Anaemias

Question 1

How are the haemoglobinopathies classified?

Answer 1

1) Qualitative abnormalities (“abnormal haemoglobins”, including sickle cell disease) - normal quantities of an abnormal haemoglobin
- amino acid substitutions in the polypeptide chains of the globin gene cause the haem ring to fall out of the haem binding pocket, making the haemoglobin unstable
2) Quantitative abnormalities (thalassaemias) - abnormal quantities of normal Hb
- mutations reduce the rate of production of one or other of the globin chains altering the ratio of alpha to non alpha

Question 2

What are the haemoglobinopathies?

Answer 2

These are diseases caused by dysfunction of genes encoding the globin chains of haemoglobin.

Normal Hb is comprised of two alpha and two non alpha globin chains. Alpha globin chains are produced throughout life, so severe mutations may cause intrauterine death.

Production of non alpha chains varies with age: faetal haemoglobin (HbF) has two gamma chains, while the predominant adult haemoglobin (HbA) has two beta chains. So disorders affecting the beta chains do not present until after 6 months of age.

Question 3

What is the pathophysiology of sickle cell disease?

Answer 3

Deoxygenated HbS causes molecules to polymerise to form pseudocrystalline structures called “tactoids”.

These distort the red cell membrane and produce sickle-shaped cells.

Polymerisation reversible when reoxygenation occurs; distortion may become permanent and the red cell irreversibly sickled.

Greater the concentration of HbS the more easily tactoids are formed (this is affected by the presence of other haemoglobins, e.g. HbC or HbF).

Question 4

Who gets sickle cell disease and what is the relationship to malaria?

Answer 4

Sickle cell disease is autosomal recessive.

Homozygotes only produce abnormal beta chains that make HbS and this results in sickle cell disease.

Heterozygotes produce a mixture of normal and abnormal beta chains that make normal HbA and HbS - this causes asymptomatic sickle trait.

Heterozygote frequency is >20% in tropical Africa. Sickle cell trait carriers are resistant to the lethal effects of falciparum malaria (survival advantage). Homozygote patients are not protected.

Question 5

How is sickle cell disease diagnosed?

Answer 5

Compensated anaemia - 60-80g/L

Blood film - sickle cells, target cells and hyposplenism (i.e. Howell-Jolly bodies). Reticulocytosis present

Definitive diagnosis by Hb electrophoresis. Shows absence of HbA and predominance of HbS

MCV is usually near normal - if very low think of sickle cell trait

Question 6

What are the complications of HbSS?

Answer 6

1) Crisis (vaso-occlusive, chest syndrome, sequestration, aplastic)
2) Renal (papillary necrosis, poor concentrating ability, nocturnal eneuresis)
3) Priapism
4) Hepatic disease (gallstones, transfusion hepatitis, liver failure due to infarct)
5) Stroke
6) Leg ulcers
7) Hyposplenism
8) Occular (background retinopathy, proliferative retinopathy, vitreous bleeds)

Question 7

What is an aplastic crisis?

Answer 7

Infection of adult sicklers with parvovirus B19 causes severe but self limiting red cell aplasia.

Produces very low Hb that can cause heart failure.

Reticulocyte count is low (unlike all other sickle crises)

Question 8

What is a sequestration crisis?

Answer 8

Thrombosis of venous outflow from an organ causes loss of function and painful enlargement.

Spleen is most common site in children.

Massive splenic enlargement may cause severe anaemia, circulatory collapse and death.

Liver may undergo sequestration in adults with pain due to capsular stretching.

Think of this is Hb drops suddenly in a sickle patient!

Question 9

What is a vaso-occlusive crisis?

Answer 9

This is a complication of sickle cell disease. Sickling is precipitated by hypoxia, acidosis, dehydration and infection. Irreversibly sickled cells have a shorter lifespan and plug vessels in the microcirculation causes a number of acute syndromes.

Vaso-occlusive crisis:

Plugging of small vessels in the bone produces acute severe bone pain
Affects areas of active marrow: hands and feet in children (dactylitis) or femora, humerus, ribs, pelvis and vertebrae in adults
Systemic response (tachycardia, sweating, fever)
Most common sickle crisis

Question 10

What is sickle chest syndrome?

Answer 10

May follow from vaso-occlusive crisis.

Most common cause of death in adult sickle disease.

Bone marrow infarction causes fat emoboli to the lungs which cause further sickling and infarction leading to ventilatory failure.

Question 11

How are sickle cell crises managed?

Answer 11

Vaso-occlusive - rehydration, oxygen, analgesia (opiates) and antibiotics. Transfuse with fully genotyped blood.

Sequestration and aplastic crisis - top up transfusion.

Regular transfusion programme to suppress HbS production and maintain HbS level below 30% may be indicated in patients with recurrent severe complications (e.g. CVA in children or chest syndromes in adults).

Hydroxycarbamide - if recurrent/ severe crises.

Exchange transfusion (simultaneous venesection and transfusion to replace HbS with HbA) if life-threatening crisis or to prepare for surgery.

Question 12

Why do patients with high levels of HbF have a milder form of disease?

Answer 12

High HbF inhibits polymerisation of HbS and reduces sickling. Patients with sickle cell disease and high HbF have milder clinical course with fewer crises.

Question 13

What are the thalassaemias?

Answer 13

Inherited impairment of haemoglobin production with partial or complete failure to synthesise a specific type of globin chain (i.e. abnormal amount of normal Hb, cf. sickle cell disease)

Alpha thalassaemia - disruption of one or both alleles on chromosome 16 producing some or no alpha globin genes

Beta thalassaemia - defective production causes disabling point mutations causing no or reduced beta chain production

Question 14

What is the pathophysiology of thalassaemia?

Answer 14

Beta thalassaemia is the most common type of thalassaemia.

Failure to synthesise beta chains.

Heterozygotes have thalassaemia MINOR - mild anaemia, little or no clinical disability, may be detected when treatment for presumed iron deficiency does not work

Homozygotes have thalassaemia MAJOR - unable to synthesise HbA or produce very little; develop profound hypochromic anaemia after the first 4-6 months

Question 15

Why do patients with sickle cell disease need vaccination?

Answer 15

Due to splenic infarction, sickle cell patients are functionally asplenic.

All patients with sickle cell should receive prophylaxis with daily folic acid and penicillin V to protect against pneumococcal infection.

They should be vaccinated against pneumococcus and Haemophilus influenzae B and hepatitis B (pneumococcus and Haemophilus are encapsulated bacteria)

Question 16

What are the diagnostic features of beta thalassaemia major?

Answer 16

Profound hypochromic anaemia
Evidence of severe red cell dysplasia
Erythroblastosis
Absence or gross reduction of the amount of haemoglobin A
Increased levels of HbF
Evidence that both parents have thalassaemia

Question 17

How is beta thalassaemia major treated?

Answer 17

Erythropoietic failure:

• Allogenic bone marrow transplant from HLA compatible siblings
• Transfusion to maintain Hb >100g/L
• Folic acid 5mg daily

Iron overload:

• Iron therapy forbidden
• Iron chelation therapy

Splenomegaly causing mechanical problems, excessive transfusion needs:
- Splenectomy

Question 18

What is the pathophysiology of alpha thalassaemia?

Answer 18

Reduced or absent alpha chain synthesis is common in Southeast Asia.

Two alpha gene loci on chromosome 16 and each individual carries four alpha gene alleles:

• If one is deleted - no clinical effect
• If two are deleted - mild hypochromic anaemia
• If three are deleted - patient has haemoglobin H disease
• If all four deleted - hydrops fetalis

HbH is a beta chain tetramer formed from the excess beta chains which are functionally useless. Patient relies on low HbA levels for oxygen transport. Treatment of HbH disease is similar to intermediate severity beta thalassaemia (e.g. folic acid, transfusion etc).

Question 19

What are the diagnostic features of beta thalassaemia minor?

Answer 19

Mild anaemia
Microcytic hypochromic erythrocytes (not iron deficient)
Some target cells
Punctate basophilia
Raised haemoglobin A2 fraction
Evidence that one parent has thalassaemia

Question 20

How is haemolytic anaemia classified?

Answer 20

1) Congenital/ Inherited:
- Hb: thalassaemias, sickle cell disease
- Enzyme: G6PD deficiency, pyruvate kinase deficiency
- Membrane: hereditary spherocytosis, hereditary eliptocytosis
2) Acquired:
i) Immune - DCT +ve
- Autoimmune - e.g. warm and cold types
- Alloimmune - antibodies produced by one individual reacts with red cells of another, e.g. transfusion reaction, HDN
- Drugs
ii) Non-immune - DCT -ve
- Red cell fragmentation - heart valves, microangiopathic haemolysis (HUS, TTP, DIC)
- Infections (e.g. Malaria), drugs, burns
- PNH

Question 21

What investigations confirm haemolysis is present?

Answer 21

Key investigations:

1) Increased reticulocyte count (also occurs with blood loss and partially treated anaemia)
2) LDH
3) Bilirubin (release of red cell components - increased unconjugated bilirubin)
4) Reduced serum haptoglobins
5) Bone marrow - erythroid hyperplasia

Question 22

If haemolysis is suspected, how can we determine whether there is an underlying red cell change?

Answer 22

On history:

Onset at an early age or a family history - intrinsic red cell defect
Ethnicity: African Americans (sickle cell), Southern Mediterranean (Thalassaemia), G6PD
In the laboratory:

Blood film - target cells (thalassaemia, sickle cell disease), spherocytes (hereditary spherocytosis, immune haemolysis), fragments (microangiopathic), polychromasia
Direct Coombs test
Hb electrophoresis (at pH 8.6 and 6.8) for thalassaemia and sickle cell disease
G6PD and pyruvate kinase screening tests
Ham’s test/ immunophenotyping

Question 23

What causes intravascular haemolysis?

Answer 23

During intravascular haemolysis, free Hb is released into plasma that is filtered by the urine as haemoglobinuria (black urine) and haemosiderinuria. A portion is bound to albumin as methemalbuinuria. Haptoglobins are also reduced.

Think “MCP”

M - Microangiopathic haemolytic anaemia (irregularly fragmented cells; maybe helmet cells on blood film seen in up to 25% of patients with DIC)

M - March haemoglobinuria

C - Chronic cold agglutinin disease

C - Cardiac valvular disease

P - Paroxysmal cold haemoglobinuria

P - Paroxysmal nocturnal haemoglobinuria

Question 24

What happens in extravascular haemolysis?

Answer 24

Usually happens in membrane defects and most cases of haemolytic anaemia.

Haemolysis is extravascular (outside the blood) and takes place in the reticuloendothelial tissues. Avoids free Hb in the plasma.

Question 25

Why does a leuco-erythroblastic picture appear on the blood film in haemolysis?

Answer 25

Bone marrow compensation for increased red cell breakdown is reticulocytosis. Nucleated red cell precursors may appear in the blood.

Activation of the bone marrow can result in neutrophilia and immature granulocytes appearing in the blood. This is called a leuco-erythroblastic picture.

Compensatory erythroid hyperplasia may give rise to folate deficiency. Blood findings will be complicated by the presence of megaloblastosis (measurement of haemolysis and serum is unreliable in haemolysis as it will be elevated).

Question 26

What is the structure of the normal red cell membrane?

Answer 26

RBC membrane is a lipid bilayer in close associations with membrane proteins.

Two types of membrane-protein interaction:

1) Membrane-integral proteins such as ion channels (band 3), which interact with each other and also the second group of proteins:
2) Proteins that form the cytoskeleton (alpha and beta spectrin)

Ankyrin is a protein that bridges beta spectrin to band 3. Mutations of the integral proteins usually result in spherocytosis, whilst abnormalities of the cytoskeletal proteins cause eliptocytosis. Gallstones are also frequent and cause episodes of cholecystitis and biliary colic.

Question 27

What is hereditary spherocytosis? How does it present?

Answer 27

Autosomal dominant (25% of cases have no family history and represent new mutations).

Most commonly caused by deficiency of beta spectrin or ankyrin.

The severity of spontaneous haemolysis varies.

Presents as asymptomatic compensated chronic haemolytic state with spherocytes present on the blood film, a reticulocytosis and mild hyperbilirubinemia. Gallstones are present in 50%.

Occasional cases are associated with more severe haemolysis. This can be due to coincidental polymorphisms in alpha spectrin or co-inheritance of a second defect involving a different protein.

Crises can occur

Question 28

What crises can occur in hereditary spherocytosis?

Answer 28

1) Haemolytic crises - the severity of haemolysis increases; rare and usually associated with infection
2) Megaloblastic crisis - follows development of folate deficiency; this may occur as a first presentation of the disease in pregnancy
3) Aplastic crisis - in association with erythrovirus infection. Causes common exanthem in children, but if individuals with chronic haemolysis become infected the virus directly invades red cell precursors and temporarily switches off red cell production. Presents with severe anaemia and a low reticulocyte count

Question 29

How is hereditary spherocytosis investigated?

Answer 29

Patient and other family members should be screened for features of compensated haemolysis. This may be all that is required to confirm the diagnosis.

Hb levels are variable, depending on the degree of compensation. MCHC is increased.

Blood film shows spherocytes but direct Coombs test is negative, excluding immune haemolysis.

Osmotic fragility test show increased sensitivity to lysis in hypotonic solutions (low sensitivity and specificity).

EMA (eosin-5-maleimide) binding test recommended now.

Question 30

How is hereditary spherocytosis managed?

Answer 30

Folic acid prophylaxis 5mg once weekly given for life.

Consideration for splenectomy which improves life but does not normalise red cell survival. Potential indications include moderate to severe haemolysis with complications (e.g. anaemia, gallstones) - splenectomy is delayed until after age 6 years due to risk of sepsis

For acute, severe haemolytic crises patients need transfusion support. Blood must be cross-matched carefully and transfused slowly as haemolytic transfusion reactions may occur.

Question 31

What is hereditary elliptocytosis?

Answer 31

Heterogeneous group of disorders that produce an increase in elliptocytic red cells on the blood film and variable degree of haemolysis.

Functional abnormality of one or more anchor proteins in the red cell membrane, e.g. alpha spectrin or protein 4.1.

Inheritance may be autosomal dominant or recessive. Less common than hereditary spherocytosis.

Variable clinical course and depends on degree of membrane dysfunction. Most present as asymptomatic blood film abnormality, but can result in neonatal haemolysis or a chronic compensated haemolytic state. Management of the latter is the same as for hereditary spherocytosis.

Question 32

What are the red cell enzymopathies?

Answer 32

Mature RBC must produce energy via ATP to maintain a normal internal environment and cell volume whilst protecting itself from oxidative stress presented by oxygen carriage.

Anaerobic glycolysis via the Ebden-Meyerhof pathway generates ATP and the hexose monophosphate shunt produces NADPH and glutathione to protect against oxidative stress.

Impact of functional or quantitative defects in the enzymes in these pathways depends on the importance of the steps affected and the presence of alternative pathways.

Generally, defects in hexose-monophosphate shunt lead to periodic haemolysis induced by oxidative stress, whilst those in the Ebden-Meyerhof pathway results in shortened red cell survival and chronic haemolysis.

Question 33

What is glucose-6-phosphate dehydrogenase deficiency?

Answer 33

Important enzyme in hexose-monophosphate shunt pathway.

Deficiency results in most common human enzymopathy, affecting 10% of worlds population. Distribution parallels malaria (heterozygotes are protected).

Enzyme has heteromeric structure made of catalytic subunits, encoded by X chromosome. Deficiency affects males and rare homozygotic females, but is carried by females. Carrier heterozygote females are usually only affected in the neonatal period or in the presence of extreme lyonisation producing selective inactivation of one of the X chromosomes.

Question 34

What are the clinical features of G6PD deficiency?

Answer 34

Acute drug-induced haemolysis
Chronic compensated haemolysis
Infection or acute illness
Neonatal jaundice: maybe a feature of the B- enzymes
Favism, i.e. acute haemolysis after ingestion of the broad beans

Question 35

What drugs can precipitate haemolysis in G6PD deficiency?

Answer 35

Analgesics: aspirin, phenacetin

Antimalarials: primaquine, quinine, chloroquine, pyrimethamine

Antibiotics: sulphonamides, nitrofurantoin, ciprofloxacin

Misc: quinidine, probenecid, vitamin K, dapsone

Question 36

What are the laboratory features of G6PD deficiency?

Answer 36

Blood film shows:

Bite cells (red cells with a “bite” of membrane missing)
Blister cells
Irregularly shaped small cells
Polychromasia reflecting the reticulocytosis
Denatured haemoglobin visible as Heinz bodies within the red cell cytoplasm, if stains with a supravital stain such as methyl violet

G6PD level:

Can be indirectly assessed by screening methods which usually depend upon the decreased ability to reduce dyes
Direct assessment of G6PD is made in those with low screen values
Care must be taken close to an acute haemolytic episode because reticulocytes may have higher enzyme levels and give rise to a false normal result

Question 37

How should the splenectomised patient be managed?

Answer 37

Vaccinate with pneumococcal, Haemophilus type B, meningococcal group C and influenza vaccines at least 2-3 weeks before elective splenectomy. Vaccination should be given after emergency surgery, but may be less effective

Pneumococcal re-immunisation should be given at least 5 yearly and influenza annually

Life long prophylactic penicillin V 500mg BD (erythromycin in penicillin allergic patients)

If septic, resuscitate and give antibiotics with pneumococcus, Haemophilus and meningococcal cover

Increased risk of malaria

Treat animal bites promptly

Question 38

What is pyruvate kinase deficiency?

Answer 38

Another red cell enzymopathy.

Second most common red cell enzyme defect.

Results in deficiency of ATP production and a chronic haemolytic anaemia. Inherited as autosomal recessive trait.

Extent of anaemia is variable; blood film shows characteristic “prickle cells: which resemble holly leaves. Enzyme activity 5-20% of normal.

Transfusion support may be needed.

Question 39

What is pyrimidine 5’ nucleotide deficiency?

Answer 39

Another RBC enzymopathy.

This enzyme catalyses dephosphorylation of nucleoside monophosphate and is important during the degradation of RNA in reticulocytes.

Inherited as autosomal recessive trait and is as common as pyruvate kinase deficiency in Mediterranean, African and Jewish populations.

Accumulation of excess ribonucleoprotein results in coarse basophilic stippling associated with a chronic haemolytic state.

Enzyme is very sensitive to inhibition by lead (this is the reason why basophilic stippling happens in lead poisoning)

Question 40

What is autoimmune haemolytic anaemia?

Answer 40

Increased red cell destruction due to red cell antibodies.

Antibodies may be IgG or IgM, and rarely IgE or A.

If an antibody avidly fixes complement it will cause intravascular haemolysis, but if complement activation is weak the haemolysis will be extravascular.

Antibody coated RBCs lose membrane to macrophages in the spleen, and hence spherocytes are present in the blood.

Question 41

What are the different types of AIHA?

Answer 41

The optimum temperature at which the antibody is active (thermal specificity) is used to classify immune haemolysis:

1) Warm antibodies - bind at 37oC; 80% of cases. Majority are IgG and often react against Rhesus antigens
2) Cold antibodies - bind at 4oC but can bind up to 37oC in some cases. Usually IgM and bind complement. Account for 20%