Haemolytic Anaemias

Question 1

What is haemolytic anaemia?

Answer 1

Haemolytic Anaemia = anaemia due to shortened RBC survival

Question 2

What are the characteristics of a normal RBC life cycle?

Answer 2

2x1011 RBC/day in the bone marrow
Red blood cells circulate for approximately 120 days without nuclei or cytoplasmic organelles.
Components needed for function and survival already are present when erythrocytes reach maturity.
Red cell deformability: squeezing through a splenic sinus.
Ability to survive also depend on cytoplasmic enzymes involved in metabolic pathways.
Removal senescent RBC by the reticular endothelium system of the liver and spleen

Question 3

Describe the steps of haemolysis.

Answer 3

1. Shortened red cell survival 30-80 days
   
   2. Compensation by Bone marrow to increase production
   3. Increased young cells in circulation = Reticulocytosis +/- nucleated RBC
   4. Incompletely compensated haemolysis: RBC production unable to keep up with decreased RBC life span = Decreased Hb
      Basically, to compensate, the bone marrow may increase its output of red cells six- to eightfold by increasing the proportion of red cells produced, expanding the volume of active marrow, and releasing reticulocytes prematurely.

Question 4

What are the clinical findings for haemolytic anaemia?

Answer 4

Clinical findings
- Jaundice due to increase of unconjugated bilirubin
- Pallor
- Fatigue
- Splenomegaly
Chronic clinical findings:
- Gallstones - pigment again as a result of bilirubin
- Leg ulcers
- Folate deficiency - (from increased use to compensate)

Question 5

What does a peripheral blood film show in haemolytic anaemia patients?

Answer 5

Peripheral blood film:– polychromatophilia (cells which are basophilic in colour due to increased RNA content), nucleated RBC, thrombocytosis; neutrophilia with left shift;
- Morphologic abnormalities provides clue to underlying disorder: Spherocytes, Sickle cell, Target cells, Schistocytes (fragmented, triangular RBC) acanthocytes

Question 6

What are the bone marrow findings in haemolytic anaemia patients?

Answer 6

Bone marrow findings: compensatory mechanism to haemolysis.
Erythroid hyperplasia of BM – with normoblastic reaction, Reversal of M:E (myeloid:erythroid) ratio
- Erythroid hyperplasia is present with a predominance of erythroid precursors. The normal myeloid to erythroid ration is 2 to 5:1. In the case of hyperplasia there is reversal of the M:E ratio of 1:4

Question 7

What is reticulocytosis and other findings in patients with haemolytic anaemia?

Answer 7

Reticulocytosis – variable on the cause of haemolysis
- Mild (2-10%) seen in haemoglobinopathies
- Moderate to marked (10-60%) seen in IHAs, HS, G6PD def.
Other findings:
- Increased unconjugated bilirubin
- Increased LDH (lactate dehydrogenase)
- Decreased serum haptoglobin protein that binds free Hb so if there is more haemoglobin
- Increased urobilinogen
- Increased urinary hemosiderin (a brown iron-containing pigment usually derived from the disintegration of extravasated red blood cells)

Question 8

What are the different classifications of haemolytic anaemia?

Answer 8

Inheritance:
- Hereditary- Hereditary spherocytosis 9HS0
- Acquired- Immunes haemolytic anaemia (IHA)
Site of RBC destruction:
- Intravascular- Haemolytic Transfusion Rxn
- Extravascular- Autoimmune haemolysis
Origin of RBC damage:
- Intrinsic (Intracorpuscular, inside the RBC)- G6PD deficiency
- Extrinsic (Extracorpuscular, outside the RBC)- Delayed haemolytic transfusion reaction

Question 9

What are the conditions under the intrinsic and extrinsic classifications of haemolytic anaemia?

Answer 9

Under the intrinsic we have:
- Membrane defects
- Enzyme defects
- Haemoglobin defects
Under the extrinsic category we have immune-mediated:
- Autoimmune- warm , cold or drug induced
- Alloimmune- HDN, haemolytic transfusion rx
And the non-immune:
- Red cell fragmentation syndrome- mechanical trauma (e.g. from an artificial valve) and macroangiopathic HA
- Drugs and chemicals- infections such as malaria, March haemoglobinuria (where haemolysis occurs as a result of repetitive mechanical motions like marching)
- Hypersplenism

Question 10

How do red cell fragmentation syndromes arise?

Answer 10

Red Cell fragmentation syndromes arise through physical damage to red cells either on abnormal surfaces (e.g. artificial heart valves or arterial grafts), arteriovenous malformations or as a microangiopathic haemolytic anaemia.

Question 11

What occurs at the site of RBC destruction?

Answer 11

The normal one occurs extravascularly while the abnormal one occurs intravascularly
Red cells are engulfed in the reticular endothelium system and break them down into globin, iron and protoporphyrin
The iron and globin are reused for the synthesis of haemoglobin
The protoporphyrin is broken down into unconjugated bilirubin which is transported to the liver to be turned into conjugated bilirubin
It is finally transported to the small intestine where it can be excreted as stercobilinogen in the faeces or urine in the form of urobilinogen
When haemolysis occurs in the vascular system, that releases haemoglobin which can be absorbed back into the circulation, which goes into the kidney and the you can find some haemoglobin in the urine
That is called haemoglobin urea
Because of the haemoglobin breakdown you will also get a pigment called haemosiderin found in the urine- termed haemosiderinuria

Question 12

What are the red cell membrane disorders?

Answer 12

The red cell membrane is made up of a lipid bilayers which has got integral peripheral proteins that traverse the lipid bilayer such as Band 3 and Glycophorin A and C
We also have the cytoskeletal proteins such as protein 4.2 and 4.1, ankyrin etc.
Defects in any of these can lead to hereditary spherocytosis (HS) or hereditary elliptocytosis
HS is a dominant autosomal condition caused by defects in the vertical interactions such as defects in the spectrin, ankyrin and protein 4.2
- So deficiency in any of these can cause HS
HE is due to defects in the horizontal interactions like mutations in the alpha spectrin or beta spectrin and also due to a loss of interaction between ankyrin and a spectrin
Also deficiency in protein4.1 can also be a cause of HE

Question 13

What are the clinical features of HS (hereditary spherocytosis)?

Answer 13

Asymptomatic to severe haemolysis
Neonatal jaundice
Jaundice, splenomegaly (because of the spleen removing the spherical cells), pigment gallstones (because of the breakdown of RBCs there’s a lot more bilirubin)
*Reduced eosin-5-maleimide (EMA) binding is a test for HS– EMA binds to band 3 of the plasma membrane
Positive family history
Negative direct antibody test

Question 14

What is the monophosphate shunt?

Answer 14

10% of red cell glucose is metabolized via the Hexose Monophosphate Shunt (HMS), otherwise known as the Pentose Phosphate Pathway (PPP).
G6PD catalyses the first step in the hexose monophosphate shunt which is necessary for producing NADPH.
Role of the HMP shunt:
- Generates NADPH which reduces glutathione
- Glutathione protects the cell from oxidative stress

Question 15

What does the glucose-6-phosphate dehydrogenase deficiency cause?

Answer 15

Effects of Deficiency.
Oxidative stress due to lack of glutathione:
Oxidation of Hb by oxidant radicals
- resulting denatured Hb aggregates & forms Heinz bodies –which bind to membrane.
Oxidised membrane proteins
- reduced RBC deformability so wont be able to go through small capillaries and return to normal form

Question 16

What is pyruvate kinase deficiency?

Answer 16

An autosomal recessive disorder
Glycolytic Pathway
Generates energy in ATP;
- to maintain red cell shape and deformability
- To regulate intracellular cation conc. via cation pumps (Na/K pump),
Deficiency in PK
- affects the above intracellular cation and more potassium leaves the cell so cell become dehydrated and can lyse quite easily
- So affects deformability and shape (seen as prickle cells)
- Causes chronic non-spherocytic haemolytic anaemia
The anaemia may worsen at times of infection or other stress; dense red cells with spicules (prickle cells) may be seen on the peripheral blood film.

Question 17

What are two globin disorders?

Answer 17

Thalassaemias - quantitative:
Defect in the rate of synthesis alpha- or beta-globin chain (structurally normal)
Variant haemoglobins: - qualitative
production of a structurally abnormal globin chain

Question 18

What are thalassaemias and how are they divided?

Answer 18

Imbalanced alpha and beta chain production
Excess unpaired globin chains are unstable
Heterogenous gp genetic disorders.
Ineffective erythropoiesis
Clinically divided:
	- Hydrop foetalis
	- β-Thalassaemia major
	- Thalassaemia intermedia
	- Thalassaemia minor

Question 19

What are the characteristics of beta thalassaemia major?

Answer 19

Clinical features
- Severe anaemia
- Progressive hepatosplenomegaly
- Bone marrow expansion (compensatory)– facial bone abnormalities
- Mild jaundice
- Iron overload which can affect the endocrine organs
- Intermittent infections as a result of spleen removal, pallor from anaemia
Peripheral blood:
- Microcytic hypochromic with decreased MCV, MCH, MCHC
- Anisopoikilocytosis; target cells, nucleated RBC, tear drop cells
- Reticulocytes >2%

Question 20

What are the characteristics of beta-thalassaemia minor?

Answer 20

Asymptomatic
Often confused with Fe deficiency
α-thal trait often by exclusion
HbA2 increased in b-thal trait – (diagnostic haemoglobin electrophoresis)

Question 21

What are the characterisitcs of alpha-thalassaemias; Hb BArts hydrops syndrome and HbH disease?

Answer 21

Hb Barts hydrops syndrome (- -/- -)
- deletion of all 4 globin genes
- incompatible with life because the alpha globin chain is required for foetal haemoglobin too
HbH disease (- α/- -)
- Deletion of 3/4 α-globin genes
- Common in SE Asia
Clin Features:
- moderate chronic HA
- Splenomegaly, hepatomegaly sometimes
- hypochromic microcytic anaemia, poikilocytosis, polychromasia, target cells
- Electrophoresis as a diagnostic
Thal trait (minor) (- α/αα; - α/- α; - -/αα)
- Deletion of one or two alpha haemoglobin genes
- Normal or mild HA
- MCV & MCH low

Question 22

What is thalassaemia intermedia?

Answer 22

Disorder with clinical manifestation between major and minor

- transfusion independent
- diverse clinical phenotype
- Varying symptoms
- Increased bilirubin level
- diagnosis – largely clinical
- e.g. include βE/mild β+ (HbE- β-thal); β0/mild β+ (one parent has loss of beta gene the other reduced beta gene)

Question 23

What are poikilocytes?

Answer 23

Poikilocytes (abnormally-shaped cells), target cells and teardrop cells, which signifies marked dyserythropoiesis.

Question 24

What is sickle cell disease?

Answer 24

SCD – refers to all diseases as a result of inherited HbS;
Hb S caused by single nucleotide substitution
- On the sixth amino acid position that changes glutamic acid to valine which makes the haemoglobin insoluble when it is deoxygenated which then polymerises and forms a sickle shaped cell
- HbSS is sickle cell anaemia (homozygous state)
- HbAS – sickle cell trait (heterozygous)

Question 25

How does Hb sickling happen?

Answer 25

Deoxygenation of SS RBC leads to intracellular Hb polymerisation, loss of deformability and morphological changes leading to irreversible sickling

Question 26

What are the clinical features of SCD?

Answer 26

Clinical:
- Painful crises- are the most frequent.
· They may be sporadic and unpredictable or precipitated by infection, acidosis, dehydration or deoxygenation (e.g. altitude, operations, obstetric delivery, stasis of the circulation, exposure to cold, violent exercise).
- Aplastic crises- occur as a result of infection with parvovirus or from folate deficiency and are characterized by a sudden fall in haemoglobin and reticulocytes, usually requiring transfusion.
- Infections due to hyposplenism
- Acute sickling (the most common cause of death in all ages):
· Chest syndrome
· Splenic sequestration
· Stroke
- Chronic sickling effects:
· Renal failure
· Avascular necrosis of the bone

Question 27

What are the laboratory features of SCD?

Answer 27

- Anaemia
	· Hb often 60-90g/L
	- Reticulocytosis
	- Increased NRBC
	- Raised bilirubin
	- Low creatinine

Question 28

How can you confirm diagnosis of sickle cell anaemia?

Answer 28

Solubility test
- Expose blood to reducing agent
- We have a positive and negative control, you can see the lines behind the negative
- Hb S precipitated so can’t see lines drawn behind tube
- Positive in trait and disease
With the HPLC (high performance liquid chromatography)
- The different bands indicate different haemoglobins
And electrophoresis