L12 Blood Microcytic anaemias and iron metabolism

Overview

This lecture covers the three causes of microcytic anaemia (iron deficiency, thalassaemia, anaemia of inflammation) and then focuses on iron metabolism: how iron is stored (ferritin) and transported (transferrin), how iron status is assessed biochemically, how absorption is regulated by the hepcidin/ferroportin axis, the causes and treatment of iron deficiency, and iron overload (haemochromatosis).

Microcytic anaemia: causes

• A red cell is essentially a bag of haemoglobin; if less Hb is made, cells are smaller and paler. Both iron and the globin chains are needed to make haemoglobin, so lack of either causes small red cells.
• Three main causes of microcytic anaemia:
  • Iron deficiency: iron is rate-limiting for haem synthesis, and haem is needed for haemoglobin.
  • Thalassaemias: deletions and genetic variants in the globin genes mean a normal amount of globin cannot be made.
  • Anaemia of inflammation: microcytic when severe, normocytic when less severe; iron is unavailable (a functional iron deficiency).
• Iron deficiency features: anaemia, small red cells (low MCV), pale red cells (low MCH), hypochromic pale cells on microscopy.
• Thalassaemia: a family of genetic diseases causing reduced production of the α or β globin chain, producing microcytic anaemia with marked variation in severity (from mildly abnormal films to films with basophilic stippling and nucleated red cells).
• Distinguishing iron deficiency from thalassaemia: the two conditions can look similarly microcytic/hypochromic on blood film morphology; serum ferritin is the key distinguishing test — low in iron deficiency, not low in thalassaemia.

Storage iron: ferritin

• Most storage iron in the body is held as ferritin, a globular protein complex of 24 subunits (Ferritin H and Ferritin L subunit types) forming a hollow nanocage.
• Fe2+ enters the ferritin shell, is converted by ferroxidase activity, and is sequestered inside as Fe3+ mineral.
• Serum ferritin estimates stored tissue iron (likened to a bank statement); very little iron is actually carried in circulating serum ferritin.
• Ferritin reference intervals: females <50 years ~20-200 µg/L; males ~20-500 µg/L. Lower limit debated: some use 15 µg/L, some 30 µg/L. Ferritin <40-50 µg/L increases the likelihood of iron deficiency.
• Likelihood of iron deficiency falls as serum ferritin rises (Guyatt 1992), for both “mixed disease” and “inflammatory” populations.
• When ferritin is <50 µg/L, dietary iron absorption increases. Normally about 5% of dietary iron is absorbed, but in severe deficiency this can rise to 50% (Galetti 2021).

Circulating iron: transferrin

• Iron in blood is bound to transferrin, the iron transporter; each transferrin molecule carries up to 2 iron atoms.
• Transferrin saturation example: 6 transferrin molecules (12 total binding sites), 4 occupied by Fe = 33% saturation (normal), i.e. one third of binding sites occupied.
• In iron deficiency:
  • Serum iron is reduced (reference interval 10-30 µmol/L).
  • Transferrin saturation is similarly reduced (reference interval 16-50%).
  • Transferrin level rises as a compensatory response but is not very useful diagnostically (reference interval 2.0-3.5 g/L).
• Ferritin in plasma does not contribute to circulating iron levels.
• Worked example (females): normal vs mild vs severe iron deficiency anaemia — Hb 130 / 110 / 70 g/L (ref. 115-155); MCV 89 / 78 / 67 fL (ref. 80-98); MCH 30 / 25 / 19 pg (ref. 27-33); serum ferritin >20 / <20 / usually <10 µg/L; serum Fe 10-30 / ~6 / ~3 µmol/L (ref. 10-30).

Total body iron distribution

• About two-thirds of body iron is in red blood cells.
• Haemoglobin (haem iron): 2.5-3.5 g. Myoglobin (haem iron): 0.15 g. Other proteins: mg amounts. Stores: 0-1 g.
• Over 80 proteins require iron (often as Fe-sulphur clusters) to function as enzymatic sites, essential to all life, e.g. in mitochondria — iron-sulphur clusters are central to electron transport chain complexes (Complex I and Complex II) supporting oxidative phosphorylation (NADH/FADH2 oxidation, proton pumping, ATP synthesis).

Causes of iron deficiency

• Blood loss: menstruation (heavy periods), GI tract cancer, other GI bleeding.
• Growth demands: pregnancy (mother and fetus); premature infants always need iron supplementation; infants and adolescents have increased iron needs for growth.
• Inadequate diet: rare as a sole cause in NZ but may be a secondary/contributory factor; common in developing nations.
• Malabsorption: small bowel malabsorption.
• Globally, iron deficiency/anaemia prevalence in women of reproductive age (15-49 years) varies markedly by region (WHO 2011 estimates): <20% in North America/Australia, 20-39.9% across much of Eurasia, 40-59.9% across much of sub-Saharan Africa.
• Causes of GI blood loss are broader than cancer alone (only ~11% of a highlighted proportion), including:
  • Mass lesions: carcinoma (any site), large polyps.
  • Inflammatory: reflux oesophagitis, Cameron lesions, erosive gastritis, gastric/duodenal ulcer, small bowel or colon ulcer, coeliac (celiac) sprue, Whipple’s disease, Meckel’s diverticulum, idiopathic ulcers, Crohn’s disease, ulcerative colitis.
  • Vascular: vascular ectasia(s), portal hypertensive lesions, watermelon stomach, haemangiomas, blue rubber bleb naevus syndrome.
  • Infectious/miscellaneous: hookworm, haemoptysis, epistaxis, strongyloidiasis, ascariasis, tuberculous enterocolitis, amoebiasis, long-distance running, factitious causes.
• Pregnancy requires an additional ~580 mg iron, mostly in the last 3 months; in the third trimester median ferritin is only 13 µg/L, with 95% below 35 µg/L. Iron requirement rises across pregnancy (baseline body iron loss ~1 mg/day, plus menstruation loss when nongravid/postpartum) peaking around 7 mg/day in the third trimester (fetus/placenta and red cell demand), then falling after delivery.
• Coeliac disease and iron deficiency: coeliac disease affects about 1% of the global population and about 1% of the NZ population.

  The transcript notes the slide states coeliac disease is "the most common inflammatory bowel disease" in NZ, transcribed exactly as shown on the slide (not corrected).

  • Malabsorption is caused by intestinal inflammation induced by hypersensitivity to gluten.
  • Iron deficiency is common in coeliac disease and vice versa; coeliac disease occurs in approximately 3% of patients with iron deficiency, so patients with iron deficiency should be tested for coeliac disease.

Treating iron deficiency

• Oral iron tablets: usually ferrous fumarate or ferrous sulphate; dose and duration depend on cause and severity; often need more than 3 months to correct iron deficiency anaemia and rebuild stores.
• Side effects: gastric irritation/indigestion/pain, loose bowel motions or constipation, black bowel motions.
• Food sources of iron and approximate absorption: liver/kidneys ~20%; red meats ~10%; white meats (fish, chicken, pork, mussels) ~5-10%; vegetable sources (beans, tofu, nuts, tahini — the best vegetarian sources) ~2-5%.
• Optimising absorption:
  • Taking iron with a glass of orange juice (80 mg vitamin C) increases absorption by about 30%.
  • Taking iron with coffee and breakfast decreases absorption by 66%.
  • Iron + vitamin C gives about 4 times more absorption than iron + coffee + breakfast (von Siebenthal, Am J Hem 2023).
  • Morning dosing is better since hepcidin levels are lower then.
  • Smaller doses can reduce side effects; taking iron on alternate days can improve absorption.
• Case example: a 62-year-old female with upper GI bleeding given IV iron, compared 14 days later. Before: HGB 55 g/L, HCT 0.182, MCV 80.5 fL, MCH 24.3 pg, MCHC 302 g/L, PLT 253×10⁹/L. After 14 days: HGB 56 g/L, HCT 0.199, MCV 104.2 fL, MCH 29.3 pg, MCHC 281 g/L, PLT 204×10⁹/L (several values flagged outside range). A double population of normal and iron-deficient cells is seen on repeat testing (dimorphic red cell picture, bimodal RBC histogram), reflecting a mix of newly-made normal cells alongside older small pale cells.
• Causes for failure to respond to oral iron treatment:
  • Wrong diagnosis: consider other causes of microcytic anaemia (thalassaemia, anaemia of inflammation).
  • Multiple causes / mixed picture: e.g. iron deficiency plus inflammation, or ongoing bleeding.
  • Malabsorption: e.g. coeliac disease.
  • Mixed deficiency: folate or vitamin B12 deficiency also present.

Regulation of iron absorption: hepcidin and ferroportin

• Iron is essential but highly toxic in its free form: too much free Fe²⁺ causes oxidative damage to cell membranes (fatty acids) and proteins. It is therefore carefully stored (ferritin) and carried (transferrin); normally there is no free/unbound iron. Regulated absorption in the small intestine prevents excessive uptake; failure of this regulation results in iron overload (haemochromatosis).
• Absorption mechanism: stomach acid converts Fe3+ to Fe2+; Fe2+ is absorbed in the duodenum and jejunum via DMT-1 (divalent metal transporter); iron then exits the enterocyte across the basolateral membrane via ferroportin (“the iron gate”) into the blood (Lo, JO Eur J Hem 2023).
• Systemic iron homeostasis is primarily regulated via the hepcidin/ferroportin axis.
  • Normal state: iron enters the circulation through open ferroportin gates from the gut and from macrophage stores, binding transferrin. Hepcidin is present at low levels and does not block iron entry. Iron-deficient patients have very low hepcidin, so the gate is fully open.
  • During infection, inflammation or iron overload: the liver secretes high levels of hepcidin, which closes ferroportin gates, stopping absorption from the gut and release from macrophages/liver stores. This produces low serum iron and low transferrin saturation.
  • When hepcidin is low, iron absorption from the gut is markedly increased (consistent with the earlier observation that iron deficiency enhances absorption).
• Why inflammation triggers hepcidin: iron is essential for life, including bacteria. During bacterial infection the body attempts to starve bacteria of iron; hepcidin secretion (name reflecting “liver + bactericidal”) lowers serum iron, restricting bacterial growth. Infection, inflammation, surgery and some cancers all trigger this same response: high hepcidin and low serum iron produce “functional” iron deficiency, i.e. anaemia of inflammation, which is often microcytic.
• After surgery (e.g. cholecystectomy), serum iron drops immediately and sharply, reaching a trough around days 1-3 before partially recovering by day 7, reflecting the acute hepcidin response.
• CRP (C-reactive protein) is used to measure the severity of inflammation when interpreting iron studies.

Interpreting iron studies with inflammation

• Effect of inflammation on iron studies (example patient, before vs during inflammation): serum iron 17 to 4 µmol/L (ref. 10-30); transferrin 3.2 to 1.9 g/L (ref. 2.0-3.5); transferrin saturation 21% to 8% (ref. 16-50%); ferritin 27 to 185 µg/L; CRP 5 to 82 mg/L.
• In inflammation/infection, hepcidin sequesters iron in macrophages and ferritin is released from cells, so inflammation “falsely” elevates the ferritin (ferritin behaves as an acute phase reactant), which can mask underlying iron deficiency and complicate interpretation.

Iron overload and haemochromatosis

• Causes of iron overload:
  • Genetic: HFE gene variant (common); other genetic variants (rare).
  • Regular chronic blood transfusions, e.g. in thalassaemia, sickle cell anaemia (SCA), haemophilia.
  • Some chronic anaemias drive increased iron absorption.
  • Excessive dietary intake (uncommon), e.g. African iron overload from beer brewed in cast iron pots.
• Haemochromatosis: caused by the HFE variant, HFE Cys282Tyr homozygosity.

  The transcript notes the source slide text "HFE Cys282Tyr homozygous (" ends with an unclosed parenthesis in the original — transcribed exactly as shown, not corrected or completed.

  • The HFE variant produces a defective HFE protein and failure to produce hepcidin.
  • Lifelong low hepcidin leads to excessive iron absorption from the gut despite adequate iron stores.
  • 1 in 200 NZ Caucasians are homozygous for HFE C282Y.
  • Clinical symptom onset is usually in the 3rd-5th decades; males present earlier than females, who are protected by menstrual blood loss.
  • Iron accumulation causes liver cell injury (eventually cirrhosis and liver cancer), joint pain, and fatigue.
• Laboratory findings in iron overload (liver biopsy shows blue-stained iron with heavy overload in hepatocytes and liver cell damage from iron toxicity): mild overload in C282Y homozygotes — ferritin ~500 µg/L, transferrin saturation 70-90%; severe overload — ferritin ~4000 µg/L, transferrin saturation 98% (reference male ferritin 20-500 µg/L).
• Treating haemochromatosis:
  • Genetic penetrance of the HFE variant is variable.
  • If iron overload is present (ferritin greater than approximately 500 µg/L), treatment is venesection (removing blood) to remove iron; this may be acceptable via blood donation (1 donation removes ~240 mg Fe), with lifelong venesection to control ferritin level.
  • Usually no treatment is needed for female C282Y homozygotes, as they experience less iron overload and disease progression than males.
  • Usual venesection target range: ferritin 50-100 µg/L.

Self-test

1. Describe why lack of either iron or globin chains causes red cells to become microcytic.
2. List the three main causes of microcytic anaemia and briefly state the mechanism of each.
3. Distinguish iron deficiency from thalassaemia using blood film appearance versus serum ferritin.
4. Describe the structure of ferritin and its role in iron storage.
5. What serum ferritin value increases the likelihood of iron deficiency, and how does the likelihood ratio for iron deficiency change as ferritin rises?
6. What proportion of dietary iron is normally absorbed, and how does this change in severe iron deficiency?
7. Describe how transferrin saturation is calculated and give the reference interval.
8. Explain why plasma ferritin does not reflect circulating iron levels.
9. List the four categories of causes of iron deficiency, with one example of each.
10. A patient with iron deficiency has a normal 1% background prevalence coeliac disease population, but is found to have coeliac disease. Explain why patients with iron deficiency should be tested for coeliac disease.
11. Describe the standard oral treatment for iron deficiency, including drug options and expected duration.
12. List two ways of enhancing dietary iron absorption and one way absorption can be reduced, based on the transcript.
13. List the four main causes for failure to respond to oral iron treatment.
14. Describe the steps of intestinal iron absorption from the gut lumen to the bloodstream, naming the transporters involved.
15. Describe how hepcidin and ferroportin control iron absorption and release in the normal state versus during infection/inflammation.
16. Explain why inflammation or infection triggers hepcidin secretion, in terms of host defence.
17. A patient has iron studies showing low serum iron, low transferrin saturation, and raised ferritin, with a raised CRP. Explain this pattern in terms of hepcidin and ferroportin, and explain why ferritin cannot be used alone to exclude iron deficiency here.
18. What genetic cause underlies most hereditary haemochromatosis, and what is its effect on hepcidin production?
19. Why do males with HFE C282Y homozygosity typically present with symptoms earlier than females?
20. List the main complications of iron accumulation in severe hereditary haemochromatosis.
21. Describe the standard treatment for iron overload in haemochromatosis, including the target ferritin range.
22. Integrative: A patient presents with microcytic anaemia. Describe how iron studies (ferritin, serum iron, transferrin saturation, CRP) would differ between iron deficiency, thalassaemia and anaemia of inflammation.

Answers

Reveal answers

1. A red cell is essentially a bag of haemoglobin; if less haemoglobin is made because either iron or globin chains are lacking, less haemoglobin fills the cell, so the cell is made smaller and paler.
2. Iron deficiency (iron is rate-limiting for haem synthesis, and haem is needed for haemoglobin); thalassaemia (genetic deletions/variants in globin genes prevent normal globin production); anaemia of inflammation (iron becomes unavailable, a functional iron deficiency, causing microcytic anaemia when severe and normocytic when milder).
3. On blood film, iron deficiency and thalassaemia can look similarly microcytic and hypochromic; they are distinguished by serum ferritin, which is low in iron deficiency and not low (normal or raised) in thalassaemia.
4. Ferritin is a globular protein complex of 24 subunits (types Ferritin H and Ferritin L) forming a hollow nanocage; Fe2+ enters the cage, is converted by ferroxidase activity, and is sequestered as Fe3+ mineral inside, forming the body’s main iron store.
5. Serum ferritin <40-50 µg/L increases the likelihood of iron deficiency; as serum ferritin rises, the likelihood ratio for iron deficiency falls steeply (Guyatt 1992), in both mixed disease and inflammatory populations.
6. Normally about 5% of dietary iron is absorbed; in severe iron deficiency this can rise to about 50%.
7. Transferrin saturation is the proportion of transferrin’s iron-binding sites occupied by iron (each transferrin molecule carries up to 2 iron atoms); e.g. 4 Fe occupying 12 total binding sites on 6 transferrin molecules = 33% saturation. Reference interval: 16-50%.
8. Plasma/serum ferritin contains very little iron itself; it reflects tissue iron stores (like a “bank statement”) rather than contributing meaningfully to the pool of circulating iron, which is carried by transferrin instead.
9. Blood loss (e.g. heavy menstruation, GI tract cancer/other GI bleeding); growth demands (pregnancy, premature infants, infants/adolescents); inadequate diet (rare alone in NZ, common in developing nations); malabsorption (e.g. small bowel malabsorption).
10. Coeliac disease causes malabsorption via gluten-induced intestinal inflammation; it occurs in approximately 3% of patients with iron deficiency (compared with about 1% background population prevalence), so iron deficiency should prompt testing for coeliac disease as a treatable cause of malabsorption.
11. Oral iron tablets, usually ferrous fumarate or ferrous sulphate; dose and duration depend on cause and severity, and often more than 3 months of treatment is needed to correct the anaemia and rebuild iron stores.
12. Taking iron with vitamin C (e.g. orange juice) increases absorption (~30%, and about 4-fold more than iron with coffee/breakfast); taking iron in the morning improves absorption since hepcidin is lower then. Taking iron with coffee and breakfast decreases absorption by about 66%.
13. Wrong diagnosis (another cause of microcytic anaemia such as thalassaemia or anaemia of inflammation); multiple causes/mixed picture (e.g. iron deficiency plus inflammation or ongoing bleeding); malabsorption (e.g. coeliac disease); mixed deficiency (concurrent folate or vitamin B12 deficiency).
14. Stomach acid converts dietary Fe3+ to Fe2+; Fe2+ enters the duodenal/jejunal enterocyte via DMT-1 (divalent metal transporter); iron then crosses the basolateral membrane into the blood via ferroportin, the “iron gate,” which is regulated (inhibited) by hepcidin.
15. Normally, hepcidin is low, ferroportin gates are open, and iron enters circulation from the gut and macrophage stores, binding transferrin. During infection, inflammation or iron overload, the liver secretes high hepcidin, which closes ferroportin gates on enterocytes, macrophages and liver, stopping gut absorption and release of stored iron, lowering serum iron and transferrin saturation.
16. Iron is essential for life, including bacteria; during infection the body attempts to starve invading bacteria of iron by raising hepcidin (name reflecting “liver + bactericidal”), which lowers serum iron and restricts bacterial growth; the same response occurs with inflammation, surgery, and some cancers.
17. This is the pattern of anaemia of inflammation/functional iron deficiency: raised CRP indicates inflammation, which raises hepcidin, closing ferroportin so iron is sequestered in macrophages (low serum iron, low transferrin saturation); ferritin is simultaneously released from cells during inflammation (acute phase reactant), so it rises and “falsely” appears adequate/elevated, masking true iron deficiency and making ferritin unreliable on its own when inflammation (raised CRP) is present.
18. Homozygosity for the HFE Cys282Tyr (C282Y) variant is the most common genetic cause; it produces a defective HFE protein, causing failure to produce hepcidin.
19. Females are protected by menstrual blood loss, which removes excess iron, delaying the accumulation of iron overload compared with males; males therefore typically present earlier (3rd-5th decade).
20. Liver cell injury progressing to cirrhosis and liver cancer, joint pain, and fatigue.
21. Venesection (removing blood) to remove excess iron, which may be done via blood donation (each donation removes ~240 mg Fe), continued lifelong to control ferritin; the target ferritin range for venesection is 50-100 µg/L. Female C282Y homozygotes usually require no treatment due to lower iron overload/disease progression.
22. Iron deficiency: low ferritin, low serum iron, low transferrin saturation, normal CRP. Thalassaemia: ferritin not low (normal/raised), serum iron and transferrin saturation not typically reduced by the underlying disease, normal CRP; genetic globin defect distinguishes it clinically. Anaemia of inflammation: ferritin raised (falsely elevated acute phase reactant) despite functional iron deficiency, low serum iron, low transferrin saturation, and raised CRP reflecting the underlying inflammatory process driving high hepcidin and ferroportin blockade.