• Megaloblastic anaemia is common worldwide, particularly in populations with dietary deficiencies or high prevalence of autoimmune disease.
• B12 deficiency is the predominant cause in developed countries; folate deficiency is more common in developing countries and in the context of malnutrition/alcoholism.
• Pernicious anaemia (the most common medical cause of B12 deficiency) has a prevalence of approximately 0.1% in the general population and up to 2% in those over 60, with a female predominance.

• In Hong Kong, B12 deficiency is the primary cause of megaloblastic anaemia. Folate deficiency causing megaloblastic anaemia is exceedingly rare — Professor Kwong's paper on HK subjects found zero cases of folate-deficiency megaloblastic anaemia ^[1][4].
• Dietary B12 deficiency is uncommon given the Cantonese diet (rich in meat, fish, shellfish), so most cases in HK are due to pernicious anaemia, gastric surgery, or terminal ileal disease.
• Strict veganism (increasingly popular) is an emerging risk factor.

The most common medical cause of vitamin B12 deficiency is pernicious anaemia (95% of cases) ^[4].

Aetiological Classification (High-Yield for SAQ)

Folate deficiency causing megaloblastic anaemia is extremely rare in Hong Kong ^[1]. In clinical practice and HKUMed exams, B12 deficiency (especially pernicious anaemia) is the primary focus.

Drugs causing megaloblastic change include immunosuppressants (MTX, AZA) and TKI (imatinib, sunitinib) ^[2]. These directly impair DNA synthesis:

• Methotrexate (MTX): Inhibits dihydrofolate reductase → blocks regeneration of THF → impaired thymidylate synthesis.
• Azathioprine (AZA): Converted to 6-mercaptopurine → inhibits purine synthesis.
• TKIs: Mechanism less clear but involve impaired haematopoiesis.

The fundamental defect is impaired DNA synthesis with relatively preserved RNA and protein (including haemoglobin) synthesis.

Why does this happen?

• B12 and folate are both required for thymidylate synthesis (converting dUMP → dTMP), which is essential for DNA replication.
• Without adequate thymidylate, cells cannot replicate their DNA properly.
• However, RNA translation and protein synthesis continue normally → the cytoplasm matures and enlarges normally while the nucleus remains immature.
• The result: large cells with immature nuclei (megaloblasts in the bone marrow).

Consequences in each cell line:

1. Erythroid: Megaloblastic erythroid precursors → large RBCs (macrocytes, specifically oval macrocytes). Many of these abnormal precursors die within the bone marrow before reaching maturity → this is called intramedullary haemolysis (also termed "ineffective erythropoiesis") ^[4].
2. Myeloid: Giant metamyelocytes and band forms → hypersegmented neutrophils (≥ 5 lobes in one neutrophil or ≥ 6 lobes = diagnostic) ^[1]. The mechanism is that the nucleus keeps attempting division but fails to complete it, creating extra lobes.
3. Megakaryocytic: Impaired platelet production → thrombocytopenia.

The net effect is pancytopenia — all three cell lines are affected because all rapidly dividing cells need DNA synthesis ^[4].

Macrocytic anaemia is classified by MCV as part of the clinical classification of anaemia: ^[5][6]

Pernicious anaemia is the most common cause of Vitamin B12 deficiency but it is not the only reason for patients becoming deficient in this vitally important vitamin ^[4].

Pathophysiology:

• Autoimmune disease targeting the parietal cells of the stomach and/or intrinsic factor → less production/function of intrinsic factor ^[4].
• Autoantibodies against parietal cells / intrinsic factor ^[4].
• T-cell directed destruction of oxyntic (parietal) cells → loss of acid secretion (achlorhydria) + loss of IF secretion ^[7].
• The resulting chronic autoimmune gastritis is confined to the body and fundus (where parietal cells reside) ^[7].

Associations:

• Associated with other autoimmune diseases: vitiligo, autoimmune thyroid disease, type 1 DM, Addison's disease ^[2].
• Epidemiology: F > M, associated with other autoimmune diseases especially thyroid disease ^[7].

B12 deficiency in pregnancy can cause neural tube defects (similar to folate deficiency) and megaloblastic anaemia in the mother. All pregnant women receive folate supplementation, but B12 status should also be checked, especially in vegetarian/vegan mothers ^[8].

In the old textbooks, CML/MPN diagnosis was based on an increase in serum B12 → because these conditions caused an increase in haptocorrin/TCIII, causing greater B12 ^[4]. This is a historical pearl — elevated B12 without deficiency symptoms should prompt consideration of myeloproliferative disease.

Metformin (widely used in type 2 DM) interferes with calcium-dependent B12-IF complex absorption in the terminal ileum. Long-term metformin use (> 4 years) can cause subclinical B12 deficiency. Guidelines recommend periodic B12 monitoring in patients on long-term metformin.

PPIs reduce gastric acid → impaired liberation of B12 from food proteins. Long-term PPI use (> 2 years) is associated with increased risk of B12 deficiency, though clinically significant deficiency is relatively uncommon.

This is the most commonly tested and clinically important distinction. Both can present with pancytopenia + macrocytosis in an elderly patient.

Bone marrow examination is not routinely needed in megaloblastic anaemia, except when laboratory findings are incompatible with PA → particularly when you are thinking of MDS (can also cause pancytopenia with macrocytosis), or when patient does not improve when you replace patient with parenteral B12 ^[4].

Should check vitamin B12, folate ± zinc, copper in all patients with suspected MDS ^[10] — because you don't want to miss a treatable nutritional deficiency masquerading as MDS.

In the workup of aplastic anaemia, serum vitamin B12 and folate levels are measured specifically for exclusion of megaloblastic anaemia ^[11][12].

Both can present with anaemia + raised LDH + raised unconjugated bilirubin. But the mechanism is different:

The critical discriminator is the reticulocyte count: if it is appropriately elevated with anaemia + ↑LDH + ↑bilirubin, think haemolysis. If it is paradoxically low despite those markers, think intramedullary haemolysis (megaloblastic anaemia, thalassaemia major, or MDS) ^[4].

Intramedullary haemolysis differential diagnosis: pernicious anaemia, thalassaemia major, MDS ^[4].

Why is the reticulocyte count so important? It tells you WHERE the problem is. Low reticulocytes = problem in the bone marrow (production failure or intramedullary destruction). High reticulocytes = marrow is working overtime, so the problem is peripheral (haemolysis, bleeding). In megaloblastic anaemia, despite markers of haemolysis (↑LDH, ↑bilirubin), the reticulocyte count is paradoxically low because the defective cells are destroyed before leaving the marrow.

State the peripheral blood smear finding results for megaloblastic anaemia: ^[1]

Markers of haemolysis: ↑LDH, ↑unconjugated bilirubin, ↓haptoglobin due to decreased survival and ineffective erythropoiesis ^[9]

The combination of ↑LDH + ↑bilirubin + ↓reticulocytes is the biochemical signature of intramedullary haemolysis (ineffective erythropoiesis). This pattern is seen in only a few conditions: pernicious anaemia, thalassaemia major, and MDS ^[4].

Investigations for megaloblastic anaemia: active B12 and folate levels ± RBC folate ^[2]

Further testing of metabolites if cannot tell B12 vs folate deficiency: ^[2]

Why does this work?

• Homocysteine accumulates when either B12 OR folate is deficient, because the methionine synthase reaction (homocysteine → methionine) requires BOTH methyl-B12 and methyl-THF as cofactor/substrate.
• MMA accumulates ONLY in B12 deficiency, because the methylmalonyl-CoA mutase reaction (MMA → succinyl-CoA) requires Ado-B12 but NOT folate.
• Therefore, MMA is the specific discriminator: if MMA is elevated, it must be B12 deficiency.

Metabolite testing (MMA, homocysteine) is used when serum B12 and RBC folate are equivocal ^[9]. In practice, this is helpful when serum B12 is in the borderline range (200–300 pg/mL).

Laboratory investigations for patients with suspected pernicious anaemia: ^[16]

Clinical approach: Order both antibodies together. If anti-IF is positive with confirmed B12 deficiency, the diagnosis of PA is secure. If anti-IF is negative but anti-parietal cell is positive with compatible clinical features, PA remains the most likely diagnosis but is not definitively confirmed — proceed to upper endoscopy for supportive evidence.

Upper endoscopy — atrophic gastritis and carcinoma of stomach ^[4][16]

Why does PA increase gastric cancer risk? Chronic achlorhydria → bacterial overgrowth in the stomach → production of carcinogenic N-nitroso compounds. Additionally, compensatory hypergastrinaemia (the body tries to stimulate acid production by releasing more gastrin) → trophic effect on gastric mucosa → ECL cell hyperplasia → potential carcinoid tumour development.

Schilling test — historical interests, obsolete in HK ^[4][16]

Old test, test how good body absorbs B12, radioactive label B12 then measure urine → don't use anymore since we have the antibodies ^[4]

Principle: Patient given oral radiolabelled B12, then a "flushing" dose of parenteral unlabelled B12 (to saturate body stores so that absorbed radioactive B12 would spill into urine). 24-hour urine collected and radioactivity measured. If low urinary excretion → malabsorption. Then repeat with added IF: if excretion normalises → IF deficiency (PA); if still low → intestinal cause.

This test is no longer available in most centres because radiolabelled B12 is no longer manufactured and serology (anti-IF/anti-parietal cell Ab) provides equivalent diagnostic information without radiation exposure.

Bone marrow examination — not routinely needed, except when laboratory findings incompatible with PA ^[4][16]

Megaloblastic BM features (when performed):

• Hypercellular marrow (in contrast to aplastic anaemia which is hypocellular)
• Megaloblastic erythroid precursors: large cells with open, lacy, immature-looking chromatin despite mature cytoplasm
• Giant metamyelocytes and band forms: myeloid precursors also affected
• Nuclear-cytoplasmic dissociation in all lineages
• NO dysplastic features (distinguishing from MDS)
• NO increased blasts (distinguishing from AML)

Usually replace over weeks, but urgent replacement is indicated in: ^[9]

Why is urgency stratified? Because megaloblastic anaemia is typically chronic and slowly progressive (B12 stores last ~3 years), patients often compensate remarkably well even with very low haemoglobin levels (you may see patients walking around with Hb of 50 g/L). The danger points are: (1) cardiac decompensation from severe anaemia, and (2) irreversible SACD from prolonged B12 deficiency.

Folate with B12 if B12 status unknown, e.g. urgent treatment needed while awaiting B12/folate result ^[9]

Use of folate alone in presence of vitamin B12 deficiency may cause worsening of neurological deficit despite partially masking its haematological deficits ^[9]

Route of administration: parenteral if urgent or impaired absorption, oral if dietary deficiency ^[9]

Duration: lifelong if underlying condition irreversible (e.g. gastrectomy, pernicious anaemia), otherwise until deficiency corrected ^[9]

Replacement therapy addresses the deficiency, but you must also treat or modify the underlying cause whenever possible:

Consider transfusion in symptomatic severe anaemia ^[9].

Folinic acid (leucovorin = 5-formyl-THF) is used, NOT folic acid, because:

• MTX inhibits dihydrofolate reductase (DHFR), blocking the conversion of dihydrofolate → THF
• Folic acid enters the folate cycle above this block (as dihydrofolate) and is therefore also blocked by MTX
• Folinic acid enters the cycle below the block (it is already a reduced folate — 5-formyl-THF) and can be directly converted to 5,10-methylene-THF for DNA synthesis, bypassing DHFR entirely

• All pregnant women should receive folic acid 400 μg/day (or 5 mg/day if high-risk for NTDs)
• B12 should be checked and supplemented in vegan/vegetarian mothers
• If megaloblastic anaemia is diagnosed in pregnancy, treat urgently — both B12 and folate if status unknown

Post-gastrectomy patients and those with coeliac disease commonly have combined B12 + iron deficiency. The MCV may be paradoxically normal (opposing effects). After B12 replacement, the MCV may initially rise (as the microcytic iron-deficient population is diluted by new macrocytic cells), then normalise with concurrent iron supplementation. Monitor iron studies and supplement accordingly.

High-dose oral B12 (1000–2000 μg daily) is an acceptable alternative even in pernicious anaemia, based on the principle that ~1% is absorbed passively. Multiple studies have shown comparable efficacy to IM injections at these doses. Sublingual preparations are also available.

The neurological complications are the most feared and clinically significant. They occur because adenosylcobalamin deficiency leads to MMA accumulation → abnormal fatty acid incorporation into myelin → demyelination. They can also relate to impaired methylation of myelin proteins (from the methionine synthase pathway).

Why does hyperhomocysteinaemia cause cardiovascular disease? Homocysteine at high concentrations is directly toxic to vascular endothelium → endothelial dysfunction → promotes atherosclerosis, platelet activation, and coagulation cascade activation. It also impairs nitric oxide-mediated vasodilation and increases oxidative stress. This is why B12/folate deficiency is now recognised as a cardiovascular risk factor.

B12 and folate are essential for DNA synthesis in all rapidly dividing cells, not just haematopoietic cells. The GI epithelium turns over every 3–5 days — making it particularly vulnerable.

Two gastric diseases associated with pernicious anaemia: ^[4]

• Atrophic gastritis → common complication of PA, autoimmune attack to stomach ^[4]
• Carcinoma of stomach → notably associated with PA, due to autoantibody attack to stomach ^[4]

Why does achlorhydria lead to cancer? The stomach needs acid for more than just digestion. Without acid: (1) bacteria colonise the stomach (normally sterile due to low pH) → they convert dietary nitrates to carcinogenic N-nitroso compounds; (2) the body tries to produce more acid by releasing more gastrin (negative feedback lost) → chronic hypergastrinaemia → trophic stimulation of ECL cells → hyperplasia → neoplasia.

Pernicious anaemia is associated with other autoimmune diseases: vitiligo, autoimmune thyroid disease ^[2][9]

These associations follow the pattern of polyglandular autoimmune syndromes (particularly Type II / APS-2: Addison's + autoimmune thyroid ± type 1 DM). PA clusters with these because they share common HLA susceptibility alleles and autoimmune predisposition.