ICID > Clinical Management > Haematology > Investigation and Management of Vitamin B12 and Folate Deficiency  

Investigation and Management of Vitamin B12 and Folate Deficiency 

1. Introduction

2. In which patiens should vitamin B12 and folate values be measured

3. Interpretation of Results

4. Treatment and monitoring

5. References

1. Introduction

Vitamin B12 and folate levels are amongst the more commonly requested haematological assays, but are often used inappropriately. These guidelines are intended to ensure appropriate use and interpretation of these investigations, together with giving brief guidance on management of patients with documented deficiency.


2. In which patients should vitamin B12 and folate values be measured?

Before requesting B12/folate levels, it is important to assess the pre-test probability of tissue deficiency e.g. is there anaemia or macrocytosis? glossitis? vegan diet? GI problems or previous gastric surgery? neurological or psychiatric condition? Some of the clinical features to guide clinicians in cases of suspected B12 or folate deficiency are shown in Table 1.

Table 1: Clinical features to guide clinicians in cases of suspected B12 or folate deficiency

Clincal Feature

Possible cause of cobalamin or folate deficiency


Exclude other causes, including heamatological disorders (e.g. malignancy, myelodysplasi, haemolysis), hypothyroidism, chronic liver disorder, etc.

Evaluation of Diet

Is patient vegan or vegetarian?

Is patient anorexic or has food fads/poor diet?

Dietary deficiency

Personal and family history or autoimmune disease

Does patient, parent or sibling have vitiligo, hypothyroidism or pernicious anaemia?

Positive family history or personal autoimmune conditons increase pre-test probability or pernicious anaemia

History of glossitis or mouth ulceration

Glossitis is common presentation of low cobalamin, and mouth ulcers may reflect folate deficiency

History of parasthesiae, unsteadiness, peripheral neuropathy (particularly proprioception)

Wide differential including cobalamin deficiency, diabetes, carpal tunnel, paraproteinanaemia, other causes. Note that neurological presentation of cobalmin deficiency may occur despite normal haematological indices

Features of malabsorption

Ask about pale stool, bowel movements at night, abdominal pain, mouth or perianal ulceration

Consider history or pancreatitis due to alcohol excess


Ask about previous stomach surgery including partial gastrectomy, bariatric surgery, or small bowel resection, which may have ocurred as part of a hemicolectomy


Steatorrhoea may be due to pancreatic disease, or small bowel disease, such as coeliac disease

Terminal ileal disease with mouth and perianal ulceration may be due to Crohn disease

Gastrectomy will result in gradual depletion of cobalamin stores, with deficiency occurring 1 - 2 years post surgery

Ask about drug history

Prolonged proton pump inhibition



Oestrogen contraceptive pill


Omeprazole or equivalent result in a gastric pH of 3.0, this may affect cobalamin release from food and cause low cobalamin levels

Metformin may cause malabsorption and low cobalamin levels

May be associated with mild reduction in cobalamin levels


Pre-pregnancy folate supplements are important to reduce risk of neural tube defects

Low cobalamin levels found in third trimester may be physiological

Neurocognitive impairment in the elderly

Low cobalamin levels of uncertain significance in the elderly may be associated with neurocognitive impairment. Low cobalamin or folate levels may reflect poor diet as part of poor general condition

If none of these are present, testing for B12 or folate levels is unlikely to be useful.

The following are generally agreed indications for the measurement of vitamin B12 and folate concentrations:

  • Macrocytic anaemia

  • Macrocytosis (particularly in patients with a mean red cell volume (MCV) >110 fl)

  • Patients with specific neuropsychiatric abnormalities (vitamin B12 only if no macrocytosis or anaemia)

Note that coexisting conditions such as iron deficiency or thalassaemia trait may mask the development or presence of macrocytosis.

2.2.1 Macrocytic anaemia

Macrocytosis in itself may or may not be present with anaemia but those patients who have macrocytosis with anaemia should have vitamin B12 and folate measurements performed.

2.2.2 Macrocytosis

The level of macrocytosis can predict the probability of vitamin B12 and folate deficiency being present. As the MCV increases above 100 fl, so the probability of vitamin B12 and folate deficiency increases. This is particularly true of patients with an MCV above 130 fl, except for those receiving hydroxycarbamide.

The probability of B12 or folate deficiency is less with MCVs between 100 and 110 fl, which is more likely to be related to other causes of macrocytosis, such as alcohol abuse, liver disease, anti-neoplastic drugs, human immunodeficiency virus infection, and also haematological disorders, such as myelodysplastic syndromes or haemolytic conditions.

Blood film comments may help to point to a diagnosis: hypersegmented neutrophils and macro-ovalocytes are associated with vitamin B12 and folate deficiency, a uniform macrocytosis with alcohol abuse, target cells with liver disease, and polychromasia with haemolysis.

2.2.3 Neuropsychiatric abnormalities (check vitamin B12 only unless co-existant macrocytosis or anaemia)

Several neuropsychiatric abnormalities have been described in association with vitamin B12 deficiency (including paraesthesia, ataxia, peripheral neuropathy, and memory loss). These may occur in the absence of either anaemia or macrocytosis. Objective signs associated with vitamin B12 deficiency include impaired vibration, touch, pain, and position sense, together with an abnormal gait.

Folate levels should not be requested in patients with neuropsychiatric abnormailities unless there is a;so macrocytosis or anaemia.

2.2.4 Severe oral ulceration

B12/folate deficiency can occasionally present with glossitis or severe oropharyngeal ulceration without changes in the blood count and respond to vitamin B12 or folate treatment.

2.2.5 Coexisting conditions

Coexisting conditions such as iron deficiency or thalassaemia trait may mask the development of macrocytosis and, therefore, in patients who have anaemia without a raised MCV, dual conditions should be considered as a possible diagnosis, and vitamin B12 and folate should be measured if the origin of the anaemia cannot be established by initial tests.

2.2.6 B12 and folate should NOT be requested under the following circumstances:

  1. Young, tired all the time, chronic fatigue

  2. On treatment with B12 or folate

  3. Repeat sample with normal previous result within 1 month (folate) or 3 months (B12) 

  4. During pregnancy unless there is macrocytosis


3. Interpretation of results

3.1 Vitamin B12

Interpretation of vitamin B12 levels can be difficult. The relationship between measured vitamin B12 levels and tissue deficiency causing megaloblastic anaemia or neuropsychiatric disorders is not always clear and it is well recognised that low vitamin B12 levels only have 50% specificity for true deficiency. To add to complexity, clinically significant vitamin B12 deficiency may be present even with vitamin B12 levels in the low normal range, especially in elderly people. Please see figure to aid interpretation of low/borderline low levels.

In difficult cases, further investigations may be appropriate – please discuss with consultant haematologist. Where there is neurological or haematological abnormality and low or borderline low B12, giving a course of treatment (see below) is unlikely to do harm: reconsider ongoing replacement if there is no response.

Megaloblastic anaemia due to pernicious anaemia (autoimmune syndrome associated with chronic gastric atrophy) is rarely seen nowadays. Other conditions associated with low B12 levels include:

  • Veganism

  • Achlorhydria (including use of proton pump inhibitors)

  • Pancreatic insufficiency

  • Gastric or ileal resection/disease

  • Primary folate deficiency

  • Parasitic competition

  • Myeloma

  • Metformin administration

Women taking oral contraceptives may show decreased blood vitamin B12 levels because of a decrease in cobalamin carrier protein, rather than a deficiency state. Vitamin B12 levels may be falsely low in pregnant women because of the increased plasma volume of pregnancy rather than actual deficiency of vitamin B12. This makes it practically very difficult to diagnose vitamin B12 deficiency in pregnancy.

3.2 Pernicious anaemia

Pernicious anaemia (PA) is commonest cause of true vitamin B12 deficiency.

Anti-gastric parietal cell antibodies are present in 85% of cases, but are very non-specific, also being found in autoimmune type A gastritis, thyroid disease, iron deficiency anaemia and 3-10% of healthy population.

In contrast, anti-intrinsic factor antibodies are present in about 50% of patients with PA, but are highly specific, especially if found in combination with gastric parietal cell antibodies.

3.3 Folate

Historically our laboratory has used red cell folate as the standard test for folic acid stores, but this assay is more complex to perform than measuring serum folate, and in line with the majority of other laboratories in the UK, we will be moving over to measuring serum folate instead of red cell folate from 1st April 2011.

Conditions associated with folate deficiency include:

  • Dietary deficiency

  • Alcoholism

  • Malabsorption

  • Haemolysis

  • Malignancy

  • Pregnancy


4. Treatment and monitoring



4.1 Vitamin B12

All patients with documented clinical vitamin B12 deficiency i.e. with anaemia. macrocytosis, glossitis should be treated. Parenteral treatment is preferred, and for patients with permanently decreased ability to absorb dietary vitamin B12 (e.g. pernicious anaemia, total gastrectomy), lifelong treatment is necessary.

We recommend that patients are initially treated with parenteral vitamin B12 (hydroxycobalamin 1mg intramuscularly) and an example schedule is given below:

  • 1mg 3 times per week for 2 weeks then

  • 1mg every 3 months (every 2 months for cases with neurological presentation).

More frequent administration should not be necessary in the maintenance phase.

For patients with low B12 levels detected during investigation of more non-specific symptoms in the absence of objective clinical parameters:

  • IFAb’s should be checked if B12 level <100ng/L and lifelong treatment initiated if positive

  • For levels between 100-147ng/L, consider repeat levels after 3 months

  • If levels have normalised, no further investigations are necessary

  • If levels are persistently low, check IFAb’s and initiate lifelong treatment if positive

  • If IFAb negative, consider 4 weeks oral B12 and repeat B12 level after 3-4 months

Figure: Algorithm for investigation and management of patients presenting with a strong clinical suspicion of B12 deficiency and objective parameters to support this. MMA, methylmalonic acid; Hcy, total homocysteine; PA, pernicious anaemia

4.2 Folate

Patients with documented folate deficiency should be treated with oral folic acid. Standard dosing is 5mg/day orally for 1-4 months, or until complete haematological recovery occurs. The oral route is sufficient even in those with malabsorption. Vitamin B12 deficiency must be excluded before giving folic acid to a patient with megaloblastic anaemia since administration of folic acid may worsen neurological complications of untreated B12 deficiency.

4.3 Monitoring

How should vitamin B12 or folate deficiency be monitored in patients who have or are receiving replacement?

We recommend:

  • Initially, a full blood count after 10–14 days to document the response and after eight weeks to confirm a normal blood count

  • Longterm:   folate—not necessary unless the cause persists

  • vitamin B12—not necessary in replaced patients

Patients with PA have an increased risk of developing gastric or colorectal adenocarcinoma, with the risk appearing greatest in the first 2 years after diagnosis, so patients should be monitored for signs or symptoms of these malignancies during this period.


5. References

  1. Galloway M, Hamilton M. Macrocytosis: pitfalls in testing and summary of guidance. BMJ 2007;335:884-6.

  2. Hvas AM, Nexo E. Diagnosis and treatment of vitamin B12 deficiency—an update. Haematologica 2006;91:1506-12.

  3. Hudson B. Vitamin B12 deficiency. BMJ 2010:340;2305

  4. Smellie WSA, Wilson D, McNulty CMA et al. Best practice in primary care pathology: review 1. J Clin Pathol 2005;58:1016-24

  5. Devalia D. Diagnosing vitamin B12 deficiency on the basis serum B12 assay. BMJ 2006;333:385-6

  6. Devalia V, et al. Guidelines for the diagnosis and treatment of cobalamin and folate disorders. British Journal of Haematology 2014;166,496-513

Patient support

Vitamin B12 deficiency anaemia—information and resources on the Patient UK website (www.patient.co.uk/health/Anaemia-(Pernicious)-and-Vitamin-B12-Deficiency.htm)

Pernicious Anaemia Society—international society providing information, help, and support (www.pernicious-anaemia-society.org)

Document Owner Jonathan Cullis 
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