Pathophysiology

Pernicious anaemia accounts for 80% of cases of megaloblastic anaemia due to impaired absorption of vitamin B12.¹

Vitamin B12 is present in meat and animal protein foods. Absorption occurs in the terminal ileum and requires intrinsic factor, a secretion of gastric mucosal (parietal) cells, for transport across the intestinal mucosa. In pernicious anaemia, intrinsic factor production is deficient. It is believed to be an autoimmune disease.

Parietal-cell antibody and antibodies to intrinsic factor are found in nearly all cases. 90% of patients have antibodies to parietal cells and their components, including antibodies to intrinsic factor and the proton pump H+/K+-ATPase.² 50% of patients have thyroid antibodies.¹

Pernicious anaemia may be associated with simple gastric atrophy in 15% of people aged 40-60 years and 20-30% of the older population. Pathology shows gastritis with all layers of the body and fundus atrophied. The antrum is spared in >80% of patients (type A gastritis).³

Helicobacter pylori infection has been mooted to be an initiating factor, with subsequent autoimmune changes affecting the gastric mucosa. Genetic susceptibility to this process has been suspected.⁴

Epidemiology¹

The incidence of the disease is 1:10,000 in northern Europe. The disease occurs in all races. The peak age is 60, although it is starting to be recognised in younger age groups. ⁵ The condition is more common in those with blue eyes, early greying, a positive family history and blood group A. The condition has a female:male ratio of 1.6:1.0.

Presentation^1,3,6

• The onset is usually insidious as B12 stores in the liver are depleted, and starts with symptoms of anaemia, i.e. lethargy and breathlessness. The anaemia may be more severe than symptoms suggest, due to physiological adaptation.
• Other symptoms may include anorexia, weight loss, diarrhoea and dyspepsia. Glossitis may be an early symptom.
• Pernicious anaemia may first present as an incidental finding during the investigation of (reversible) diarrhoea.⁷
• Neurological involvement may be present even in the absence of anaemia. This is particularly common in patients over the age of 60. The peripheral nerves are most commonly involved, followed by the spinal cord (subacute degeneration of the cord).
• Peripheral loss of vibratory sense and position are early indications of central nervous system (CNS) involvement, accompanied by reflex loss and mild-to-moderate weakness. Later stages may be characterised by spasticity, Babinski's responses and ataxia.
• Other uncommon neurological symptoms include impairment of pain, temperature and touch sensations. The legs and feet are involved earlier and more consistently than the hands.
• Yellow-blue blindness may occur.
• Psychiatric symptoms (usually more prominent in advanced cases) may include depression, paranoia (megaloblastic madness), delirium, confusion and dementia.³
• Signs may include anaemia and jaundice.
• Severely anaemic patients may present with heart failure, often triggered by an infection. Hepatomegaly and splenomegaly⁸ may be present.

Differential diagnosis^1,3

Causes of vitamin B12 deficiency

• Poor-quality diet, vegetarian diet.
• Gastric causes - gastrectomy, congenital intrinsic factor deficiency.
• Intestinal causes - stagnant loop, congenital selective malabsorption, ileal resection, inflammatory bowel disease.
• Infestation - fish-tapeworm.
• Metabolic causes - transcobalamin II deficiency, nitrous oxide anaesthesia.
• Drugs causing decreased B12 levels - oral calcium-chelating agents,³ aminosalicylic acid,³ biguanides.

Causes of megaloblastic anaemia

• Folate deficiency - poor diet, goat's milk,⁹ gluten-induced enteropathy, tropical sprue, pregnancy, prematurity, chronic haemolytic anaemias (e.g. sickle cell anaemia), malignant disease, increased renal loss (congestive cardiac failure, dialysis), drugs (anticonvulsants, sulfasalazine).

Causes of macrocytosis^1,6

• Alcohol excess - the most common cause of macrocytosis in the UK;¹⁰ may co-exist with folate deficiency in spirit drinkers¹¹ (not seen in beer drinkers due to high folic acid content in beer¹²).
• Liver disease.
• Severe hypothyroidism.
• Reticulocytosis (e.g. post acute blood loss or haemolytic anaemia).¹³
• Other blood disorders - red-cell aplasia, aplastic anaemia, myeloid leukaemia, myelodysplastic disorders.
• Changes in plasma proteins (e.g. increased paraprotein secondary to multiple myeloma) may cause a spurious rise in mean cell volume (MCV) without the presence of macrocytes.¹⁴
• Drugs that affect DNA synthesis, e.g. azathioprine, hydroxyurea.

Investigations^1,6

Tests commonly performed in primary care

Full blood count:

• This may show low haemoglobin and increased MCV, although macrocytosis can precede the development of anaemia. Severe cases may show a pancytopenia.
• The reticulocyte count may be low for the degree of anaemia (1-3% only).
• The MCV may be normal if there is associated iron deficiency.

• This may show macrocytic red cells, neutrophils with hypersegmented nuclei and Howell-Jolly bodies (residual fragments of the nucleus causing spherical blue-black inclusions on red blood cells seen on Wright-stained smears.¹⁵
• Associated iron deficiency may result in the MCV being normal, in which case two types or red blood cells may be seen (a dimorphic blood film).¹⁶
• The ferritin level should be checked if such a picture is seen.

• There may be an increase in plasma unconjugated bilirubin due to increased destruction of red-cell precursors in the marrow. Liver and thyroid function tests and protein electrophoresis may help in the differential diagnosis of macrocytosis.
• Serum vitamin B12 is the most commonly used method of establishing B12 deficiency. In general, levels <111 pmol/mL reliably indicate deficiency. Neurological deficiency or anaemia is usually evident in patients with levels <89 pmol/mL. False positives (low levels in the absence of deficiency) can occur with pregnancy, folate deficiency, myeloma,¹⁷ and excessive vitamin C intake.
• False negatives (normal levels in the presence of deficiency) may occur in true deficiency, liver disease, lymphoma, autoimmune disease and myeloproliferative disorders. In borderline cases or where B12 deficiency is clinically suspected, other tests must be carried out. Tissue deficiency of B12 results in raised levels of serum methylmalonic acid and this is a useful test where false positive of negative values are suspected. Other tests include transcobalamin II B12 content and plasma total homocysteine.¹⁸
• Folic acid levels should be measured to exclude deficiency, which may co-exist with B12 deficiency. Red-cell folate is a better guide to deficiency than serum folate.¹ B12 deficiency may result in increased serum folate levels but reduced red-cell folate levels, because of the effect on intracellular folate metabolism.¹⁹ Combined deficiency usually results in both reduced serum folate and vitamin B12 levels.

Tests which may be performed in secondary care

The Schilling test:

The purpose of this test is to differentiate between patients whose B12 deficiency is due to pernicious anaemia and those who have an intestinal lesion causing malabsorption. It measures the absorption of B12 with and without intrinsic factor.

• The patient must not take B12 for five days prior to the test.
• Radioactive B12 is given orally, followed in one to six hours by a parental B12 'flushing' dose (1,000 micrograms) to avoid liver storage of radioactive B12.
• The percentage or radiolabelled material in a 24-hour urine collection is then measured (normally >9% of the dose given).
• Reduced urinary excretion (<5%) in the presence of normal kidney function supports the diagnosis of decreased absorption of vitamin B12.
• Repeating the first test (Schilling I) using radiolabelled cobalt attached to intrinsic factor from a hog (Schilling II) will confirm if absorption is increased, thus supporting the diagnosis of pernicious anaemia.³

The Schilling test has its limitations:

• Radiolabelled vitamin B12 is difficult to obtain, it is complicated to perform and test results can be difficult to interpret in (often elderly) patients with renal insufficiency.²⁰
• Because the Schilling test does not measure absorption of food-bound B12, the test will not detect defective liberation of food-bound B12 in the elderly patient.²¹ Furthermore, the test result often does not contribute much to the ultimate management of the patient.

If a Schilling test is felt inappropriate, in elderly patients with a low vitamin B12 level and negative intrinsic factor antibodies, response to vitamin B12 may be adequate to confirm a diagnosis of pernicious anaemia if:

• The person feels better in 1-2 days.
• The reticulocyte count increases in 2-3 days and peaks in 5-8 days.
• The red blood cell count increases within 1 week and normalizes in 4-8 weeks.
• The MCV increases for 3-4 days (due to the increased reticulocyte count), then decreases, reaching the normal range in 25-78 days.
• Haemoglobin level increases by 2-3 g/dL every 2 weeks.
• White blood cell and platelet counts normalise in 7-10 days.

Associated diseases¹

• Vitiligo
• Myxoedema
• Hashimoto's disease
• Addison's disease
• Giant cell myocarditis
• Hypoparathyroidism
• Diabetes mellitus

Management¹

• For patients with no neurological involvement, treatment is with six injections of hydroxocobalamin, 1 mg in 1 mL at intervals of between 2-4 days.
• Subsequently, 1 mg is usually given at intervals of three months. There is as yet no evidence-based guidance as to the optimum regime but the National Institute for Health and Clinical Excellence (NICE) is considering releasing guidance in due course. It should be remembered that serum B12 is not always an accurate reflection of deficiency at a cellular level.²⁴ It is perhaps for this reason that some patients become symptomatic if the frequency of their injections is reduced, despite having normal serum B12 levels.
• For patients with neurological involvement, referral to a haematologist is recommended. Initial treatment is with hydroxocobalamin 1 mg on alternate days until there is no further improvement, after which 1 mg should be given every 2 months for life.¹
• Care should be taken not to give folic acid (instead of B12) to any patient who is B12-deprived, as this may result in fulminant neurological deficit.³
• Oral iron therapy should be given before B12 if iron deficiency is diagnosed by an absence of stainable Fe in the bone marrow or other parameters (e.g. serum ferritin <449 pmol/mL).³

When to refer¹

Referral to a gastroenterologist should be considered for any patient with pernicious anaemia who has gastric symptoms and/or co-existent iron deficiency. Patients with pernicious anaemia have a 2-3 times increased incidence of gastric carcinoma and gastric polyps compared with matched controls.

Complications¹

• Heart failure - this may be secondary to anaemia, or rarely, myocarditis.
• Angina.
• Neuropathy - subacute combined degeneration of the cord, optic atrophy, neurosis, depression and dementia.
• Gastric carcinoma.
• Infertility (rare).⁶
• Iron deficiency anaemia - secondary to the achlorhydria which results from gastric mucosa atrophy.⁶

Prognosis⁶

Before the advent of treatment with B12, the disease was fatal, hence the name 'pernicious'. However, pernicious anaemia responds rapidly to replacement therapy and most patients have a normal lifespan with little morbidity. If the deficiency has been severe and prolonged, any neurological complications may be irreversible.¹

Document references

1. Anaemia - B12 and folate deficiency, Clinical Knowledge Summaries (April 2008)
2. Burman P, Mardh S, Norberg L, et al; Parietal cell antibodies in pernicious anemia inhibit H+, K+-adenosine triphosphatase, the proton pump of the stomach. Gastroenterology. 1989 Jun;96(6):1434-8. [abstract]
3. Merck Manual;; Pernicious Anaemia
4. Lahner E, Annibale B; Pernicious anemia: new insights from a gastroenterological point of view. World J Gastroenterol. 2009 Nov 7;15(41):5121-8. [abstract]
5. Malizia RW, Baumann BM, Chansky ME, et al; Ambulatory dysfunction due to unrecognized pernicious anemia. J Emerg Med. 2010 Apr;38(3):302-7. Epub 2007 Dec 3. [abstract]
6. Conrad ME; Pernicious Anaemia, eMedicine, Aug 2009
7. Marty H; Pernicious anemia as cause of secondary sterility. Schweiz Med Wochenschr. 1984 Feb 4;114(5):178-9. [abstract]
8. Pruthi RK, Tefferi A, Petitt RM; Reversible marked splenomegaly in pernicious anemia. Ann Intern Med. 1993 Nov 15;119(10):1053.
9. Schick P; Megaloblastic Anemia, eMedicine, Aug 2009
10. Koivisto H, Hietala J, Anttila P, et al; Long-term ethanol consumption and macrocytosis: diagnostic and pathogenic implications. J Lab Clin Med. 2006 Apr;147(4):191-6. [abstract]
11. Min H, Im ES, Seo JS, et al; Effects of chronic ethanol ingestion and folate deficiency on the activity of 10-formyltetrahydrofolate dehydrogenase in rat liver. Alcohol Clin Exp Res. 2005 Dec;29(12):2188-93. [abstract]
12. Mayer O Jr, Simon J, Rosolova H; A population study of the influence of beer consumption on folate and homocysteine concentrations. Eur J Clin Nutr. 2001 Jul;55(7):605-9. [abstract]
13. Ward PC; Investigation of macrocytic anemia. Postgrad Med. 1979 Feb;65(2):203-7, 209, 212-3. [abstract]
14. Hoffbrand V, Provan D; Clinical review. ABC of clinical haematology: Macrocytic anaemias.; BMJ 1997;314:430 (8 February)
15. Howell-Jolly Bodies; Clinical Chemistry and Hematology Wadsworth Centre
16. Red blood cell morphology; Faculty of Health Sciences, Stellenbosch University, 2010
17. Vlasveld LT; Low cobalamin (vitamin B12) levels in multiple myeloma: a retrospective study. Neth J Med. 2003 Aug;61(8):249-52. [abstract]
18. Wickramasinghe SN; Diagnosis of megaloblastic anaemias. Blood Rev. 2006 May 19. [abstract]
19. Haltmayer M, Mueller T, Poelz W; Erythrocyte mean cellular volume and its relation to serum homocysteine, vitamin B12 and folate. Acta Med Austriaca. 2002;29(2):57-60. [abstract]
20. Oh R, Brown DL; Vitamin B12 deficiency. Am Fam Physician. 2003 Mar 1;67(5):979-86. [abstract]
21. Andres E, Loukili NH, Noel E, et al; Vitamin B12 (cobalamin) deficiency in elderly patients. CMAJ. 2004 Aug 3;171(3):251-9. [abstract]
22. Hematopathology; Pictures of giant metamyelocytes
23. Ye W, Nyren O; Risk of cancers of the oesophagus and stomach by histology or subsite in patients hospitalised for pernicious anaemia. Gut. 2003 Jul;52(7):938-41. [abstract]
24. Turner MR, Talbot K; Functional vitamin B12 deficiency. Pract Neurol. 2009 Feb;9(1):37-41. [abstract]

Internet and further reading

• Lanska DJ; Chapter 30 Historical aspects of the major neurological vitamin deficiency Handb Clin Neurol. 2009;95:445-76. [abstract]

Acknowledgements