A full blood count which shows anaemia and macrocytosis should prompt the practitioner to look for a deficiency of vitamin B12 or folate. Tests commonly used for the detection of these vitamin deficiencies are serum folate, red cell folate and serum B12 concentrations. Serum folate becomes subnormal in the early stages of negative folate balance, before reduction of folate stores. Red cell folate is a direct measure of tissue folate stores. Measurement of both serum and red cell folate yields the maximum information, but in practice, red cell folate needs to be assayed only when there is macrocytosis and the serum folate is low. Serum B12 is a sensitive index of deficiency, but a low level does not necessarily indicate deficiency. Following the identification of folate or B12 deficiency, a cause must be sought.


Laboratory tests for folate and vitamin B12 are essential for the diagnosis of a deficiency of these vitamins, and for the investigation of some forms of anaemia. Untreated, deficiency of folate or B12 may lead to severe anaemia, and in B12 deficiency, crippling neurological disease. The clinical indications for testing are broad (Table 1). Often the indication for testing is an abnormality found in a full blood examination, such as unexplained anaemia or macrocytosis. Neurological conditions associated with B12 deficiency include peripheral neuropathy and subacute combined degeneration of the spinal cord. Deterioration in cognitive ability may also occur. Serum B12 should therefore be checked, even in the absence of haematological abnormality, in patients with some unexplained neurological or neuropsychiatric abnormalities.

Investigation of folate and vitamin B12 status

Three tests are in common use:

  • serum folate
  • red cell folate
  • serum vitamin B12

In addition, investigations should always include a full blood examination with assessment of a blood film. The blood tests must always be taken before specific therapy begins. After such therapy, it may be impossible to identify the underlying deficiency.

Serum and red cell folate
The usual first test for folate deficiency is assay of serum folate (reference range 7-40 nmol/L*). The margin of safety between folate intake and requirement is small so the serum folate concentration may become subnormal after only 3 weeks of negative balance (folate intake less than folate consumption). This is a stage which precedes, and may not necessarily ever progress to, body folate depletion with subsequent haematological changes. Subnormal serum folate concentration may imply body deficiency, but the serum folate level depends on recent dietary intake and is not strictly a diagnostic test for body folate depletion. For example, a low serum folate concentration without body depletion occurs with recent alcohol abuse (Table 2).

* Reference ranges for serum folate, red cell folate and serum vitamin B12 are specific to the methodology used by individual laboratories and may not be identical to the ranges given in the text.

Table 1

Indications for which testing for folate and B12 deficiency might be considered

Unexplained anaemia


Suspected malabsorption

Some neurological diseases e.g. peripheral neuropathy

Some psychiatric disorders e.g. unexplained memory loss or dementia

Malnutrition including subjects on restrictive diets e.g. vegetarians

Haematological disease associated with increased cell turnover

Alcohol abuse

Drug therapy e.g. anticonvulsants

Family history of pernicious anaemia


Red cell folate (reference range 360-1400 nmol/L) is a direct measure of tissue folate stores. It falls after about 4 months of negative folate balance. Red cell folate will differentiate between negative folate balance and body folate depletion. Low serum with normal red cell folate suggests negative folate balance. Subnormal values for both tests indicate tissue depletion.

A low concentration of red cell folate usually implies significant depletion of folate stores. Subnormal values may occur also in severe vitamin B12 deficiency, and return to normal following vitamin B12 therapy alone. A falsely normal result may occur in a folate deficient patient who has received a blood transfusion (Table 2).

Measurement of serum folate only will not differentiate between negative folate balance and tissue folate depletion. Measurement of red cell folate only may miss the early stage of negative folate balance. Serum and red cell folate yield complementary data and together the maximum information. However, in practice, it is usual to 'screen' with the serum folate assay and to proceed to red cell folate only if the serum folate is subnormal. In a folate deficient patient recently given folic acid, only red cell folate will detect deficiency.

Table 2

Problems in interpretation of serum and red cell folate levels in relation to body folate stores

False normal False low
Serum folate Patient given folic acid

Negative dietary folate balance

Recent alcohol intake

Red cell folate Following blood transfusion

Primary B12deficiency

Serum folate assay is technically easy to perform, and is reasonably reproducible, but falsely elevated values may occur in folate deficient patients who have been given folic acid. For technical reasons, measurement of red cell folate is not as reliable or reproducible as serum folate.

Serum vitamin B12
The most widely used test for B12 deficiency is the serum B12 assay (reference range 150-600 pmol/L). This is a sensitive index for the detection of B12 deficiency, but a low concentration does not necessarily indicate tissue deficiency. Conditions where the serum concentrations are low without tissue deficiency include pregnancy, folate deficiency, iron deficiency, simple atrophic gastritis, vegetarian diet, women taking oral contraceptives, and certain rare inherited disorders of B12 metabolism (Table 3). Concentrations below 150 pmol/L are sometimes seen in people (often elderly) who have no neurological or haematological abnormalities, eat a mixed diet and absorb B12 normally. Long-term observation indicates that the majority of these people do not develop clinical or haematological features of B12 deficiency. The cause of their low B12 is unknown, but low values should never be regarded as normal for the elderly.

The clinical significance of serum B12 levels that are only mildly reduced or in the region of 150-200 pmol/L may be difficult to determine. The presence of any haematological or neuropsychiatric evidence of B12 deficiency would indicate true B12 depletion and the need for B12 therapy. Although the serum B12 assay is generally reliable and reproducible, a result which does not appear compatible with the clinical findings should always be confirmed by repeat testing. A low serum B12 result should never be ignored.

Blood count and film examination
Changes in the blood count and film are relatively late manifestations of folate or B12 deficiency, but are often the first clues to the deficiency. Folate and B12 deficiency cannot be differentiated as the haematological changes are identical. The degree of anaemia varies, but macrocytosis (raised mean cell volume - MCV) and hypersegmented neutrophils are important features. Many laboratories do not regard macrocytosis as significant until the MCV is >100 fl, but in normal people the MCV should not be >95 fl, and a mild increase above this size may be the earliest haematological sign of deficiency. If there is associated iron deficiency or thalassaemia, the MCV will often not be raised despite severe folate or B12 deficiency. In addition, while macrocytosis is an important feature of folate or B12 deficiency, it occurs in many other conditions such as alcohol abuse, liver disease, hypothyroidism and with some drugs including oral contraceptives. Macrocytosis may be physiological in pregnancy and the newborn.

Table 3
Problems in interpretation of serum B12 levels in relation to body B12 stores
False normal False low

Patient given B12

Myeloproliferative disease Primary folate deficiency
Hepatoma Iron deficiency
Acute liver disease Inherited disorders of B12 metabolism
Inherited disorders of B12 metabolism Some normal subjects
Oral contraceptives

Bone marrow biopsy
In the past, bone marrow was commonly examined to confirm megaloblastic anaemia when folate or B12 deficiency was suspected. Nowadays, with the ready availability of vitamin assays, this expensive and invasive procedure is less frequently used. It is of most value in differentiating macrocytosis due to myelodysplasia (preleukaemia) and erythroleukaemia from megaloblastic anaemia.

Identifying the deficient vitamin in macrocytic anaemia
In the investigation of patients with macrocytic anaemia, it is essential to assay both serum B12 and serum and red cell folate in view of the reciprocal changes which may take place in the tests (Table 2). As red cell folate may fall moderately in patients with B12 deficiency, this test alone will not differentiate folate from B12 deficiency. Serum folate levels may be elevated, normal, or occasionally reduced in B12 deficiency. Furthermore, serum B12 levels may be reduced moderately in patients with folate deficiency. These parallel changes make it difficult to distinguish combined deficiency of the two vitamins which may occur in malnutrition or with intestinal disorders.

Metabolic assays
The role of folate and B12in metabolic pathways generates various metabolites. Assay of these metabolites has been used in the diagnosis of folate and B12 deficiency. Serum homocysteine (Hcy) is raised in both folate and B12 deficiency. Although a sensitive index, it has limited specificity because elevations occur in other inherited and acquired disorders, particularly renal impairment. In view of the interest in elevated Hcy concentrations as a risk factor for cardiovascular disease, some laboratories now offer Hcy assays routinely. This assay may help when vitamin measurements are in the indeterminate range or do not correlate with the clinical findings. A normal Hcy level excludes significant folate or B12 deficiency.

Establishing the cause of the deficiency
After folate or B12 deficiency has been identified by suitable tests, a cause for the deficiency must be sought. Folate deficiency is commonly the result of under nutrition or malnutrition in association with increased demand (e.g. pregnancy). It is important to assess dietary folate intake and to exclude gluten enteropathy (coeliac disease). Dietary faddism or reliance on `junk' foods devoid of green vegetables, an important source of folate1, renders members of all socio-economic groups susceptible to deficiency.

Pernicious anaemia is the most important syndrome of B12 deficiency. The diagnosis identifies the need for lifelong B12 treatment and the maintenance of a high index of suspicion for complications such as carcinoma of the stomach. In the past, the diagnosis was usually established by assessing B12 absorption with the Schilling test, but this is not often used nowadays. However, valuable information can be obtained from blood tests for intrinsic factor antibodies (IFA), parietal cell antibodies (PCA) and serum gastrin assay. The presence of IFA is virtually diagnostic of pernicious anaemia, but they are detected in only about 50% of cases. PCA and elevated serum gastrin are common, but they are not diagnostic. In patients in whom IFA and PCA are not detected and serum gastrin is not elevated, the cause of the B12 deficiency is unlikely to be pernicious anaemia. It is controversial as to whether further investigation is warranted before treating these patients. The Schilling test is useful in this group to distinguish intrinsic factor deficiency from intestinal malabsorption.


Jack Metz

Head of Haematology, Royal Melbourne Hospital, Melbourne