Endgames Case Report

A woman with macrocytic anaemia and confusion

BMJ 2014; 349 doi: http://dx.doi.org/10.1136/bmj.g4388 (Published 08 July 2014) Cite this as: BMJ 2014;349:g4388
  1. Alan Sugrue, senior house officer,
  2. Aoife Egan, specialist registrar,
  3. Anthony O’Regan, consultant respiratory physician
  1. 1Galway University Hospital, County Galway, Ireland
  1. Correspondence to: A Sugrue alansugrue{at}gmail.com

An 82 year old woman with a history of type 2 diabetes presented with unsteady gait and confusion of two months’ duration. Her diabetes was controlled by metformin (500 mg, three times a day) and she had no other comorbidities. She was a non-smoker and did not drink alcohol. Dietary history showed an adequate intake of meat, vegetables, and calories.

On examination, her vital signs were stable. She was disorientated in time but not in person or place. Cranial nerve examination was normal but examination of the peripheral nervous system showed ataxia and impaired proprioception of the lower limbs bilaterally. The rest of her examination was normal. Her score on the mini-mental state examination at presentation was 18 out of 30.

Blood tests showed: haemoglobin 55 g/L (reference range 120-150), white blood cell count 3.9×109/L (4-10), neutrophils 2.4×109/L (2-7), lymphocytes 1.4×109/L (1-3), red blood cell count 1.22×1012/L (3.8-4.8), mean cell volume 133 fL (84-96), platelets 127×109/L (150-400), bilirubin 28 µmol/L (1-19) (all other liver function tests were normal), lactate dehydrogenase 31.7 µkat/L (2.3-3.6). Her haptoglobin was <0.08 g/L (0.58-2.43) and vitamin B12 and serum folate were 61.2 pmol/L (138-651.5) and 13.6 nmol/L (6.8-38.5), respectively. Absolute reticulocyte count was 6×109/L (50-100). Ferritin was raised at 605.8 pmol/L ng/mL (22.5-449.4). Antiparietal cell antibodies and anti-intrinsic factor antibodies were negative. A peripheral blood smear showed hypersegmented neutrophils and oval macrocytes. A megaloblastic picture was seen on bone marrow biopsy, with no evidence of dysplastic features.

No abnormality was seen on computed tomography of the brain, and oesophagogastroduodenoscopy and biopsy showed no evidence of gastritis.


  • 1. What are the causes of red cell macrocytosis?

  • 2. What is the most likely cause of macrocytosis in this patient?

  • 3. What are the causes of this condition?

  • 4. How should this condition be investigated?

  • 5. How should this patient be managed?


1. What are the causes of red cell macrocytosis?

Short answer

Red cell macrocytosis can be divided into two categories: megaloblastic and non-megaloblastic. Megaloblastic macrocytosis is generally secondary to vitamin B12 or folate deficiency. Non-megaloblastic macrocytosis is commonly caused by excess alcohol intake, hypothyroidism, myelodysplasia, or drugs.

Long answer

Red cell macrocytosis is defined as a mean cell volume greater than 100 fL. The most common causes of macrocytosis are excess alcohol intake, liver disease, hypothyroidism, and vitamin B12 or folate deficiency. However, the likelihood of these different causes being encountered in clinical practice depends on the surrounding population and demographics. For example, in the past decade the incidence of macrocytosis associated with drugs, particularly zidovudine (nucleoside for treatment of HIV), has increased.1 2

Broadly speaking, macrocytosis can be divided into megaloblastic and non-megaloblastic (box). Megaloblastic macrocytosis is differentiated from non-megaloblastic by the occurrence of abnormal erythrocyte precursors in the bone marrow, particularly ones that are larger than normal and have nuclei that appear more immature than their cytoplasm.3 The underlying mechanism behind these megaloblastic changes is disrupted DNA synthesis in the cell. Non-megaloblastic anaemia does not have a common pathogenic mechanism, and causes include excess alcohol intake and liver disease.

Causes of megaloblastic macrocytosis versus non-megaloblastic macrocytosis

Megaloblastic macrocytosis
Vitamin B12 deficiency
  • Dietary deficiency

  • Lack of intrinsic factor

  • Pernicious anaemia

  • Gastric surgery or ileum resection

  • Small bowel bacterial overgrowth

  • Fish tapeworm disease (Diphyllobothrium latum)

  • Familial selective vitamin B12 malabsorption (Imerslund-Gräsbeck syndrome)

  • Drug induced vitamin B12 malabsorption

Folate deficiency
  • Dietary deficiency

  • Pregnancy

  • Infancy

  • Alcoholism

  • Drug induced folate deficiency

  • Extensive intestinal resection, jejunal resection

Drugs and toxins
  • Folate antagonists (such as methotrexate, proguanil, pyrimethamine, and trimethoprim)

  • Purine antagonists (such as 6-mercaptopurine, dacarbazine, and fludarabine)

  • Pyrimidine antagonists (such as cytosine arabinoside, 5-fluorouracil, capecitabine, and gemcitabine)

  • Alkylating agents (such as cyclophosphamide)

  • Zidovudine

  • Hydroxyurea

  • Nitrous oxide

  • Alcohol

Non-megaloblastic macrocytosis
  • Alcoholism

  • Liver disease

  • Myelodysplastic syndrome

  • Hypothyroidism

  • Reticulocytosis

Mean cell volume greater than 100 fL may not reflect a pure red cell macrocytosis, and a mixed iron and vitamin B complex deficiency should be considered. In addition, cold agglutinins, hyperglycaemia, and marked leucocytosis can cause false increases in mean cell volume,4 and the normal value for mean cell volume in newborn babies is greater than 100 fL until about 1.5 months of age.

2. What is the most likely cause of macrocytosis in this patient?

Short answer

Macrocytosis was secondary to severe vitamin B12 deficiency with subsequent haemolysis and pancytopenia. The most likely cause is drug (metformin) induced vitamin B12 malabsorption because she had negative tests for pernicious anaemia, an adequate dietary intake, no history of gastric or ileal resections, and no evidence of an infectious cause. In addition, bone marrow examination confirmed a megaloblastic process.

Long answer

The patient’s vitamin B12 level was very low, and given the other normal test results, this suggests that vitamin B12 deficiency led to macrocytosis, anaemia, and her clinical symptoms of confusion, as is well described in the literature.5 6

Hypersegmented neutrophils are considered almost pathognomonic for megaloblastic anaemia. This phenomenon is variably defined, but in general it refers to the presence of more than five nuclei in 4-5% of neutrophils. It is important to appreciate that although hypersegmentation of neutrophils is strongly associated with vitamin B12 or folate deficiency, it is not specific. It can also occur in patients receiving chemotherapeutic agents such as hydroxyurea and 5-fluorouracil or steroids.7 The mechanism behind hypersegmentation is unclear. Our patient also had raised lactate dehydrogenase and bilirubin, along with a low haptoglobin. This suggests the occurrence of haemolysis. This has been reported in about 10% of people with vitamin B12 deficiency.8 Haemolysis in vitamin B12 deficiency has been linked to ineffective erythropoiesis, with subsequent intramedullary destruction of red blood cells.9 However, it has also been associated with high homocysteine levels.10

The patient had pancytopenia on presentation, although her absolute neutrophil count was within the normal range. She subsequently underwent bone marrow aspiration to investigate this and to assess whether the macrocytosis was megaloblastic, in keeping with vitamin B12 deficiency, or whether there was another haematological cause. The bone marrow confirmed hypercellularity in keeping with B12 deficiency. Bone marrow aspiration is usually not necessary in patients with vitamin B12 deficiency, but we wanted to ensure that there was no other haematological cause of pancytopenia in our patient. Vitamin B12 deficiency rarely presents with pancytopenia, although it has been described in paediatric populations11 12 13 and in adults,14 15 particularly those taking metformin.16 17

3. What are the causes of this condition?

Short answer

The most common cause of vitamin B12 deficiency worldwide is autoimmune gastritis (pernicious anaemia). Other gastric abnormalities to consider include total or partial gastrectomy or ileal resections. Drugs are being increasingly implicated, particularly proton pump inhibitors and metformin. Rarer considerations include dietary deficiency (particularly in vegans), fish tapeworm (Diphyllobothrium latum), and hereditary causes.

Long answer

Autoimmune gastritis (pernicious anaemia) is the most common cause of severe deficiency of vitamin B12.18 A high prevalence of pernicious anaemia is not only reported in Europe,19 but is also highly prevalent in studies of megaloblastic anaemia in Africa,20 21 the Middle East,22 India,23 and Asia.24 Pernicious anaemia is caused by an underlying autoimmune destruction of gastric parietal cells, which leads to loss of intrinsic factor. Loss of intrinsic factor impairs absorption of vitamin B12 across the terminal ilium, thereby causing deficiency. The immune response is directed at the H/K-ATPase in the gastric cells and the resultant cellular damage results in achlorhydria. Loss of gastric parietal cells can also occur in people who have had gastrectomy (total or subtotal) or gastric bypass, and this also leads to insufficient intrinsic factor. Another cause is dietary deficiency of vitamin B12. This is particularly common is adults who eat a strict, restricted diet, such as vegans. This can also occur in infants who are exclusively breast fed by vitamin B12 deficient mothers.25

Drugs can also reduce vitamin B12 levels—metformin and proton pump inhibitors are two common drugs that have been shown to have this effect.26 27 28 29 30 Proton pump inhibitors (such as omeprazole) reduce the secretion of gastric acid and this affects the release of vitamin B12 from food. The exact mechanism of how metformin reduces vitamin B12 remains unknown. Many hypotheses have been proposed—altered bowel motility, bacterial overgrowth, or a direct inhibitory effect on vitamin B12 absorption. This last hypothesis, which has gained the most standing, is based on the theory that metformin affects the calcium dependent uptake of vitamin B12-intrinsic factor complexes in the terminal ileum. This is supported by the observation that calcium supplements reverse the deficiency in people with vitamin B12 deficiency as a result of metformin use.31

Infectious causes such as fish tapeworm (D latum) are also associated with low vitamin B12 values. D latum has a unique affinity for vitamin B12 and competes with the host for this vitamin. Rarer causes include nitric oxide, which inactivates methionine synthase, thereby inactivating vitamin B12. Methionine synthase catalyses the regeneration of methionine from homocysteine in the S-adenosylethionine cycle. A reduction in this enzyme reduces the production of methionine and tetrahydrofolate, which is required in DNA synthesis. Lastly, Imerslund-Gräsbeck syndrome (also known as juvenile megaloblastic anaemia) is a familial form of vitamin B12 deficiency caused by mutation of the gene encoding cubulin (a receptor that binds vitamin B12 to intrinsic factor).

In our patient, the most likely cause of vitamin B12 deficiency was the use of metformin, given that she had negative tests for pernicious anaemia, an adequate dietary intake, no history of gastric or ileal resections, no evidence of an infectious cause, and a bone marrow biopsy result that confirmed a megaloblastic process.

4. How should this condition be investigated?

Short answer

After a thorough history and clinical examination, blood should be taken for vitamin B12 and folate measurements in addition to intrinsic factor and parietal cell antibodies. Blood film should be assessed for hypersegmented neutrophils.

Long answer

After a methodical history taking and physical examination—to look for underlying symptoms of anaemia and possible clues to its cause—the first key tests to perform are assays for vitamin B12 and folate. The choice of method for measuring vitamin B12 is controversial. Levels of vitamin B12 tend to vary between method used (chemiluminescence or radioassay), and agreement between commercial assays is poor. Furthermore, the testing of vitamin B12 involves the use of competitive binding luminescence assays and results may not always accurately reflect actual vitamin B12 stores.32 Consequently, a clear gold standard test is lacking.10 Coexisting treatments and medical conditions that falsely lower serum B12 values include use of the oral contraceptive pill, multiple myeloma, pregnancy, and folate deficiency. Clinicians must be aware that higher than normal levels of vitamin B12 have been reported in myeloproliferative disorders, owing to increased concentrations of haptocorrin (a circulating binding protein of vitamin B12).33 Given these limitations, clinicians should be careful when interpreting vitamin B12 values and consider the variation that can occur with each patient. It is important to be aware that a normal value doesn’t exclude vitamin B12 deficiency and a high value may be pathological.

Given the difficulty in interpreting vitamin B12 values, it is recommended that clinicians use the following guidelines to help interpret values34:

  • Greater than 220 pmol/L: deficiency is unlikely

  • 150-220 pmol/L: deficiency is possible

  • Less than 150 pmol/L: deficiency is likely.

A value of less than 150 pmol/L has a reported specificity of 95.2-100% for detecting vitamin B12 deficiency.35 In our patient the vitamin B value of 61.2 pmol/L was consistent was vitamin B12 deficiency. Because of the inconsistencies in current vitamin B12 assays, the measurement of metabolites, particular methylmalonic acid and homocysteine, can complement the assessment of vitamin B12 deficiency. These metabolites are more specific and should be measured when vitamin B12 deficiency is suspected but vitamin B12 values are borderline. These metabolites can also differentiate vitamin B12 deficiency from folate deficiency. Both metabolites are raised in vitamin B12 deficiency but only homocysteine is raised in folate deficiency. The usefulness of newer metabolites such as holotranscobalamin is being further evaluated.36 When should a clinician test for these metabolites? It is now accepted that testing should be considered in people with borderline vitamin B12 values, unexplained neurological problems or macrocytosis, and in those in whom there is a high degree of suspicion of vitamin B12 deficiency.18

It is also important to measure folate because folate deficiency can coexist with vitamin B12 deficiency. Folate values are also controversial because serum levels reflect short term folate balance only. It is recommended, however, to measure serum folate as the initial screening test,18 and to follow it with the red cell folate test if uncertainties arise because of borderline values or if the clinician is considering both vitamin B12 and folate deficiency in the patient. Of note, patients with vitamin B12 deficiency can also have high serum folate values and low red cell folate levels.

After a low vitamin B12 value is confirmed, testing to identify a cause can begin. Examination of the patient’s diet will confirm whether there is adequate intake of vitamin B12. If this is the case, then a problem with malabsorption must exist. Tests for pernicious anaemia involve the detection of anti-intrinsic factor antibodies or anti-parietal cell antibodies. The presence of anti-intrinsic factor antibodies almost always confirms the diagnosis, with a sensitivity of 50-70% and a specificity approaching 100%.18 In those that do not have anti-intrinsic factor antibodies it may be wise to consider measuring gastrin levels.

5. How should this patient be managed?

Short answer

All patients with documented B12 deficiency can be treated parenterally or orally. The parenteral route is the preferred one in patients with pernicious anaemia. If the cause cannot be reversed, lifelong treatment is necessary, and a standard treatment regimen is cyanocobalamin 1 mg every day for one week, followed by 1 mg every week for four weeks, then 1 mg a month.34

Long answer

Parenteral vitamin B12 is recommended and intramuscular or deep subcutaneous injections can be used. The parenteral route is the preferred method for patients with pernicious anaemia. The recommended dose is 1 mg every day for one week, followed by 1 mg every week for four weeks. If the underlying cause of B12 deficiency cannot be reversed, then 1 mg every month for the remainder of the patient’s life is necessary. Cyanocobalamin is not a pure form of vitamin B12. It is referred to as a vitamer, and active vitamin B12 components are produced when it is metabolised. There is a new trend to give oral and nasal preparations, and preliminary data suggest that they could be just as effective.37 38 39 Although the possibility of erratic absorption needs to be considered,40 oral and nasal routes could play a role in medical treatment after correction to normal levels, especially in those who need lifelong treatment. Care should be taken in the replacement of folate in people with a concomitant vitamin B12 deficiency because the administration of folate without replacement of vitamin B12 can worsen neurological sequelae. It is imperative that potassium levels are monitored closely in people with severe vitamin B12 deficiency who undergo replacement because dramatic proliferation of bone marrow cells increases the uptake of potassium, which can result in life threatening hypokalaemia.41 Other possible electrolyte disturbances include hypomagnesaemia and low iron levels, so these should be watched closely.

Anaemia from vitamin B12 deficiency develops over a long period of time and may be severe. Transfusion of packed red cells is usually unnecessary, however, unless the severe anaemia exacerbates myocardial ischaemia or heart failure. Preventive measures include ensuring adequate dietary intake and prophylactic vitamin B12 supplementation after gastric surgery. Adequate and effective replacement will see resolution of blood counts in two months and an improvement in neurological symptoms at six months. Guidelines on the management of metformin induced B12 deficiency are lacking. Some clinicians withdraw metformin, treat the deficiency, and use an alternative glucose lowering drug, whereas others take a more pragmatic approach and continue to prescribe metformin and replace vitamin B12 parenterally with hydroxycobalamin.36 37 In our patient, we elected to discontinue metformin because we had the option of using an alternative glucose lowering agent, gliclazide (a sulfonylurea) Routine screening of vitamin B12 levels in patients with diabetes who are treated with metformin should be considered.42

Patient outcome

Metformin was discontinued and vitamin B12 was replaced through intramuscular injections using the following regimen: 1 mg every day for one week, 1 mg every week for four weeks, then 1 mg once a month for three months. At her outpatient review six months after starting treatment, the patient’s haemoglobin was 112 g/L, with mean cell volume 95.0 fL, white blood cell count 8.9×109/L, neutrophils 7.0×109/L, and lymphocytes 2.9×109/L. The patient’s gait had normalised and the mini-mental state examination score had improved to 23 out of 30.


Cite this as: BMJ 2014;349:g4388


  • Competing interests: We have read and understood BMJ policy on declaration of interests and declare: None.

  • Provenance and peer review: Not commissioned; externally peer reviewed.

  • Patient consent obtained.


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