Haematology JC043: Pallor: Diagnosis Of Anaemia, Nutritional Anaemia, Anaemia Of Systemic Diseases

Question 1

Clinical presentations of Anaemia

Answer 1

No specific clinical presentation but symptomatology tell **onset + **severity of problem

1. Acute + Severe (e.g. Torrential GI bleeding, Very severe haemolytic anaemia)
   - **SOB
   - **Palpitation
   - ***Dizziness / Syncope (∵ CVS compromise)
   - Symptoms associated with cardiac ischaemia
2. Chronic + Insidious (e.g. MDS, Aplastic anaemia)
   - **Asymptomatic
   - **Fatigue
   - ***↓ Exercise tolerance
   - Pale
3. Symptoms associated to cause of anaemia
   - Menorrhagia (lead to Fe deficiency anaemia)
   - Tarry stool, Rectal bleeding
   - Bone pain (e.g. Multiple myeloma)

Question 2

***Physical examinations of Anaemia

Answer 2

No unique physical signs for Anaemia

Some examples:
1. Pallor
2. **Jaundice (suggest haemolysis —> ∵ ↑ unconjugated bilirubin)
3. **Uraemic look / Cafe au lait complexion (anaemia due to chronic kidney disease)
4. Early greying of hairs + glossitis (pernicious anaemia)
5. **Shock / tachycardia (acute blood loss)
6. Isolated splenomegaly (hypersplenism)
7. **Lymphadenopathy, ***Hepatosplenomegaly (haematological malignancy)
8. Abnormal abdominal mass (watch out for carcinoma)

Question 3

Causes of Anaemia

Answer 3

1. Decreased RBC production
   - Nutritional (Fe, B12, Folate (Folate deficiency anaemia not seen in HK))
   - **Bone marrow disorder (e.g. Aplastic anaemia, Pure red cell aplasia, Neoplastic diseases (haematological / non-haematological))
   - **Bone marrow suppression (e.g. Drugs, Chemotherapy, Irradiation (e.g. pelvic bone))
   - **Reduced trophic hormones (e.g. Erythropoietin (chronic renal failure), Thyroid hormone (not severe))
   - **Anaemia of chronic disease
2. Increased RBC destruction
   ***Intrinsic RBC defect
   - Enzymopathy / deficiency (e.g. G6PD deficiency)
   - Haemoglobinopathy (e.g. Thalassaemia, other Hb defects) —> unstable Hb
   - Membranopathy (e.g. Hereditary spherocytosis, Elliptocytosis)

**Extrinsic RBC defect
- Liver disease + **Hypersplenism
- Infections (e.g. Malaria)
- **Autoimmune haemolytic anaemia (warm / cold, drug-induced)
- **Microangiopathy (e.g. Prosthetic heart valve leak, TTP, DIC)
- Transfusion reactions (e.g. ABO incompatibility —> intravascular haemolysis)

3. Blood loss
   - GI tract
   - Genital tract (e.g. Menorrhagia)
   - Soft tissue (e.g. Coagulopathy e.g. retroperitoneal haematoma)

Question 4

Classification of Anaemia based on MCV

Answer 4

Low (<80 fL)
1. **Fe deficiency
2. **Thalassaemia
3. Sideroblastic anaemia (congenital / drug-induced) (uncommon)

Normal (80-99 fL)
1. **Anaemia of chronic disease
2. **Anaemia of renal disease (only in chronic kidney disease, ↓ in erythropoietin)
3. ***Acute blood loss (if chronic —> Fe deficiency anaemia)
4. Dimorphic anaemia (concomitant Fe deficiency + Vit B12 deficiency: some RBC big some RBC small —> blood film important)

High (>100 fL)
1. **Aplastic anaemia
2. Chronic liver disease
3. Chemotherapy
4. Alcohol usage
5. **Vit B12 deficiency anaemia (Folate deficiency anaemia not seen in HK, folate deficiency common in alcoholic, rapid cell turnover e.g. Psoriasis, haematological malignancy —> never severe enough to cause anaemia)
6. **Haemolytic anaemia (∵ Polychromasia i.e. Reticulocytosis)
7. **Myelodysplasia (MDS)

Question 5

Morphology of RBCs

Answer 5

1. Normal
2. Rouleax (↑ globulin)
3. Agglutination
4. Polychromasia (Reticulocytosis)
5. Hypochromic microcytic anaemia (Fe deficiency: Hypochromic microcytic + ***Pencil cell)
6. ***Macroovalocyte (big RBC oval in shape: in Vit B12 deficiency anaemia)
7. **Spherocytes (loss of central pallor: in **Haemolytic anaemia / ***Hereditary spherocytosis)
8. Elliptocytes (hereditary)
9. Malaria (intracellular organism)
10. Schistocytes (fragmented RBC)
11. Tear-drop cells (fibrosis in marrow, not necessarily Primary myelofibrosis e.g. metastatic cancer to marrow, chronic irradiation)
12. ***Echinocytes (in severe systemic diseases e.g. renal failure)
13. ***Spur cells / Acanthocytes (much longer processes than Echinocytes, ∵ abnormal lipid metabolism / deposition in RBC membrane e.g. severe alcoholic liver cirrhosis)
14. ***Bite cells (indicate oxidative haemolysis e.g. G6PD deficiency)
    (15. Target cells (Thalassaemia, other pathologies) (Self notes))

Question 6

Rouleaux formation

Answer 6

• Stacks of coins
• ↑ level of globulin
  —> Neoplastic / Mon-neoplastic
  —> Not necessarily Multiple myeloma
  —> Others: Infections, Autoimmune disease

Question 7

Agglutination

Answer 7

Cold agglutinin disease
- Autoimmune Ab at low temperature —> clumping of RBC + haemolysis

Question 8

Iron cycle

Answer 8

Almost a closed loop under physiological condition

Closed loop:
Main bulk of Fe: RBC (2g)
—> die
—> taken up by Macrophage (1g)
—> digest + release Fe
—> Fe bound to **Transferrin (Diferric transferrin)
—> Diferric transferrin taken up by **Bone marrow for making RBC
—> new RBC made

Daily intake: daily requirement 1mg
Daily loss: 1mg in faeces (mucosa shedding)
—> ∴ most Fe deficiency anaemia NOT due to deficient Fe intake (∵ very little Fe dietary intake)

Factors that preturb the cycle:
1. Blood loss in the cycle (e.g. menstruation, peptic ulcer, upper / lower GI bleeding)
—> Fe loss —> Fe deficiency anaemia

Question 9

Absorption of non-heme iron in Duodenal mucosa

Answer 9

Fe in Food usually in **Ferric state (Fe3+)
—> **Gastric acid convert Ferric to **Ferrous (Fe2+) through DCYTB (duodenal cytochrome B)
—> Fe2+ absorbed via **DMT1 (Divalent metal transporter 1) (Apical) into cytoplasm
—> processed by Hephaestin
—> Fe2+ transported out via **Ferroportin (Basolateral)
—> Fe2+ converted back to Fe3+ by Hephaestin
—> Fe3+ bind to **Transferrin
—> ***Diferric transferrin

Fe in Heme format
—> absorbed via Putative heme transporter into cytoplasm

Problem in converting Fe3+ to Fe2+ by acid:
1. **Gastrectomy
2. **Prolonged PPI use
3. Autoimmune gastritis
—> Deficiency in Fe intake —> If prolonged —> Might cause ***Fe deficiency anaemia

Ferroportin (site of action of Hepcidin) expression:
1. Duodenal mucosa
2. Macrophage
—> **Hepcidin (mediator of inflammation)
—> negative regulator of Ferroportin
—> **Anaemia of chronic disease

Question 10

Erythropoiesis: Hb synthesis

Answer 10

Diferric transferrin
—> bind to **Transferrin receptor of RBC membrane
—> endocytosis
—> cleavage —> Transferrin released back into circulation + Transferrin receptor back into cell membrane
—> **Ferric ion taken up by mitochondria
—> **ALAS-2: Enzyme making **Heme (+ Globin —> Hb)

Defect in ALAS-2 (by drugs / MDS etc.)
—> Defective heme synthesis
—> **Fe accumulation in mitochondria of RBC
—> **Sideroblast (Erythroid precursors)

Sideroblastic anaemia:
1. **MDS
2. **Congenital lack of ALAS-2
3. Drug-induced

Question 11

Examples of Fe restricted erythropoiesis

Answer 11

1. Absolute Fe deficiency
   - **Blood loss via GI tract, genital tract —> **Fe deficiency anaemia
   - **↓ Fe absorption due to gastric surgery, malabsorption, atrophic gastritis (long term) —> **Fe deficiency anaemia
   - ↑ Demand in pregnancy
   - Inadequate intake (rare except in anorexia nervosa)
2. Fe-sequestration syndromes (↑ Hepcidin)
   - ***Anaemia of chronic disease
3. Functional Fe deficiency
   - ***Use of erythropoiesis stimulating agents without Fe supplement in chronic kidney failure

Question 12

Anaemia of chronic disease

Answer 12

Chronic diseases:
1. Infections
2. Cancer
3. Autoimmune diseases
4. Chronic rejection after solid-organ transplantation
5. Chronic kidney disease and inflammation (not just Erythropoietin deficiency)

Question 13

Proposed mechanisms of Anaemia of chronic illness

Answer 13

Inflammation / Infection
—> Cytokines release / LPS (from bacteria)
—> act on Liver
—> ***Liver produce Hepcidin
—> Hepcidin suppress Ferroportin

1. Ferroportin on Duodenal mucosa
   —> ***↓ Fe absorption in GI tract
2. Ferroportin on Macrophage
   —> Macrophage cannot release Fe after engulfing RBC
   —> ***↓ Fe utilisation by retention in macrophages

Overall mixed situation: ↓ Fe absorption (Fe deficiency) + ↓ Fe utilisation (even might have plenty Fe store)
—> cannot make RBC

Fe deficiency anaemia and Anaemia of chronic illness —> ***Closely linked —> Diagnostic difficulty

Question 14

***Fe deficiency anaemia vs Anaemia of chronic disease

Answer 14

Fe deficiency anaemia and Anaemia of chronic illness —> ***Closely linked —> Diagnostic difficulty

Fe deficiency anaemia:
- Clinical pictures suggesting Fe deficiency (e.g. menorrhagia)
- Could be **severe (depend on chronicity)
- Hypochromic microcytic
- **↓ Serum Fe
- **↑ Transferrin (TIBC)
- **↓ Transferrin saturation (Serum Fe / TIBC)
- ***↓ Ferritin (reflect Fe store but a lot of confounding factors, e.g. Ferritin an APR)
- ↑ Soluble transferrin receptor
- Normal serum Hepcidin

Anaemia of chronic disease:
- Clinical pictures suggesting **Chronic disease but not Fe deficiency (e.g. RA, chronic illness, infection, malignancy)
- Generally **modest anaemia (never severe: Hb ~9-10)
- Normochromic normocytic (occasionally Hypochromic microcytic)
- **↓ Serum Fe
- **↓ / no change Transferrin (TIBC)
- **↓ Transferrin saturation (Serum Fe / TIBC)
- **Normal / ↑ Ferritin (reflect Fe store but a lot of confounding factors)
- Normal soluble transferrin receptor
- ***↑ serum Hepcidin

Question 15

Absorption of Vit B12

Answer 15

Vit B12 in food (dairy product, animal product)
—> combine with **Intrinsic Factor in stomach (produced by **Parietal cells)
—> **Vit B12-Intrinsic Factor Complex
—> travel along small intestine
—> Complex absorbed in **Terminal ileum
—> Essential Vit (cannot be produced by human alone)

Vegan / Vegetarians:
- A lot of food products now fortified with Vit B12
- Fermented products (e.g. Bread) also contain Vit B12
- Intestinal microbiota can make Vit B12 (but already in large intestine so no use)

Question 16

Vit B12 deficiency anaemia

Answer 16

Pathophysiology:
1. Deficiency / Defective **Intrinsic Factor / release in stomach (e.g. **gastrectomy) (body store of Vit B12 enough for 1/2 years)

2. Defective **absorption of Vit B12-IF complex at terminal ileum (e.g. **ileectomy)

Causes:
1. Medical
- Autoimmune (i.e. ***Pernicious anaemia) (95% of all medical causes)
- Drugs (Metformin, PPI) (never severe enough to cause anaemia)
- Strict vegetarian / vegan (never severe enough to cause anaemia)

2. Surgical
   - **Gastrectomy
   - **Ileectomy

Question 17

Cellular functions of Vit B12

Answer 17

Vit B12:
- Binds to **Transcobalamin 2 / Holotranscobalamin (Active fraction of Vit B12, recognised and taken up by every tissue)
(some may bind to Transcobalamin 3 / Haptocorrin, but not recognised by tissue —> inactive, not bound to tissue)
—> Recognised by **TC2 receptor
—> Endocytosis
—> Lysozyme release Vit B12
—> Vit B12 function

1. Metabolism
   - Methylmalonyl CoA changed in Mitochondria —> Succinyl CoA (important for ***TCA cycle)
2. DNA synthesis
   - Convert Homocysteine —> Methionine
   —> generate THF (Tetrahydrofolate) from MethylTHF
   —> ***THF for DNA synthesis

Significance:
1. NOT measure total Vit B12 (∵ those bound to TC3 are inactive) —> only measure **Vit B12-TC2 (active fraction)
—> Transcobalamin 3 / Haptocorrin produced by **myeloid cells (↑ when ↑ in myeloid cells e.g. myeloproliferative neoplasms e.g. CML)

2. At tissue level (whether tissue deficient in Vit B12), can measure **Homocysteine / **Methylmalonyl CoA —> ↑ in Vit B12 deficiency

Question 18

Clinical presentation of Pernicious anaemia

Answer 18

Pernicious anaemia: a type of Vit B12 deficiency anaemia
- only refers to Anaemia results from lack of **IF
- Autoimmune process affecting mucosa in stomach
—> Ab against **Parietal cell + Ab against ***IF
—> block Vit B12 from binding to IF

1. Asymptomatic, Incidental finding of ***macrocytic anaemia (∵ chronic problem)
2. Anaemia
3. ***Early greying of hairs (∵ autoimmune process)
4. ***Mild jaundice (∵ Intramedullary haemolysis)
5. ***Painful Glossitis: beefy-red, smooth tongue (∵ autoimmune process)
6. ***Angular stomatitis
7. **Neuropathy (in severe cases, Subacute **Combined Degeneration of cord (corticospinal tract + peripheral nerve affected), ∵ Vit B12 also needed in neural tissue)

Question 19

Laboratory abnormalities in Pernicious anaemia / Vit B12 deficiency anaemia

Answer 19

1. ***Pancytopenia
   - ∵ Vit B12 also important for making WBC, platelets
2. ***Macrocytic anaemia
3. ***Intramedullary haemolysis (premature haemolysis of RBC precursor / other precursor cells)
   - ↑ Unconjugated Bilirubin + LDH
4. **Hypersegmented neutrophils + **Macroovalocytes on blood film

Question 20

Investigations for Suspected Pernicious anaemia

Answer 20

1. Serum Vit B12 level
2. Serum ***Transcobalamin 2 (Holotranscobalamin)
3. Serum + Red cell Folate level (not essential)
4. ***Anti-parietal cell Ab
   - sensitive but less specific, also detectable in small proportion of normal individuals (i.e. false positive)
5. ***Anti-IF Ab
   - specific but less sensitive, some patients with PA may be negative (i.e. false negative)
6. Upper endoscopy
   - **Atrophic gastritis
   - Surveillance for **Carcinoma of stomach
7. Schilling test
   - historical interests, obsolete in HK
   - take radioactive Vit B12 and see how much absorbed / excreted
8. Bone marrow examination
   - not routinely needed, except when laboratory findings incompatible with PA

Question 21

Management of Pernicious anaemia / Vit B12 deficiency anaemia (CL Lai)

Answer 21

IM Vit B12 (cannot give oral since already cannot absorb in stomach)