JC48 (Medicine) - Hemolytic anaemia, Aplastic anaemia, Inherited anaemia

Question 1

Inherited causes of haemolytic anaemia

Answer 1

RBC Membrane defect:

• Hereditary spherocytosis
• Hereditary elliptocytosis

RBC enzyme deficiency

• G6PD deficiency
• Pyruvate kinase/ glycolytic enzyme deficiency
• Pyrimidine 5’ nucleotidase deficiency

Haemoglobin defect

• Thalassaemia
• Abnormalities: e.g. Sickle cell disease

Question 2

acquired causes of haemolytic anaemia

Answer 2

Immune

1. Autoimmune - Warm or Cold
2. Alloimmune: Transfusion reaction or Hemolytic disease of Newborn
3. Drug induced hemolysis

Non-immune:

• Mechanical: Prosthetic valves, microangiopathic (DIC, TTP)
• Infection: bacteria (C. perfringens) and parasites (malaria)
• Chemical: Wilson’s disease, Heat, venom, Oxidative drugs, chemicals >>> damage Hb, cell membrane

Acquired intrinsic red cell defect:
- Paroxysmal nocturnal hemoglobinuria

Question 3

Paroxysmal nocturnal hemoglobinuria

Pathogenesis

Answer 3

PIGA gene mutation > deficiency of glycophosphatidylinositol > Deficiency of CD55, CD59 > RBC membrane defect > complement mediated lysis of RBC

Question 4

Features of haemolytic anaemia on Lab tests: CBC, Serum, PBS

Answer 4

Evidence of RBC destruction:

• High LDH
• High unconjugated bilirubin
• Low haptoglobin
• High methaemalbumin

RBC changes:

• Fragmentation (microangiopathic hemolysis)
• Agglutination (cold agglutinin disease)
• Spherocytes (hereditary or immune-mediated)
• Positive DAT

Bone marrow compensation:

• Reticulocytosis/ Polychromasia on PBS
• Bone marrow erythroid hyperplasia

Question 5

History taking for hemolytic anaemia

Answer 5

1. Age, Sex, Ethnicity
2. Infections (HIV, C. perfringens, Mycoplasma), Travel History (Malaria)
3. Drug use (L-dopa, methyldopa, mefenamic acid, penicillin, quinidine)
4. Haematological malignancies, autoimmune diseases
5. Transfusion history
6. Metabolic disease/ Wilson disease/ IEMs (enzyme defects)
7. Surgical history: prosthetic valves/ filters
8. Gallstone history (portal hypertension, hypersplenism)

Question 6

General clinical features of hemolytic anaemia

Answer 6

Pallor

Jaundice (no obstructive jaundice signs as unconjugated bilirubin only)

Haemoglobinuria (intravascular hemolysis/ G6PD…etc)

Splenomegaly

Question 7

Immune haemolytic anaemia

• Cause
• Categorize causative antibodies
• Pathogenesis of drug-induced haemolysis

Answer 7

Autoimmune antibodies against self RBC

Autoimmune:

• Warm IgG
• Cold IgM

Alloimmune: previous sensitization to foreign antigen

• Hemolytic disease of newborn: Rh- or ABO-incompatibility between mother/ child
• Hemolytic transfusion reaction: ABO-incompatibility

Drug induced:

• Alteration of RBC antigen causing AutoAb cross-reaction with normal antigens
• Hapten reaction: associate RBC structures forming part of antigen

Question 8

Compare underlying causes of Warm and Cold type autoimmune haemolytic anaemia

Answer 8

Warm antibodies (80%): binds best at 37oC, majority IgG, often against Rhesus antigens

• Idiopathic (50%)
• Preceding viral infection: usually in children
• Autoimmune disease, eg. SLE, ALPS
• Immunodeficiencies, eg. CVID
• Lymphoproliferative disease, eg. CLL, NHL

Cold antibodies (20%): binds best at 4oC, usually IgM and binds complement

• Idiopathic
• Infections, esp M. pneumoniae, EBV
• Lymphoid malignancies, eg. monoclonal gammopathies, NHL, CLL

Question 9

Investigations for diagnosis of AIHA

Answer 9

CBC - Anaemia, usually normocytic
PBS - Reticulocytosis + spherocytosis
Serum - High unconjugated bilirubin, High LDH, Low hepatoglobin
Direct antiglobulin test/ Coombs’ test: positive

Question 10

Compare direct and indirect antiglobulin tests (/)

Answer 10

Direct Coombs test: useful in detecting prior Ab binding to RBC using anti-IgG, anti-C3

• Autoimmune hemolytic anaemia
• Hemolytic disease of newborn
• Transfusion reactions

Indirect Coombs test: useful in detecting autoreactive Ab in serum

• Antibody screening in pre-transfusion tests
• Screening for HDN during pregnancy

Question 11

Clinical presentation of warm AIHA (/)

Answer 11

Typical S/S of extravascular haemolytic anaemia

Presents as part of:
- Evans syndrome: co-occurrence of ≥2 immune cytopaenias, most often AIHA + ITP
- Lymphoproliferative disease: most commonly in CLL, present with systemic symptoms (eg. LOW/LOA, fever, LN)

Question 12

Clinical presentation of cold AIHA (/)

Answer 12

Asymptomatic when not exposed to cold

Cold-induced symptoms:
→ Acrocyanosis
→ Livedo reticularis
→ Raynaud phenomenon
→ Cutaneous ulcer/necrosis
→ Pain/discomfort on swallowing cold food/liquids

Haemolytic anaemia: generally extravascular
→ Variable severity from compensated haemolysis to severe haemolytic anaemia requiring transfusion
→ Precipitant: cold ambient temperature, febrile illness

High risk of VTE

Lymphoid malignancy S/S

Mycobacterium pneumoniae infection/ Infectious Mononucleosis S/S

Question 13

Management of warm AIHA

Answer 13

1. Folate supplement
2. Decrease further hemolysis/ immunosuppress:
   - Oral prednisolone
   - Rituximab
   - Splenectomy
   - Alternative immunosuppressants: azathioprine, cyclophosphamide
3. Transfusion: test for allo-Ab (T/S)
4. Treat underlying causes (e.g. lymphoproliferative diseases)

Question 14

Management of Cold AIHA

Answer 14

1. Folate supplement
2. Avoid cold temperature: eg. avoid cold liquid, warm clothing, pre-warmed IV fluid
3. Therapy for anaemia:
   - Transfusion with prior T/S
4. Decrease antibody production

• Rituximab/bortezomib-based chemotherapy for idiopathic cases
• Plasmapheresis, IVIg
• Treat underlying lymphoid malignancy

Question 15

Aplastic anaemia

• Definition
• Causes

Answer 15

Bone marrow hypoplasia/ aplasia causing pancytopenia

• *Primary aplastic anaemia:**
• Primary idiopathic (80%)
• Inherited bone marrow failure syndromes: Fanconi anaemia, Shwachman-Diamond syndrome, Dyskeratosis congenita
• *Secondary aplastic anaemia:**
• Drugs: alcohol, NSAIDs
• Toxins: benzenes, pesticides
• Ionizing radiation
• Infection: sero-negative hepatitis, HIV, EBV
• Acquired clonal abnormalities (PNG, MDS)

Question 16

List drugs that cause secondary aplastic anaemia (/)

Answer 16

Cytotoxic drugs: anticipated effect with nadir d7-10
Antibiotics, eg. chloramphenicol, sulphonamide
DMARDs, eg. penicillamine, gold
NSAIDs, eg. phenylbutazone, indomethacin, diclofenac
Thionamides, eg. carbimazole, propylthiouracil
Anticonvulsants, eg. carbamazepine, phenytoin

Alcohol

Question 17

Idiopathic aplastic anaemia

• Pathogenesis

Answer 17

Immune-mediated, T-cell suppression of marrow stem cells

Autoimmunity due to over-expression of HLA-DR2, polymorphisms in perforin gene or TNF-α

Question 18

Clinical features of Aplastic anaemia

Answer 18

Pancytopenia: recurrent infections, mucocutaneous bleeding, anaemic symptoms
→ Infections: typically bacterial (sepsis, pneumonia, UTI) but invasive fungal infection is deadly

NO lymphadenopathy, NO hepatosplenomegaly → suggestive of haematological malignancies

± Dysmorphic features in the young, eg. thumb abnormality in Fanconi syndrome

± Haemolytic anaemia, thrombosis → suggestive of associated PNH

Question 19

Aplastic anaemia

First-line investigations and expected results

Answer 19

□ CBC: pancytopenia with normocytic/macrocytic anaemia, reticulocytopenia

□ PBS: NO abnormal cells

□ R/LFT, haemolysis markers, serum B12, RBC folate to r/o alternative causes

□ BM examination: required for diagnosis
→ Profoundly hypocellular marrow with decrease in all elements and replacement by fat/stromal cells
→ Morphologically normal residual haematopoietic cells without megaloblastic haematopoiesis
→ NO BM infiltration by fibrosis/malignancy

□ Other Ix:
→ Autoimmune markers
→ Flow cytometry (↓CD55/59) for PNH
→ Chromosome breakage with diepoxybutane for Fanconi anaemia esp for children

Question 20

Severity grading for aplastic anaemia
Purpose of grading?

Answer 20

Non-severe:
BM cellularity < 25% + peripheral pancytopenia not as severe as ‘severe grade’

Severe:
BM cellularity <25% + 2 out of 3 of following:
1. Neutrophil < 0.5
2. Platelet < 20
3. Reticulocytes < 20

Very severe:
BM cellularity <25% + 2 out of 3 of following:
1. Neutrophil < 0.2*******
2. Platelet < 20
3. Reticulocytes < 20

Purpose: Severe and very severe need cytotoxic drug treatment

Question 21

Treatment of aplastic anaemia **

Answer 21

Allogeneic HSCT: 1st line for young adult + matched donor or refractory disease

Immunosuppressive therapy: Tx for >50y or ineligible for HSCT:
- eltrombopag (TPO-agonist) + antithymocyte globulin (ATG) + Cyclosporine A + steroids

Manage cytopaenia:
- Transfusion for packed cells, platelet concentrate +/- iron chelation/phlebotomy

Infection prophylaxis: antifungals, antibiotics

Question 22

List inherited marrow failure syndromes causing anaemia and pancytopenia

Answer 22

Anaemia only

• Diamond-Blackfan anaemia
• Congenital dyserythropoietic anaemia

Pancytopenia:

• Fanconi anaemia
• Dyskeratosis congenita
• Schwachman-Diamond syndrome

Question 23

Compare the inheritance patterns between 5 inherited marrow failure syndromes (/)

Answer 23

Question 24

Compare between pancytopenia vs anaemia only inherited marrow failure syndromes:

• Consequence of BM failure
• Presence of malignancies
• Presence of short telomeres and chromosomal instability
• Number of genes involved

Answer 24

Question 25

Fanconi anaemia (FA) (/) - Genetic cause - Clinical features - Diagnosis - Treatment

Answer 25

Cause: mutation in ≥17 FA genes (FANCA-Q, majority AR but FNACB/R XLR) → ↓repair of DNA crosslinks → ↑genomic instability → premature loss of HSCT → High risk of CA - MDS (6000×), AML (700×), SCCs Clinical features: Congenital malformations - Skin: hyper-/hypopigmentation, **café-au-lait spots** - **Thumb/radial abnormality**: absent/hypoplastic/bifid thumb, absent/hypoplastic radii - Axial skeleton: **microcephaly, triangular facies, short stature** - VACTERL association: vertebral anomalies, anal, congenital heart (usu VSD), trachea-esophageal, renal, limb defects - BM failure with pancytopenia Diagnosis: peripheral blood T cells for **chromosomal breakage** following exposure to diepoxybutane (DEB) Tx: allogeneic HSCT with androgen Tx

Question 26

Dyskeratosis Congenita (short telomere syndrome) (/) - Genetic cause - Clinical features - Diagnosis - Treatment

Answer 26

Cause: mutation in telomere-related genes → premature cell death or genomic instability Clinical features: → Mucocutaneous findings: classical triad of nail dystrophy, lacy reticular hyperpigmentation of upper chest and neck, oral leukoplakia → BM failure → Lung fibrosis → Cancer predisposition: H&N SCC, gastro/oesophageal, anorectal, skin...etc Dx: clinical + telomeric length analysis + BM hypocellularity Mx: supportive + allogeneic HSCT if available

Question 27

Diamond-Blackfan anaemia (/) - Genetic cause - Clinical features - Diagnosis - Treatment

Answer 27

Cause: genetic mutation affects ribosome synthesis → ↓activation of TP53 tumour suppressor pathway Clinical features: Congenital anomalies: mainly in H&N + UL areas - Craniofacial: hypertelorism, microcephaly, congenital cataract/glaucoma, micro-ophthalmos, blue sclera, high-arched palate, ear malformation - Thumb: bifid, duplication, hypoplasia, absence, flat hypoplastic thenar eminence - Urogenital: dysplastic/horseshoe kidney, duplex ureter, RTA - Others: A/VSD, hypogonadism, mental retardation Pure red cell aplasia (PRCA) - isolated anaemia Diagnosis: isolated macrocytic anemia with onset \<1y, no other cytopenias, reticulocytopenia, normal BM cellularity Tx: transfusion + oral steroid from 6-12mo

Question 28

History taking for inherited anaemia \*

Answer 28

Family history - Pattern of inheritance Age of onset Drug Hx Symptoms of haemolysis Transfusion requirement Complications

Question 29

List 5 inherited RBC membrane defects

Answer 29

Hereditary spherocytosis Hereditary elliptocytosis and hereditary pyropoikilocytosis Southeast Asian ovalocytosis Hereditary acanthocytosis Hereditary Stomatocytosis

Question 30

Outline the underlying membrane proteins associated with inherited RBC defects (/)

Answer 30

Question 31

Hereditary spherocytosis Definition Inheritance, genetic defects, pathogenesis

Answer 31

Definition: hereditary **RBC membrane defect** characterized by spherocytes on RBCs **AD inheritance** **Ankyrin- spectrin complex defect** \>\> abnormal vertical cytoskeleton-membrane interaction \>\> primary loss of membrane (due to microvesiculation) → progressive spherocytosis Poor deformability → **inability to pass through splenic microcirculation** → confers liability to haemolysis

Question 32

Hereditary spherocytosis Clinical presentation

Answer 32

Chronic haemolytic anaemia: moderate, with **jaundice** Crises of anaemia exacerbation/ Aplastic crisis: → Haemolytic crisis due to ↑severity of haemolysis ma/w infections → Megaloblastic crisis due to folate deficiency, a/w during pregnancy → **Aplastic crisis\*\*** due to _parvovirus B19_ infection → severe anaemia with low reticulocyte count Complications of haemolysis: - Neonatal **jaundice** - **splenomegaly** - **pigmented gallstones \*\***

Question 33

Investigations and typical findings of Hereditary Spherocytosis

Answer 33

**CBC**: anaemia, MCV variable, MCHC ≥36g/dL Blood film: **spherocytes, polychromasia** **Bone marrow**: erythroid hyperplasia Markers of extravascular haemolysis: - ↑LDH, ↑unconj bilirubin Flow cytometric analysis - ↓ **eosin 5’ melamide (EMA) binding\*\*** on red cell skeletal proteins * *Osmotic fragility test\*\*** (± pre-incubation) when EMA binding N/A: - ↑fragility compared to normal RBCs when exposed to hypotonic solutions

Question 34

Treatment of Hereditary spherocytosis

Answer 34

\*\*no specific treatment\*\* * *Folic acid** supplementation * *Transfusion** if severe anaemia * *Splenectomy** for severe haemolysis * *EPO** for infants * *Cholecystectomy** if symptomatic gall-stones (Allogeneic HSCT: NOT used due to unfavourable risk-benefit ratio)

Question 35

Categorize haemoglobin disorders with examples

Answer 35

Question 36

List Red Cell Enzymopathies

Answer 36

G6PD deficiency Pyruvate kinase (PK) deficiency Deficiencies of other glycolytic pathways \>\>\> Congenital non-spherocytic haemolytic anaemia

Question 37

PK deficiency enzymopathy (/) - Inheritance - Pathogenesis - Clinical features - Dx - Tx

Answer 37

AR inheritance Pathogenesis: haemolysis due to PK def, tolerated due to ↑O2 delivery from ↑2,3-BPG Clinical features: lifelong condition but age of presentation varies → Chronic, Coombs’-negative haemolytic anaemia: usually from birth → Haemolysis Cx: NNJ, gallstones, jaundice, variable splenomegaly, folate deficiency → Iron overload due to ineffective erythropoiesis or transfusional iron overload Dx: by testing PK activity in RBC haemolysate or genetic testing Mx: transfusion ± chelation, folate supplementation, splenectomy

Question 38

G6PD deficiency - Epidemiology - Inheritance - Pathogenesis

Answer 38

- most common RBC enzymopathy, affects 400M worldwide - Inheritance: **X-linked recessive** - Mutation: **most commonly G6PD-Canton** in China Haemolysis: occurs during exposure to oxidative stress → insufficient regeneration of glutathione due to deficient G6PD activity → oxidant accumulation within RBC → oxidization of Hb and other proteins → formation of Heinz bodies (clumps of denatured Hb) and bite cells as Heinz bodies are removed by macrophages in RES → rigid non-deformable RBC destroyed in RES (extravascular)

Question 39

5 classes of G6PD (/)

Answer 39

- Class I: G6PD activity \<10%, a/w chronic haemolytic anaemia - **Class II: G6PD activity \<10%,** a/w intermittent haemolysis only with NNJ, Favism, drug-induced intravascular haemolysis * *\*\*\*\*\* most common in Chinese with Canton variant \*\*\*\*\*\*** - Class III: G6PD activity 10-60%, a/w intermittent haemolysis only - Class IV (normal): G6PD activity \>60%, a/w no clinical significance - Class V: G6PD activity \>100%, a/w no clinical significance

Question 40

Clinical presentation of G6PD deficiency

Answer 40

Asymptomatic between episodes of haemolysis * *Acute haemolytic anaemia trigger by infection, critical illness, drugs \>\> Intravascular hemolysis** - classically sudden onset of jaundice, pallor, dark urine - abrupt ↓Hb by 3-4g/dL ± abdominal, back pain (due to haemoglobinuria) **Neonatal jaundice**

Question 41

Triggers of acute hemolysis episodes of G6PD deficiency (/)

Answer 41

Question 42

Investigations for G6PD deficiency - Acute hemolytic attacks - Screening tests in newborn - Confirmatory tests

Answer 42

Non-spherocytic intravascular haemolysis during attack → **CBC/PBS: polychromasia with bite cells, hemighosts, ghost cells, Heinz bodies** → **Intravascular haemolysis:** ↑LDH, ↑unconj bilirubin, ↓haptoglobin, ↑methaemalbumin, haemoglobinuria/haemosiderinuria ± AKI **Screening test:** **absence of fluorescence in UV light** or failure to reduce methaemoglobin → indicate failure to generate NADPH (semiquantitative) **Confirmatory test: by G6PD assay** - test activity directly

Question 43

Management of G6PD deficiency

Answer 43

**□ Avoid haemolysis triggers** □ **Transfusion ± aggressive hydration** for acute intravascular haemolysis (Folate supplementation: NOT necessary unless in class I variant a/w chronic haemolysis)