Haem: Haemolytic Anaemia Flashcards
What is the normal lifespan of a RBC?
120 days
How may haemolysis be classified?
Intravascular - within circulation
Extravascular - removal/destruction by reticuloendothelial system
Inherited or Acquired
What are causes of extravascular haemolytic anaemias?
Autoimmune
Alloimmune
Hereditary spherocytosis
What are causes of intravascular haemolytic anaemia?
Malaria G6PD deficiency Mismatched blood transfusion (ABO) Cold antibody haemolytic syndromes Drugs Microangiopathic haemolytic anaemia e.g. haemolytic uraemic syndrome, thrombotic thrombocytopenic purpura Paroxysmal nocturnal haemoglobinuria
What is the most common cause of intravascular haemolytic anaemia globally?
Malaria - plasmodium falciparum malaria (in severe forms depicted as black water fever).
What cause of intravascular haemolysis is linked to malaria?
G6PD deficiency - this is one of the most common genetic disorders worldwide
Describe the pathology of autoimmune haemolytic anaemia.
Antibodies fix and bind to RBCs. These are usually cold antibodies, particularly IgM.
Give an example of a drug that causes HA.
Dapsin
Describe the pathology of MAHA.
MAHA is characterised by damage to the endothelium and in turn to RBCs as they traverse the endothelium. MAHA includes HUS (common cause of acute renal failure in childhood) and TTP.
Describe the pathology of paroxysmal nocturnal haemoglobinuria.
PNH is due to an acquired genetic defect in synthesis of glycosylphosphatidylinositol anchor which is one of the GPI anchors. The GPI anchor is required for one cells to attach proteins to their surface.
How may hereditary haemolytic anaemias be categorized?
Disorders of the:
- Membrane - cytoskeletal proteins, cation permeability
- Red cell metabolism
- Haemoglobin - thalassaemia, sickle cell syndromes, unstable Hb variants
What is the most common cause of hereditary haemolytic anaemia?
Defects of haemoglobin.
RBCs have restricted metabolism that relies particularly on _______ ______ to meet energy needs by generating ____ . Defects in key metabolic pathways in RBCs, particularly _______ , may result in haemolysis.
RBCs have restricted metabolism that relies particularly on anaerobic glycolysis to meet energy needs by generating ATP. Defects in key metabolic pathways in RBCs, particularly ATP, may result in haemolysis.
Give examples of HHA due to Hb defects.
Quantitative defects - Thalassaemia
Qualitative defects - Sickle Cell
Structural defects - unstable Hb variants (rare)
What type of inheritance does hereditary spherocytosis have?
Autosomal dominant
What are the main consequences of haemolytic anaemias?
Anaemia (+/-)
Erythroid hyperplasia with increased rate of RBC production and circulating reticulocytes
Increased folate deman
Susceptibility to effect of parvovirus B19
Propensity to gallstones (cholelithiasis)
Increased risk of:
Iron overload
Osteoporosis
What may parvovirus B19 infection in hereditary spherocytosis result in?
Aplastic crisis
What stain is used to stain for Iron?
Perl stain/Prussian blue stain
What may further increase risk of cholelithiasis in chronic haemolytic anaemia?
Coinheritance of Gilbert syndrome.
What is Gilbert’s syndrome?
A benign variant where bilirubin conjugation is impaired resulting in higher level of unconjugated bilirubin and increased propensity to perform gall stones in the context of chronic haemolytic anaemia.
Gilbert syndrome is prevalent so co-existance with HA is quite common. It is due to a genetic morphism with a change to the promoter region. Instead of normal 6TA repeats in TATA box, which is an important regulatory element in the gene promoter, there is an extra dinucleotide on each allele with the genotype TA7/TA7 which is associated with reduction in transcription of UGT 1A1 gene, thus reduction of enzyme at protein level in the liver and less efficient bilirubin conjugation.
What are the clinical features of HA?
- Pallor
- Jaundice - due to increased generation of bilirubin
- Splenomegaly - spleen has important role in destruction of RBCs in patients with extravascular haemolysis or where there is extramedullary haemopoeisis with enlargement of the spleen and sometimes liver.
- Pigmenturia - refers to the appearance of abnormal urine colour.
- Family history
What are the laboratory features of HA?
- Anaemia - variable feature
- Increased reticulocytes - almost always present except when there is parvovirus infection
- Polychromasia - cells which take up eosinophilic and basophilic dye and have a bluish/purpleish appearance. These correspond to reticulocytes which can only be seen on blood film using a special stain.
- Hyperbilirubinaemia
- Increased LDH - if there is increased RBC destruction, RBC enzymes will be released into the plasma. One of these is LDH glycolytic enzyme. This is a sensitive marker of intravascular haemolysis
- Reduced/absent haptoglobins - when there is intravascular haemolysis. Haptoglobins bind to Hb and the binding capacity is exceeded with haemolysis
- Haemoglobinuria
- Haemosiderinuria - requires a special stain for iron such as Prussian blue stain of urine. Detects iron that is excreted from tubular cells. Implies indirectly the presence of intravascular haemolysis.
Absence or defects in which membrane proteins may lead to haemolytic anaemia?
Band 3, Ankryin, Band 4.2. GPI anchor.
Lipid bilayer rests upon a cytoskeletal scaffold made of spectrin. There are a number of proteins that link the lipid bilayer to the spectrin cytoskeleton. These include Band 3 (major anion transporter in RBCs and in kidneys). A number of other proteins, absence of which can produce haemolytic anaemia including Ankyrin and band 4.2.
What is the cause of paroxysmal nocturnal haemoglobinuria?
GPI anchor deficiency.
GPI anchor deficiency causes paroxysmal nocturnal haemoglobinuria. This is produced by biosynthesis, a highly conserved mechanism for attachment of a variety of biomolecules to the cell surface. This includes complement regulatory proteins – these protect the RBCs from complement damage. As a result of GPI anchor deficiency, the RBCs are destroyed by complement leading to intravascular haemolysis.
What 2 main HAs are due to red cell membrane disorders?
Hereditary spherocytosis
Hereditary elliptocytosis
What protein defects lead to hereditary spherocytosis?
Proteins involved in vertical interactions - these proteins link the lipid bilayer and cytoskeleton
Band 3
Protein 4.2
Ankyrin
Beta-spectrin
What protein defects lead to hereditary elliptocytosis?
Proteins involved in horizontal interactions.
Alpha-spectrin
Beta-spectrin
Protein 4.1
What is the most common genetic defect of the RBC cytoskeleton?
Hereditary spherocytosis
Describe the inheritance pattern of hereditary spherocytosis.
Family history in 75% - typically autosomal dominant
25% recessive or de novo mutation
What is a hallmark feature of hereditary spherocytosis?
Increased sensitivity of in vitro RBCs to lysis in hypotonic saline (osmotic fragility test)
What 2 tests are used for hereditary spherocytosis?
Osmotic fragility test
Reduced binding of eosin-5-maleimide - dye binding test
Describe the features of hereditary spherocytosis on a blood film.
RBCs lack normal area of central pallor arising from biconcave shape.
Cells are smaller than normal and more densely stained.
Hb conc is increased leading to hyperchromic cells.
Polychromasia indicative of young blood cell population
How does the dye binding test for HS work?
Use flow cytometry to look at mean cell fluorescence after incubation with dye eosin-5-myeloidmide. Mean cell fluorescence reduced with HS. Cut off of 0.8 as discriminant for HS. About 97% sensitive. Reliable and rapid.
What is Hereditary Pyropoikilocytosis?
A homozygous state of HE. Whereas heterozygous are seldom affected clinically in homozygous, this may result in severe haemolytic anaemia that sometimes requires transfusion in newborns.
What features are seen on the blood film of hereditary pyropoikilocytosis?
Fragmentation of RBCs
Budding of membranes of cells
Why is G6PD deficiency prevalent in areas of malaria endemicity?
G6PD deficiency protects against malaria
What is the inheritance mode of G6PD deficiency?
X-linked - clinical effects seen predominantly in hemizygous males and homozygous females
What is the role of G6PD?
G6PD catalyses the first step in the pentose phosphate (hexose monophosphate) pathway - generates NADPH required to maintain intracellular glutathoine (GSH)
G6PD in RBC plays relatively minor role in glycolysis, much of which occurs via another anaerobic pathway. Main function of G6PD is to generate NADPH which is required to maintain intracellular levels of glutathione which in turn are needed to protect the RBC against oxidative stress. Deficient G6PD increased vulnerability to OS –> results in several phenotypes. Most important phenotype is acute haemolytic anaemia that is triggered either by oxidative drugs or other agents, or during infection
What are the clinical effects of G6PD deficiency?
Neonatal jaundice
Acute haemolysis (triggered by oxidants/infections)
Chronic haemolytic anaemia (rare)
Neonatal jaundice is common manifestation of G6PD deficiency.
G6PD deficiency worldwide most common cause of kernicteius (brain damage due to severe neonatal jaundice). In most causes that jaundice doesn’t reflect haemolysis, it reflects impairment of bilirubin conjugation in the liver.
Most individuals of G6PD deficiency are _________ . Exposure to _______ ______ , _______ or ______ ______ may cause _______ haemolysis.
Most individuals of G6PD deficiency are asymptomatic. Exposure to oxidant drugs, infection or fava beans may cause intravascular haemolysis. May lead to acute haemolytic crisis.
Describe the blood film seen with G6PD deficiency.
Many contracted cells.
Some nucleated RBCs.
Some cells have parts of cytoplasm removed - known as bite cells.
Hb is retracted to one side of the cell - hemighost cells
These features of characteristic of G6PD deficiency during an acute haemolytic episode. During steady state, the blood film is usually unremarkable.
What are heinz bodies characteristic of?
If you stained blood with methylviolet you see peripheral inclusions called Heinz bodies. These are formed by denatured Hb. Characteristic of oxidative haemolysis. Often only seen transiently because cells containing Heinz bodies are removed quickly by spleen.
What drugs may provoke haemolysis in G6PD deficiency?
Anti-malarials
Primaquine
Antibiotics
Sulphonamides
Ciprofloxacin
Nitrofurantoin
Other drugs
Dapsone
Vitamin k
Fava beans
MothballsWhat is the most common defect in the glycolytic pathway that leads to HA?
Pyruvate kinase deficiency
What are morphological features on the blood film for Pyruvate kinase deficiency?
Morphological features (similar to other glycolytic disorders): short projections (echinocytes), cells shrink in size due to dehydration and come to resemble spherocytes / echninospherocytes. Number of echinocytes markedly increased post-splenectomy.
Deficiency of what enzyme causes defect in nucleotide metabolism?
Pyrimidine 5’-nucleotidase deficiency
Describe the pathology of pyrimidine 5’-nucleotidase deficiency.
Pyrimidine nucleotide is toxic to the RBC but the RBC needs to retain and salvage purine nucleotides so has a selective mechanism for eliminated pyrimidine nucleotides which depends on the enzyme pyrimidine 5’-nucleotidase.
Deficiency of this enzyme, which is rare, causes morphological picture where there prominent basophilic stippling which are basophilic inclusions in the RBCs. There are few conditions where you see basophilic stippling – sometimes see if in lead poisoning (which is a potent inhibitor of the enzyme) though with lead poisoning you don’t see the same degree of haemolysis.
What causes basophilic stippling?
Pyrimidine 5’-nucleotidase deficiency
Lead poisoning
What are the first line investigations for unexplained haemolysis?
Direct antiglobulin test - detect presence of immunoglobulins on the RBC, important to exclude autoimmune haemolysis
Urinary haemosiderin/haemoglobin - Intravascular haemolysis tested for by looking visually at haemoglobinuria or directly at urine microscopically to look at haemosiderin. Intravascular haemolysis: increased LDH level and low or absent haptoblobin level
Osmotic fragility - for membrane defects such as HS
G6PD +/- PK activity
Haemoglobin separation A and F% - Haemoglobin disorders excluded by haemoglobin separation by electrophoresis or high performance liquid chromatography in conjunction with blood count.
Heinz body stain - for evidence of oxidative haemolysis
Ham’s test/Flow cytometry of GPI-linked proteins - for paroxysmal nocturnal haemoglobinuria
Thick and thin blood film - malaria
What are the two patterns of haemolysis seen in G6PD and PK deficiency?
G6PD - usually episodic
PK - causes chronic haemolytic anaemia
What are the principles of management of HA?
- Folic acid supplementation
- Avoidance of precipitating factors e.g. oxidants in G6PD deficiency
- Red cell transfusion/exchange
- Immunisation against blood borne viruses e.g. hepatitis A and B
- Monitor for chronic complications
- Cholecystectomy for symptomatic gallstones
- Splenectomy if indicated
What conditions have indication for splenectomy?
Substantial benefit in:
- PK deficiency and some other enzymopathies
- Hereditary spherocytosis
- Severe elliptocytosis/pyropoikilocytosis
- Thalassaemia syndromes
Immune haemolytic anaemia
What are the risks following splenectomy?
Risk of sepsis by capsulated bacteria e.g. Pneuomococcus. Need to give penicillin prophylaxis and immunisation.
What are the criteria for splenectomy?
Criteria
- Transfusion dependence
- Growth delay
- Physical limitation Hb < 8g/dl
- Hypersplenism
Age:
- not < 3 years
- before 10 years to maximise prepubertal growth
What test is used to diagnose unstable Hb variants?
Heat stability and isopropanol precipitation tests
Cellulose acetate electrophoresis
Electrospray ionisation - mass spectrometry
What is haemoglobin hammersmith?
- Described by Sir John Dacie in 1967 at the Hammersmith Hospital
- Severe electrophoretically silent
- Heinz body haemolytic anaemia
- Mutation disrupts haem contact
- Reduced oxygen affinity
