Overview
This lecture covers macrocytic anaemia: how to recognise it on a blood screen report, the causes (megaloblastic versus non-megaloblastic), and how to work each up. The megaloblastic half centres on vitamin B12 and folate, their metabolism, why both are needed for DNA synthesis, and how impaired DNA synthesis produces the characteristic blood and marrow findings plus (for B12) neurological disease. The non-megaloblastic half covers liver disease, alcohol, reticulocytosis, myelodysplastic syndrome, drugs and myeloma. Diagnosis, treatment and prevention are covered for both deficiencies.
Recognising macrocytic anaemia on a blood screen
Worked example blood count (patient value, then reference interval):
- Haemoglobin 52, RI 115-155 g/L
- MCV 123, RI 80-98 fL
- MCH 38.5, RI 27-32 pg
- Platelet count 80, RI 150-400 x10
- Neutrophils 1.4, RI 2.0-7.5 x10
- Lymphocytes 2.1, RI 1.0-4.0 x10
- Monocytes 0.4, RI 0.2-1.0 x10
The pattern is a raised MCV and MCH with anaemia, here also with thrombocytopenia and neutropenia.
Blood film findings in B12 or folate deficiency: oval macrocytosis of red cells, and hypersegmentation of some neutrophils. The normal film by comparison shows round red cells.
Classification of the causes
Megaloblastic anaemia
- Vitamin B12 deficiency
- Folate deficiency
Non-megaloblastic anaemia
- Liver disease
- Alcohol
- Increased red cell production (reticulocytosis)
Others
- Myelodysplastic syndrome (a marrow cancer common in the elderly)
- Drugs
- Myeloma
The lecture’s summary flow chart splits macrocytic anaemia into the megaloblastic anaemias (B12 deficiency, folate deficiency) and others (liver disease, alcohol, reticulocytosis, drugs, marrow diseases including myelodysplasia and myeloma, others). B12 deficiency branches further into pernicious anaemia (autoimmune atrophic gastritis) and other causes of B12 deficiency.
Megaloblastic anaemia: definition and mechanism
- Megaloblastic anaemia describes a group of anaemias caused by impaired DNA synthesis, characterised by abnormal findings in peripheral blood (macro-ovalocytes) and bone marrow (megaloblastic hyperplasia).
- Transcription, translation and protein synthesis proceed even though DNA synthesis is affected.
- Retarded DNA synthesis leads to accumulation of dead and dying megaloblasts in the marrow, causing anaemia.
- Folate and vitamin B12 deficiencies are the most common causes.
- B12 and folate are needed for nucleic acid (thymidine) synthesis; deficiency causes delayed and abnormal maturation in the marrow, hence macrocytosis.
- All tissues are affected, but blood abnormalities are the usual means of diagnosis.
Normal erythroblast maturation involves two simultaneous processes: nuclear (DNA) growth and division, and protein synthesis with accumulation of haemoglobin (seen as the changing cytoplasmic colour through maturation). In megaloblastic development nuclear growth is delayed and abnormal while protein synthesis continues, so the red cell ends up containing more Hb than normal.
Warning
The transcript flags that slides 6 and 7 each carry two unlabelled inset photographs of a person inflating a large red balloon-shaped object. The apparent purpose is an analogy for the two maturation processes, but the connection is not labelled on the slides.
Detailed pathophysiology of megaloblastic anaemia
- Central abnormality is defective DNA synthesis, leading to intramedullary apoptosis of haematopoietic precursor cells and so to ineffective erythropoiesis.
- Both folate and B12 deficiency cause thymidylate deficiency. DNA contains two purine bases (adenine, guanine) and two pyrimidine bases (thymine, cytosine); with insufficient thymine or thymidylate, uracil is incorporated at the thymine position.
- Repair enzymes detect the misincorporated uracil, attempt repair and are unsuccessful.
- This leads to destruction of DNA strands and p53-mediated cellular apoptosis.
- The result is asynchronous maturation between nucleus and cytoplasm: the nucleus, devoid of DNA, does not mature fully, while the cytoplasm matures normally because RNA production is unaltered and haemoglobin synthesis progresses.
Vitamin B12 (cobalamin)
An important cause of macrocytic anaemia. Not very common, but not to be missed: failure to treat can result in irreversible neurological damage.
Forms. Biologically active: adenosylcobalamin and methylcobalamin. Inactive forms found in pharmacological preparations: cyanocobalamin and hydroxocobalamin.
Sources. Produced by bacteria, so found in meat, eggs, dairy and fish (from animals containing B12-producing bacteria). Not present in clean fruit or vegetables. High levels in some seaweeds (nori) and some mushrooms (shiitake, chanterelles). Non-animal sources listed: nori, golden chanterelle, shiitake, black trumpet mushroom, and plants grown on organic fertiliser.
Absorption pathway
- B12 is released from food protein complexes in the stomach.
- B12 binds haptocorrin (B12-HC), also called R-protein, which binds about 70% of cobalamin.
- Stomach parietal cells secrete intrinsic factor (IF).
- Intrinsic factor binds B12 in the small bowel.
- B12-IF is taken up by bowel receptors in the terminal ileum.
- B12 is then stored in the liver, and B12 bound to transcobalamin (TC) or haptocorrin enters the portal circulation.
Abbreviations: IF = intrinsic factor, TC = transcobalamin, HC = haptocorrin.
Functions of vitamin B12
B12 is essential for DNA synthesis and for methylation of proteins and DNA. These pathways also involve folate, so B12 and folate are often tested together.
1. Synthesis of methionine from homocysteine (cytosol)
- Mediated by methyltetrahydrofolate homocysteine methyl transferase (methionine synthase), with methylcobalamin as cofactor.
- During this reaction methyl-THF is converted to THF.
- THF is necessary for formation of methylene-THF, which is a cofactor in the synthesis of deoxythymidine monophosphate (dTMP) from deoxyuridine monophosphate (dUMP).
- dTMP is required for DNA synthesis.
2. Conversion of methylmalonyl-CoA to succinyl-CoA (mitochondrion)
- Requires adenosylcobalamin and methylmalonyl-CoA mutase.
- B12 deficiency is associated with increased levels of methylmalonic acid and propionic acid.
- This is thought to cause synthesis of abnormal myelin lipids, with consequent myelin degeneration and neurological abnormalities.
The folate/B12 metabolic map
Nuclear/DNA synthesis arm: folic acid → dihydrofolate (H2-folate/DHF) → tetrahydrofolate (H4-folate/THF) → methylene-H4-folate → converts deoxyuridine to thymidine → DNA synthesis.
Methylation arm: dietary folate → methyl-H4-folate; methyl-H4-folate plus homocysteine, via methionine synthase with methyl-B12, gives methionine → S-adenosylmethionine (SAM) → methylation of proteins and DNA → S-adenosylhomocysteine (SAH) → homocysteine, closing the cycle back with THF.
Mitochondrial arm: methylmalonyl-CoA, via methylmalonyl-CoA mutase with adenosyl-B12, gives succinyl-CoA which enters the TCA cycle; methylmalonic acid accumulates when this step fails.
Nitrous oxide (N2O) inhibits this system.
Causes of vitamin B12 deficiency
- Autoimmune atrophic gastritis (pernicious anaemia), the most common cause
- Gastrectomy (no intrinsic factor)
- Disease or resection of the terminal ileum, for example Crohn disease
- Strict vegan diet (no dairy)
Pernicious anaemia
Autoantibody-mediated atrophy of the gastric mucosa with destruction of parietal cells, that is autoimmune atrophic gastritis = pernicious anaemia. Destruction of parietal cells results in reduced or absent acid production (hypochlorhydria or achlorhydria) and loss of intrinsic factor. Antibodies found: anti-parietal cell antibodies and anti-intrinsic factor antibodies. Histology shows chronic atrophic gastritis.
Dietary deficiency
A strict vegan diet does not provide sufficient B12, as it contains no animal proteins (meat, fish, eggs, milk). B12 stores in normal individuals are sufficient for 2-4 years.
Oxford Study (men), serum B12 by diet group:
| Category | Omnivores | Vegetarians | Vegans |
|---|---|---|---|
| Sufficient >150 pmol/L | 99% | 76% | 27% |
| Borderline 118-149 pmol/L | 1% | 17% | 21% |
| Deficient <118 pmol/L | 0.4% | 7% | 52% |
Clinical features of B12 deficiency
- Stores in a healthy individual last 2-4 years, so onset of megaloblastic anaemia is usually slow and the diagnosis is often missed.
- Anaemia and related symptoms; sometimes also neutropenia and thrombocytopenia.
- Neurological symptoms and signs: peripheral neuropathy, sensory loss, abnormal balance, cognitive impairment, memory loss, dementia, psychosis.
- Glossitis (beefy red and painful tongue).
Pathophysiology of the neurological changes
- Cobalamin deficiency causes subacute combined degeneration (SACD) of the posterior and lateral grey columns of the spinal cord, due to methionine deficiency, because methionine is needed for production of myelin.
- Myelin deficiency causes demyelination and gliosis of the grey column.
- Further worsening is caused by the neurotoxicity of methylmalonic acid.
- Elevation of TNF alpha and epidermal growth factor (EGF) also contributes.
Diagnosis and treatment of B12 deficiency
Diagnosis. Serum B12 level (<200 pg/mL), but there is a high rate of borderline results. Confirmatory tests are not particularly good: serum methylmalonic acid (MMA) elevated; serum homocysteine elevated.
Treatment.
- If unable to absorb the vitamin (pernicious anaemia, gastrectomy, loss of terminal ileum): inject IM 1000 ug vitamin B12 as hydroxocobalamin, lifelong, every 2-3 months.
- If deficiency is due to diet (for example vegans): oral supplement 1000-2000 ug.
Folate (folic acid) deficiency
Causes a megaloblastic anaemia, the same as B12 deficiency. In cells, folate is important for DNA synthesis and methylation.
Functions of folate
- Serine-glycine conversion
- Histidine catabolism
- Purine synthesis
- Thymidine and methionine synthesis
Forms. There are many different folates in food. The most abundant form in serum is methyltetrahydrofolate. This differs from folic acid, the form used for food fortification, which is easily absorbed.
Causes of folate deficiency
Folate deficiency is now uncommon, and soon will become very rare in NZ with mandatory fortification of bread.
Dietary
- Lack of leafy greens
- Alcoholics, poor diet
- Poor food choices or ignorance of food needs
- Elderly: small meals, difficulty accessing shops
Increased demand
- Pregnancy
- Disorders with increased cell turnover: all haemolytic anaemias, severe (widespread allergic) dermatitis, some malignancies
- Peritoneal and haemodialysis, folate lost into dialysis fluid
Malabsorption (not common)
- Malabsorption affecting small bowel, for example coeliac disease
Drugs interfering with folic acid metabolism
- Phenytoin
- Chemotherapy (methotrexate)
Clinical manifestations of folate deficiency
Similar to B12 deficiency: macrocytic anaemia (sometimes also neutropenia and thrombocytopenia); glossitis; neuropsychiatric features that are less clear cut than for B12, namely depression and possibly neuropathy.
Diagnosis and treatment of folate deficiency
Diagnosis. Serum folate <2 ng/mL. Intra-erythrocyte folate (red cell folate) <100-160 mg/L, which is more reliable because it is not affected by diet.
Treatment. Treat the underlying cause and supplement with additional vitamin orally. Dose 0.8 mg/day if absorption is normal, for example in pregnancy; 5 mg/day if malabsorption.
Non-megaloblastic macrocytosis
Liver disease
- Usually mild macrocytosis, MCV 98-105 fL
- Target cells often seen on the film
- May be accompanied by mild to moderate anaemia and mild to moderate thrombocytopenia
- Occurs in chronic liver disease, cirrhosis, and especially alcoholic liver disease
Alcohol and alcoholic liver disease
- Alcohol increases both MCH and MCV
- Therefore it must affect red cell development in the marrow
- Folate deficiency contributes in some cases, where it reduces absorption and impairs folate metabolism
- Graphs of MCV and MCH against number of drinks show both rising with increasing intake, compared across control subjects, non-alcoholic liver disease and alcoholic liver disease groups
Increased reticulocytes
- Reticulocytes (1-day-old red cells) are large, approximately 24% larger than mature red cells
- They show polychromasia on a regular blood film, appearing as bluish cells containing RNA
- Reticulocytosis can occur after a major bleed, after treatment of a deficiency state, or in a haemolytic anaemia
Illustrative laboratory results across two dates (reference intervals in brackets): Hb 143 → 77 g/L (130-175); Hct 0.41 → 0.23 (0.40-0.52); MCV 88 → 107 fL (80-99); MCH 30 → 37 pg (27-33); reticulocyte count 256 x10^9/L (20-100).
Warning
The transcript flags that this slide carries no explanatory bullet text. It appears to illustrate macrocytic anaemia due to haemolysis via the change in lab values over time, but the slide does not state this explicitly.
Myelodysplastic syndrome
- A common marrow cancer of the elderly caused by the accumulation of genetic damage
- Mutations affect blood stem cells, leading to abnormal (dysplastic) cells
- Macrocytosis is very common
- Typical film feature: a mixture of large, well-haemoglobinised cells and pale, poorly-haemoglobinised cells, plus abnormal (“weird”) neutrophils
Case. 72-year-old man. Hb 75 (RI 130-175 g/L); MCV 114 (RI 80-99 fL); B12, folate and liver tests normal; reticulocytes 31 (RI 20-100 x10^9/L); neutrophils 0.4 (RI 1.9-7.5 x10^9/L); platelets 15 (RI 150-400 x10^9/L). Diagnosis: severe bone marrow failure, probably advanced stages of myelodysplastic syndrome.
Drugs
Drugs that affect DNA synthesis and drugs that affect folate metabolism.
- Antimetabolites: 6-mercaptopurine and 5-fluorouracil inhibit DNA synthesis directly. Methotrexate inhibits dihydrofolate reductase, the enzyme required for generation of tetrahydrofolate from dihydrofolate.
- Trimethoprim, pentamidine and pyrimethamine also inhibit dihydrofolate reductase.
- Others: zidovudine, antiepileptics, oral contraceptives and nitrous oxide.
Case: overdose of hydroxyurea, a mild chemotherapy drug, described as a record-breaking case. Results (reference intervals in brackets): RBC 0.75 x10^12/L (4.30-6.00); Hb 37 g/L (130-175); Hct 0.11 (0.40-0.52); MCV 152 fL (80-99); MCH 49 pg (27-33); platelets 52 x10^9/L (150-400); WBC 2.5 x10^9/L (4.0-11.0); neutrophils 1.5 x10^9/L (1.9-7.5).
Prevention
- Fortification of food
- Prophylaxis to at-risk individuals: pregnant women, elderly, children, individuals with haemolytic crisis
Self-test
- Define megaloblastic anaemia, including its characteristic peripheral blood and bone marrow findings.
- List the causes of macrocytic anaemia under the three groupings used in the lecture, with the specific entities in each.
- Describe the steps by which uracil misincorporation leads to ineffective erythropoiesis in megaloblastic anaemia.
- Explain why the nucleus and cytoplasm mature asynchronously in a megaloblastic erythroblast.
- Describe the steps of dietary vitamin B12 absorption from stomach to storage, naming the binding proteins involved at each stage.
- Name the two biologically active forms of cobalamin and the two inactive forms found in pharmacological preparations.
- Describe the methionine synthase reaction and explain how it links to thymidine synthesis.
- Describe the methylmalonyl-CoA mutase reaction and explain how its failure produces neurological damage.
- List the causes of vitamin B12 deficiency, identifying the most common one.
- Explain the mechanism of pernicious anaemia and name the two autoantibodies detected.
- List the neurological symptoms and signs of B12 deficiency.
- Describe the pathophysiology of subacute combined degeneration in cobalamin deficiency.
- What is the serum cut-off for diagnosing vitamin B12 deficiency, and which two confirmatory tests are used and in which direction do they move?
- Distinguish the treatment of B12 deficiency due to malabsorption from that due to diet, giving route, dose and frequency.
- List the biological functions of folate.
- List the causes of folate deficiency grouped by mechanism.
- Distinguish the clinical manifestations of folate deficiency from those of B12 deficiency.
- What are the diagnostic cut-offs for serum folate and red cell folate, and why is red cell folate more reliable?
- State the folate treatment doses for normal absorption and for malabsorption.
- Describe the blood film and count features of macrocytosis due to liver disease, including the typical MCV range.
- Explain why reticulocytosis raises the MCV, and list three situations in which reticulocytosis occurs.
- Describe the blood film features that distinguish myelodysplastic syndrome from megaloblastic anaemia.
- List the drug classes that cause macrocytosis and state the mechanism for methotrexate and trimethoprim.
- A 72-year-old man has Hb 75 g/L, MCV 114 fL, neutrophils 0.4, platelets 15 x10^9/L, reticulocytes 31 x10^9/L, with normal B12, folate and liver tests. What is the likely diagnosis and what is the reasoning?
- List the two approaches to prevention and the at-risk groups who should receive prophylaxis.
- Integrative: a patient has a raised MCV. Explain how the history, blood film and a short panel of blood tests would let you work systematically through the megaloblastic and non-megaloblastic causes.
Answers
Reveal answers
- A group of anaemias caused by impaired DNA synthesis, characterised by macro-ovalocytes in peripheral blood and megaloblastic hyperplasia in the bone marrow.
- Megaloblastic: B12 deficiency, folate deficiency. Non-megaloblastic: liver disease, alcohol, increased red cell production (reticulocytosis). Others: myelodysplastic syndrome, drugs, myeloma.
- Folate or B12 deficiency causes thymidylate deficiency, so uracil is incorporated where thymine should be; repair enzymes detect the error, attempt repair and fail; DNA strands are destroyed and p53-mediated apoptosis follows; this intramedullary apoptosis of precursors gives ineffective erythropoiesis.
- DNA synthesis is impaired so the nucleus does not mature fully, while RNA production is unaltered and haemoglobin synthesis progresses, so the cytoplasm matures normally. The cell ends up larger and containing more Hb than normal.
- B12 is released from food protein complexes in the stomach; it binds haptocorrin (R-protein, binding about 70% of cobalamin); parietal cells secrete intrinsic factor, which binds B12 in the small bowel; B12-IF is taken up by receptors in the terminal ileum; B12 bound to transcobalamin or haptocorrin enters the portal circulation and is stored in the liver.
- Active: adenosylcobalamin and methylcobalamin. Inactive (pharmacological preparations): cyanocobalamin and hydroxocobalamin.
- Methyltetrahydrofolate homocysteine methyl transferase (methionine synthase), with methylcobalamin as cofactor, converts homocysteine to methionine; in doing so methyl-THF is converted to THF. THF is needed to form methylene-THF, the cofactor for conversion of dUMP to dTMP, and dTMP is required for DNA synthesis.
- Methylmalonyl-CoA is converted to succinyl-CoA by methylmalonyl-CoA mutase with adenosylcobalamin as cofactor. B12 deficiency blocks this, raising methylmalonic acid and propionic acid, which is thought to cause synthesis of abnormal myelin lipids with myelin degeneration and neurological abnormalities.
- Autoimmune atrophic gastritis (pernicious anaemia), the most common cause; gastrectomy (no intrinsic factor); disease or resection of the terminal ileum such as Crohn disease; strict vegan diet.
- Autoantibody-mediated atrophy of the gastric mucosa destroys parietal cells, causing reduced or absent acid production (hypochlorhydria or achlorhydria) and loss of intrinsic factor. Antibodies: anti-parietal cell and anti-intrinsic factor.
- Peripheral neuropathy, sensory loss, abnormal balance, cognitive impairment, memory loss, dementia, psychosis.
- Cobalamin deficiency causes methionine deficiency; methionine is needed to produce myelin, so myelin deficiency leads to demyelination and gliosis of the posterior and lateral grey columns of the spinal cord. Methylmalonic acid neurotoxicity worsens it, and raised TNF alpha and EGF also contribute.
- Serum B12 <200 pg/mL, though there is a high rate of borderline results. Confirmatory tests, which are not particularly good, are serum methylmalonic acid (elevated) and serum homocysteine (elevated).
- Malabsorption (pernicious anaemia, gastrectomy, loss of terminal ileum): IM injection of 1000 ug hydroxocobalamin, lifelong, every 2-3 months. Dietary (for example vegans): oral supplement 1000-2000 ug.
- Serine-glycine conversion, histidine catabolism, purine synthesis, and thymidine and methionine synthesis.
- Dietary: lack of leafy greens, alcoholics with poor diet, poor food choices or ignorance of food needs, elderly with small meals and difficulty accessing shops. Increased demand: pregnancy, disorders with increased cell turnover (all haemolytic anaemias, severe widespread allergic dermatitis, some malignancies), peritoneal and haemodialysis. Malabsorption: small bowel disease such as coeliac. Drugs: phenytoin, methotrexate.
- They are similar: macrocytic anaemia, sometimes neutropenia and thrombocytopenia, and glossitis occur in both. The neuropsychiatric features in folate deficiency are less clear cut than in B12 deficiency, being depression and possibly neuropathy.
- Serum folate <2 ng/mL; intra-erythrocyte (red cell) folate <100-160 mg/L. Red cell folate is more reliable because it is not affected by diet.
- 0.8 mg/day if absorption is normal, for example pregnancy; 5 mg/day if there is malabsorption. Also treat the underlying cause.
- Usually mild macrocytosis with MCV 98-105 fL, target cells often seen, and it may be accompanied by mild to moderate anaemia and mild to moderate thrombocytopenia. It occurs in chronic liver disease, cirrhosis and especially alcoholic liver disease.
- Reticulocytes are 1-day-old red cells and are approximately 24% larger than mature red cells, so an increase in their number raises the mean cell volume; they show polychromasia (bluish, RNA-containing cells) on the film. Reticulocytosis occurs after a major bleed, after treatment of a deficiency state, and in haemolytic anaemia.
- Myelodysplasia shows a mixture of large, well-haemoglobinised cells and pale, poorly-haemoglobinised cells, plus abnormal (weird) neutrophils. Megaloblastic anaemia shows oval macrocytosis with hypersegmentation of some neutrophils.
- Antimetabolites (6-mercaptopurine and 5-fluorouracil, which inhibit DNA synthesis directly; methotrexate), dihydrofolate reductase inhibitors (trimethoprim, pentamidine, pyrimethamine), and others including zidovudine, antiepileptics, oral contraceptives and nitrous oxide. Methotrexate and trimethoprim both inhibit dihydrofolate reductase, the enzyme required to generate tetrahydrofolate from dihydrofolate.
- Severe bone marrow failure, probably advanced myelodysplastic syndrome. The reasoning is pancytopenia (anaemia, neutropenia, thrombocytopenia) with macrocytosis, a reticulocyte count that is not raised, and normal B12, folate and liver tests, so the megaloblastic and common non-megaloblastic causes are excluded, in an elderly man in whom myelodysplasia is common.
- Fortification of food, and prophylaxis to at-risk individuals: pregnant women, elderly, children, and individuals with haemolytic crisis.
- History targets diet (strict vegan), alcohol, gastric or ileal surgery or disease such as Crohn or coeliac, pregnancy, dialysis and drugs such as phenytoin, methotrexate, trimethoprim or nitrous oxide. The film separates the groups: oval macrocytes with hypersegmented neutrophils point to megaloblastic anaemia; target cells point to liver disease; polychromasia points to reticulocytosis; a mixture of well and poorly haemoglobinised cells with abnormal neutrophils points to myelodysplasia. Tests: serum B12 (with MMA and homocysteine if borderline) and serum or red cell folate for the megaloblastic causes, liver tests for liver disease, and reticulocyte count for haemolysis or blood loss. If all are normal in an elderly patient with cytopenias, consider marrow disease such as myelodysplasia or myeloma.