
draft. SN reviewed and edited the case report section. All authors reviewed and edited the
manuscript and approved the final version of the manuscript.

Ethical approval: Patient consent obtained in writing

Acknowledgements: None
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Abstract

Pernicious anaemia is a common autoimmune disorder with a prevalence of approximately

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4% amongst Europeans. Untreated it can result in permanent neurological disability or

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death. Central to the diagnosis is establishing the presence of vitamin B12 deficiency.

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Concern has been raised recently regarding false-normal results obtained with competitive-
binding vitamin B12 assays performed on automated biochemistry platforms in patients

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with pernicious anaemia due to the presence of interfering anti-intrinsic factor antibodies in
the patient sample. We report a case in which diagnosis of pernicious anaemia was delayed

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due to false-normal vitamin B12 results. Questioning the results in light of high pre-test
probability, and knowledge of the role of functional markers of vitamin B12 deficiency
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enabled the correct diagnosis to be made so that effective treatment could be initiated. It is
crucial that those who frequently request vitamin B12 are aware of the potential problems
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with the available assays and how these problems can be addressed. We suggest that all
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patients with normal vitamin B12 levels where there is a high clinical suspicion for deficiency
such as a macrocytic anaemia, neurological symptoms or megaloblastic bone marrow
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should have a functional assay of vitamin B12 (plasma homocysteine or methylmalonic acid)
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checked to further investigate for vitamin B12 deficiency.

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Introduction

Pernicious anaemia is a common autoimmune disorder with a prevalence of approximately

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4% amongst Europeans [1]. If left untreated it can result in permanent neurological

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disability or even death [2]. Central to the diagnosis is establishing the presence of vitamin

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B12 (also known as cobalamin) deficiency. Concern has been raised in recent years
regarding false-normal results obtained with competitive-binding vitamin B12 assays

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performed on automated clinical chemistry platforms in patients with pernicious anaemia
due to the presence of interfering anti-intrinsic factor antibodies in the patient sample [3].

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We report a case in which diagnosis of pernicious anaemia was delayed due to false-normal
vitamin B12 results. Questioning the results in light of high pre-test probability, and
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knowledge of the role of functional markers of vitamin B12 deficiency enabled the correct
diagnosis to be made so that effective treatment could be initiated.
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Case Report
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A 48 year old woman was referred by her GP to the haematology department with a
macrocytic anaemia of unknown cause that had been present for 5 years. Haemoglobin at
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this time was 80 g/L (130-170) with a mean cell volume of 123 fL (80-100). Past medical
history included hypothyroidism and idiopathic intracranial hypertension for which she was
receiving levothyroxine replacement and acetozolamide respectively. There was no history
of alcohol excess. Clinical examination was unremarkable with no palpable
lymphadenopathy or hepatosplenomegaly. Folate had been checked several times over the
preceding 5 years and concentrations were normal throughout. Vitamin B12 concentrations
had also been checked many times during this period and on each occasion the result was
>1500 ng/L.

Prior to haematological review the patient was admitted to hospital as an emergency with
severe symptoms of general malaise and was found to have a worsening of her macrocytic
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anaemia with haemoglobin of 44 g/L in addition to low platelets and low white cell count.
This was preceded by significant weight loss. LDH at this time was found to be 11,500 U/L
(225-425) with normal immunoglobulins and connective tissue screen. Bone marrow biopsy
demonstrated megaloblastic features, with immature cells and hypolobulated

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megakaryocytes. The patient was treated with blood transfusions and was discharged home

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pending results of further investigations.

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She was followed up in outpatients and required red cell transfusions every 3-4 weeks.

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Cytogenetic analysis was normal and JAK2 mutation analysis was negative. In light of the
bone marrow findings and unusually high vitamin B12 concentrations consistently >1500

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ng/L functional markers of vitamin B12 [plasma total homocysteine and urinary

methylmalonic acid (MMA)] were requested. Homocysteine was significantly elevated at

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167 umol/L (reference range 0-16). Urinary excretion of MMA was determined by urine
organic acid analysis (a semi-quantitative assay) and found to be markedly increased. A
serum sample for vitamin B12 was sent for analysis on a different analytical platform and
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was found to be low with a concentration of 173 ng/L (reference range 190-660). This
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suggested assay interference leading to an initially false-normal result. Intrinsic factor

antibodies were measured and found to be strongly positive at >100 u/mL (reference range
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0-6) consistent with a diagnosis of pernicious anaemia.

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The patient was treated with intra-muscular vitamin B12 injections. Her haematological
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indices responded promptly with normalisation of haemoglobin, mean cell volume, white
cell count and platelets.

Discussion

Pernicious anaemia most often occurs in those older than 30 and affects both sexes equally
[4]. Highest prevalence is seen in northern Europeans, particularly those from the UK.
Patients commonly present with symptoms related to anaemia such as pallor, fatigue,
lethargy and dizziness. Anorexia is a common finding, and clinical examination may reveal
glossitis. Neurological abnormalities frequently occur in pernicious anaemia and result from
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vitamin B12 deficiency. This will often manifest as a peripheral neuropathy with
paraesthesia and subsequent development of weakness and ataxia [5].

Most routine biochemistry laboratories measure vitamin B12 using competitive-binding

assays. These have replaced the older microbiologic and radio-isotope dilution assays that

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were used previously. Competitive-binding assays are favoured over the other formats as
they can more easily be automated for high-throughput analysis which is important given

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how frequently vitamin B12 is requested (for instance the biochemistry laboratory at

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University Hospital Southampton processes over 100 samples each day for vitamin B12).
However, it has recently been demonstrated that the competitive-binding assays are

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susceptible to significant interference due to anti-intrinsic factor antibodies in patients with
pernicious anaemia potentially leading to false-normal results when in fact the patient is
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vitamin B12 deficient [3]. Possible problems with these assays were initially identified as far
back as the year 2000 [6]. This is problematic as it is this population of patients for whom
accurate assessment of vitamin B12 status is most important in terms of securing the
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correct diagnosis to enable effective treatment. This can lead to delay in diagnosis with
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potentially serious consequences for the patient. The problem is large as false-normal
vitamin B12 results are obtained in 22-35% of patients with confirmed pernicious anaemia
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[3].
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This potentially serious failure of competitive-binding assays could easily go undetected and
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as such there are a number of steps that need to be taken by those that request vitamin B12
to mitigate against this risk. Firstly, it is important to consider the degree of clinical
suspicion for a particular patient. If, for instance, a patient has a macrocytic anaemia or
signs or symptoms of a peripheral neuropathy then a normal cobalamin result should be
questioned. Clinicians in this situation are encouraged to contact their biochemistry
laboratory to discuss the possibility of assay interference. Measurement of plasma total
homocysteine can be very useful in this situation; levels are markedly elevated in over 95%
of patients with true vitamin B12 deficiency [7][8] and it represents a useful test as it is
relatively widely available. However it should be noted that this is not specific for vitamin
B12 deficiency as concentrations can also be increased in folate deficiency, pyridoxine
deficiency, renal failure or with inborn errors of homocysteine metabolism. MMA is also
increased in over 95% of patients who are vitamin B12 deficient and is more specific as it is
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not increased in folate or pyridoxine deficiency. However, MMA can also be elevated in
renal failure as well as small bowel bacterial overgrowth [8]. As serum MMA is not widely
available in the UK as a test for nutritional B12 deficiency plasma total homocysteine is the
most appropriate first-line follow-up test. Sending the sample to an alternative laboratory

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that analyses vitamin B12 on a different automated platform can help as the various

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available assays are not all affected in the same way by anti-intrinsic factor antibodies.

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Holo-transcobalamin represents the active fraction of plasma cobalamin and may be more

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specific for vitamin B12 deficiency. Importantly the assay does not employ intrinsic factor
antibody binding and should not be affected by the presence of intrinsic factor antibodies in
the patient’s serum.

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An additional clue with this patient was the finding of a vitamin B12 concentration
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consistently above the assay range, >1500 ng/L, prior to any supplementation. This is hardly
ever seen in patients who are not receiving vitamin B12 replacement and is, in itself, highly
suggestive of assay interference. However, more dangerous is the more common scenario
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where there is a false-elevation of vitamin B12 which places the result within the reference
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range.
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This case demonstrates the importance of questioning laboratory results that are
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unexpected or are incongruous with the clinical situation. Specifically, it is crucial that those
who frequently request vitamin B12 are aware of the potential problems with the available
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assays and how these problems can be addressed. We suggest that all patients with a
normal vitamin B12 concentration in whom there is a high clinical suspicion for deficiency
such as a macrocytic anaemia, neurological symptoms or megaloblastic bone marrow
should have a plasma total homocysteine concentration (or MMA where available) checked
as a further screen for vitamin B12 deficiency. This will prevent any delay in the diagnosis of
this common, easily treatable disorder enabling prompt initiation of treatment and the
avoidance of long-term neurological sequelae.
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References

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