W3L5 - Haemolytic Anaemia: Introduction & Intrinsic Defects

Question 1

Haemolytic Anaemia

Answer 1

Physiological haemolysis removes senescent RBC as part of the normal turnover of cells
Haemolytic anaemia refers to the anaemia caused by the increased & premature destruction of RBCs
Typically, initially normocytic normochromic
Complicated by
- increased erythropoiesis
- specific pathophysiology, e.g. spherocytes formation may ↓ MCV
Anaemia may not be evident if the rate of haemolysis is compensated (balanced) by increased erythropoiesis

Question 2

Senescent Cell-Specific Antigen

Answer 2

Part of physiological haemolysis
Changes in the conformation of the membrane domain of band 3 lead to the appearance of a senescent cell-specific antigen
Recognised by specific, autologous immunoglobulin G
This marks senescent erythrocytes for removal by macrophages

Question 3

Sites of Haemolysis

Answer 3

Intravascular
- occurs within the vasculature; i.e. while cells are circulating in the blood
Extravascular haemolysis
- occurs outside the vasculature; i.e. after cells have been removed from the circulating blood

Question 4

Intravascular Haemolysis

Answer 4

Occurs within the circulation
Minority of cases (~10%)
‘Free’ Hb binds to haptoglobin or haemopexin
Transport proteins take Hb to liver for degradation
Excess Hb oxidises to methaemoglobin (MetHb, Fe3+ ) as no longer ‘protected’ by cell
MetHb dissociates to haemin and globin
Globin chains are broken down & AAs recycled
Haemin binds to haemopexin or albumin
Degraded in liver

Question 5

Intravascular Haemolysis - What happens if Mechanisms are Overwhelmed

Answer 5

Free haemoglobin in the blood (plasma)
Haemoglobin filtered through the kidney => hb in the urine (haemoglobinuria)
Also methaemoglobinuria (Fe3+ in Hb in urine)
Haemosiderin may form from the haemoglobin present and be detected in the urine (haemosiderinuria)
- as excess haemoglobin is filtered by the kidney some is reabsorbed in the proximal convoluted tubule
- the iron is removed and converted to hemosiderin
- the tubule cells of the proximal tubule with the hemosiderin are lost into the urine
- can be demonstrated with Prussian blue stain

Question 6

Mechanisms of Intravascular Haemolysis

Answer 6

Activation of complement on RBC membrane
Physical or mechanical trauma to RBC
‘Toxic’ plasma factors which act to disrupt the integrity of the cell

Question 7

Extravascular Haemolysis

Answer 7

Occurs outside of circulation
Occurs within macrophages (spleen, liver, BM)
Majority of cases (~90%)
Hb degraded within phagocyte to haem & globin
Haeme degraded to iron, biliverdin and CO
Biliverdin converted to bilirubin, binds to albumin, & degraded by liver
Eventually degraded to urobilinogen & excreted in faeces

Question 8

Causes of Extravascular Haemolysis

Answer 8

Inherited RBC Defects
- e.g. Thalassaemias
Acquired RBC Defects
- e.g. Spur cell anaemia
Autoimmune haemolytic anaemia (AIHA)

Question 9

Difference between Extravascular and Intravascular Haemoglobin Degradation

Answer 9

Extravascular haeme degeneration differs from intravascular as it has:

• no release of Hb into plasma
• no haemoglobinaemia
• no haemoglobinuria
• no haemosiderinuria

Question 10

Clinical Features of Haemolytic Anaemia

Answer 10

Primary clinical signs due to anaemia
- e.g. fatigue, pallor
Other clinical signs associated with increased RBC destruction
- e.g. jaundice, darkened urine (IV haemolysis)
- pigment gallstones may occur
Other changes:
- extra-medullary haematopoietic masses
- splenomegaly
- increased active (‘red’) marrow (may result in bone pain)
- tissue necrosis due to hypoxia

Question 11

Laboratory Investigation of Haemolytic Anaemia

Answer 11

Haematological findings typically reflect:
- increased destruction of RBCs
- increased activity of bone marrow (erythropoiesis)
Steps:
1. Full blood count
2. Peripheral blood film
3. Bone marrow
4. Other laboratory test
Initial aim to distinguish intravascular from extravascular haemolysis

Question 12

Laboratory Investigation of Haemolytic Anaemia - FBC and Peripheral Blood Film

Answer 12

FBC:
- decreased Hb (anaemia)
- variable MCV (macrocytic if reticulocytosis)
- altered sub-populations of RBC
Peripheral blood film:
- poikilocytosis e.g. spherocytes, schistocytes
- polychromasia (+++)
- nRBCs
- increased reticulocytes

Question 13

Laboratory Investigation of Haemolytic Anaemia - Reticulocytosis and Bone Marrow

Answer 13

Reticulocytosis
- takes ~48h
- reflects increased erythropoiesis
- peripheral blood
Bone Marrow:
- erythroid hyperplasia
- orderly RBC maturation
- normal RBC morphology
- decreased M:E ratio

Question 14

Osmotic Fragility

Answer 14

Gives an indication of the surface area:volume ratio of cells
Principle
- small amount of blood is mixed with large excess of buffered saline solution at various concentrations
- the fraction of RBC lysed at each saline solution is determined colorimetrically
- usually performed at room temperature
RBC that are spherocytic take up less water in hypotonic solution before rupturing

Question 15

Intrinsic Haemolytic Anaemias

Answer 15

Reflect a deficit in membrane, enzymes, or protein characteristic of RBC that shorten the lifespan of the cell
May be inherited or acquired
Include:
- hereditary spherocytosis
- hereditary elliptocytosis
- hereditary acanthocytosis
- glucose-6-phosphate dehydrogenase deficiency
- pyruvate kinase deficiency
- paroxysmal nocturnal haemoglobinuria

Question 16

Hereditary Spherocytosis

Answer 16

Membrane defect of main structural protein (spectrin)
Abnormal interactions between cytoskeletal proteins & membrane => microvesicles of membrane bud off
Loss of RBC shape: biconcave to spherical
Mild to moderate haemolysis
Osmotic fragility: increased
DAT: negative
Reticulocytosis

Question 17

Hereditary Elliptocytosis

Answer 17

Defect in glycophorin C
Defective spectrin dimer dimer association
~1 in 4000 persons affected
Milder than hereditary spherocytosis
Cells acquire elliptical shape in circulation
Elliptocytes >25% & often >60%
Reticulocytes ~4%

Question 18

Hereditary Stomatocytosis (Hydrocytes)

Answer 18

Membrane is abnormally permeable to Na+ & K+
Gain of Na+ > loss of K+ => excess H2O enters the cell
=> overly hydrated cells = ‘overhydrated hereditary stomatocytosis (OHS)’
Mild-moderate haemolytic anaemia
- 10-15% stomatocytes
- macrocytosis
- positive osmotic fragility test
- right shift of osmolarity curve

Question 19

Hereditary Stomatocytosis (HSt)

Answer 19

HSt can be classified into:

• syndromic forms that show extra-hematologic signs
• non-syndromic forms with selective involvement of the erythroid system

Question 20

Hereditary Stomatocytosis (HSt) - Syndromic Forms

Answer 20

Stomatin deficient cryohydrocytosis with mental retardation, seizures and hepatosplenomegaly
Phytosterolemia nonleaky stomatocytosis with macrothrombocytopenia
Dehydrated hereditary stomatocytosis with perinatal edema and/or pseudohyperkalemia

Question 21

Hereditary Stomatocytosis (HSt) - Non-Syndromic Forms

Answer 21

Overhydrated hereditary stomatocytosis
Cryohydrocytosis
Dehydrated hereditary stomatocytosis
Familial pseudohyperkalemia

Question 22

Hereditary Acanthocytosis

Answer 22

Inherited abnormality of lipids
Absence of
- serum β-lipoprotein
- low serum cholesterol
- low triglyceride
- increased ratio of cholesterol to phospholipid
Absence of anaemia
Mild haemolysis
Clinical features:
- steatorrhea
- retinitis pigmentosa
- neurological abnormalities

Question 23

Enzyme Deficiences

Answer 23

Mature RBCs cannot synthesise proteins
- limits life-span of normal RBC
- shortened life-span with enzyme deficiency
RBC depend on anaerobic glycolysis for ATP production by the Embden-Meyerhof pathway and Hexose monophosphate shunt
- maintains reduced GSH levels
- protects RBC from oxidative damage

Question 24

Glucose-6-Phosphate Dehydrogenase Deficiency

Answer 24

Enzyme of hexose monophosphate shunt
Deficiency or functional abnormality => inability to resist oxidative damage
Sex linked inheritance
Blood film
- Heinz bodies
- blister cells
- bite cells
- eccentrocytes/‘hemighost cells’
- fragments (crisis)
Screening tests
- fluorescent screening test
- ↓ NADPH, ↓ fluorescence
Definitive test
- G6PD enzyme assay

Question 25

Pyruvate Kinase Deficiency

Answer 25

Enzyme of glycolytic pathway
Deficiency =>
- decreased ATP levels
- Na/K ATPase pump activity
- K+ loss, cell dehydration
- echinocyte formation
- splenic phagocytosis by macrophages
Variable anaemia
RBC Mx: echinocytes “prickle cells”
Autosomal recessive inheritance
Reticulocytosis (polychromasia) is prominent
Bone marrow
- erythroid hyperplasia

Question 26

Paroxysmal Nocturnal Haemoglobinuria

Answer 26

Rare acquired stem cell disorder (~1 in 100,000 people)
Deficiency of glycosylphosphatidylinositol (GPI)
- GPI anchors surface proteins to membrane
- => absence of GPI linked proteins e.g. CD55, CD59
- CD 55: decay accelerating factor (DAF)
- CD 59: membrane inhibitor of reactive lysis (MIRL)
=> RBCs have abnormal sensitivity to complement
- inability to inhibit non specific C activation
Haemolysis occurs while sleeping (haemoglobinuria)
DAT & OF normal
Haemoglobinuria/Haemosiderinuria
Ham’s Test; RBC lysis at low pH