Haemolytic Anaemias Flashcards
What is anaemia ?
Anaemia: ↓ Hb = ↓ RBCs = ↓ oxygen-carrying capacity = cannot meet physiological needs
What is haemolytic anaemia ?
Anaemia which is due to ⬆ breakdown of RBCs
(↓ RBC survival)
Describe the variation in blood Hb concentration
↑ Hb level in neonates
↓ Hb in infants
↑ 1 years old-adulthood (20 years old aprox)
↑ Hb in males
Describe the normal RBC life cycle .
- RBC produced in bone marrow.
- 7.8 microns diameter - flexible=squeeze through 3.5micron capillaries ( = they deform their shape, cytoplasmic enzymes)
- Circulate for 120 days (no nuclei/cytoplasmic organelles(mitochondria)).
- RBC enzymes produce energy to maintain RBC shape.
- RES removes RBCs after 120 days (reticuloendothelial system=liver/spleen)
What is Haemolysis ?
- Shortened RBC lifespan from 120 days -> 30-80 days
- BM compensates by ⬆ RBC production
=⬆ immature RBCs (⬆ reticulocytes & nucleated RBCs)
What is compensated haemolysis?
⬆ RBC production(BM) compensates for ↓ RBC life span (⬆ RBC destruction) = normal Hb levels
What is incompletely compensated haemolysis?
insufficient RBC production to balance out ⬇ RBC life span (↑ haemolysis) = ↓ Hb
What are some clinical findings with haemolysis?
Jaundice
Pallor + Fatigue
Splenomegaly
Normal urine
Why does haemolyisis cause jaundice ?
- RBC Hb broken down -> haem + globin.
- Haem broken down -> protoporphyrin (contains iron).
- Protoporphyrin broken down -> bilirubin (unconjugated in plasma) = yellow skin + eyes
What is Pallor and why does haemolysis cause this ?
Pale appearance linked with fatigue.
-Caused by ↓ Hb = insufficient oxygen to the tissues.
What is splenomegaly and how is it caused ?
Spleen = Organ that removes old/damaged RBCs cells
Splenomegaly = Spleen enlarges in haemolysis
What happens to the urine in haemolysis?
Haemolysis = ↑ urobilinogen = normal/dark urine
What is haemolytic crisis ?
Haemolytic crisis = ↑ anaemia + jaundice with infections/precipitants.
What is aplastic crisis?
Aplastic crises = Anaemia involving other cells as well as RBCs
Reticulocytopenia (↓ reticulocytes) and parvovirus infection.
More chronic clinical findings of haemolysis
- Gallstones - pigment stones (due to ⬆ breakdown of RBC -> BILIRUBIN)
- Leg ulcers - due to vascular stasis/local ischaemia
- Folate deficiency - due to ⬆ demand due to RBC being broken down and more required
Lab investigation for Haemolytic Anaemia - Bone marrow findings
-As a compensatory mechanism to haemolysis, erythroid hyperplasia occurs in BM - normoblasts formed, Myeloid:Erythroid ratio reversal (normal M:E = 2:1-5:1).
Haemolytic anaemia = erythroid hyperplasia = M:E = 1:2-1:5
-Reticulocytosis - variable.
Mild reticulocytosis = 2-10% of reticulocytes ⬆ in BM = haemoglobinopathy.
Moderate-Marked reticulocytosis = 10-60% reticulocytes ⬆ in BM = Immune Haemolytic Anaemia, Hereditary Spherocytosis, G6P Dehydrogenase deficiency
Typical Lab Findings for Haemolytic Anaemia
- ⬆ reticulocyte count (reticulocytosis)
- ⬆ unconjugated bilirubin in plasma - ⬆ RBC breakdown
- ⬆ LDH (lactate dehydrogenase) -released from lysed RBC
- ⬇ serum haptoglobin = protein that binds free haemoglobin. More Hb is being released into plasma, haptoglobin binds to Hb = ⬇ haptoglobin in serum
- ⬆ urobilinogen. bilirubin is released + metabolised
- ⬆ urinary hemosiderin
Abnormal blood film
What can we see on the blood film for HA?
-Reticulocytes
-Polychromatophilic cells = RBCs basophilic in colour due to ⬆ RNA content, + bigger than normal RBCs
-Nucleated RBCs - being broken down
-Poikilocytes - Different shapes of RBC
-Spherocytes, Sickle cell, Target cells, Schistocytes (fragmented, triangular rbc)
acanthocytes
Classification of Haemolytic Anaemia - 3 categories
- Inheritance - Inherited/Acquired
- Site of RBC destruction - Intravascular/Extravascular(outside vessels,within reticuloendothelial system=spleen + liver)
- Origin of RBC damage - Intrinsic/Extrinsic
Inheritance classification of haemolytic anaemia
- Hereditary - Hereditary spherocytosis
- Acquired - Immune Haemolytic Anaemia
Site of RBC destruction classification of haemolytic anaemia
Intravascular - e.g. Haemolytic transfusion reaction.
Thrombotic thrombocytopenic purpura
(Blood disorder which causes clots forming in blood vessels -low RBC, platelets due to breakdown )
Extravascular = outside vascular system, within reticuloendothelial system (liver + spleen) - e.g. Autoimmune haemolysis (antibodies are directed against a person’s own RBC causing them to burst)
Origin of RBC damage classification of haemolytic anaemia
Intrinsic (within RBC) - e.g. G6PD deficiency
(Genetic disorder that affects mostly males, G6PD enzyme not enough - red bloods cells don’t work properly
Extrinsic (outside RBC) - e.g. infection
Delayed haemolytic transfusion reaction (present with RBC haemolysis following transfusion)
Describe Acquired RBC haemolysis
Immune:
- Autoimmune (immune system)
- Alloimmune (immune response to non self antigens)
Non-immune:
-Paroxysmal Noctural haematuria (rare acquired disease which destroys RBC) - not by immune system
Go through the different types/causes of Hereditary RBC haemolysis:
RBC enzymopathies :
- G6PD deficiency
- PK deficiency
RBC membrane disorders:
- Hereditary spherocytosis (sphere shaped RBC instead of biconcave)
- Hereditary elliptocytosis (elliptical rather than biconcave)
Haemoglobinopathies:
-Sickle cell diseases
-Thalassaemia
(no/little haemoglobin production)
Where is the normal site of RBC destruction?
Normal RBC destruction = Extravascular haemolysis
- Reticuloendothelial Macrophages engulf abnormal RBC and break it down into globin + iron + protoporphyrin.
- Iron + globin are reused to make Hb
- Protoporphyrin -> bilirubin. Bilirubin becomes unconjugated bilirubin and is taken to liver where it becomes bilurubin glucoronides (conjugated) and is excreted in faeces as stercobilinogen/urine as urobilinogen
Describe intravascular RBC breakdown
Intravascular haemolysis = Abnormal
RBC breakdown within vascular system = Hb is released into circulation (free Hb). Some Hb enters kidney + urine (haemoglobinuria). + Hb breakdown forms haemosiderin (also enters urine=haemosiderinuria)
What is the pathway that protoporphyrin undergoes following extravascular haemolysis?
Protoporphyrin → Bilirubin
Bilirubin = unconjugated in blood plasma, transported to liver
Bilirubin is carried to liver + gut
Bilirubin is conjugated to stercobilinogen in faeces
OR
Bilirubin is reabsorbed + travels to kidneys = urobilinogen in urine.
Describe the pathway of iron following extravascular RBC breakdown
The iron is transported in blood by transferrin.
(transferrin = iron transport protein)
Describe the pathway followed by globin following RBC being broken down extravascular
The globin will be broken down into amino acids
What is haemoglobinuria ?
The presence of excess haemoglobin in the urine
What is haemosiderinuria
Presence of hemosiderin in the urine.
Haemosiderin = the protein compound which stores iron in tissues.
When Hb breaks down, it releases iron.
What is Methaemalbumin ?
Methaemalbumin = degraded haemoglobin enters the plasma and binds to albumin.
What is haptoglobin ?
Haptoglobin = Protein encoded by HP gene.
In blood plasma, haptoglobin binds to free haemoglobin.
Describe the normal structure of a red cell membrane
- Lipid bilayer
- Integral proteins - protein band 3, glycophorin A, glycophorin C
- Membrane skeleton
-Cytoskeletal proteins - spectrin alpha, spectrin beta, ankyrin, protein 4.2, protein 4.1, actin
protein defects = hereditary spherocytosis/hereditary elliptocytosis
What are some defects which may arise in the vertical interaction and what can this cause?
Causes hereditary speherocytosis.
Defects can occour in the following proteins :
- Spectrin
- Band 3
- Protein 4.2
- Ankyrin
What are some proteins in which defects in horizontal interactions may result in and what can this cause ?
Causes hereditary elliptocytosis.
Defects can occour in the following proteins :
- Protein 4.1
- Glycophorin C
- Spectrin - HPP
- Loss of interaction b/w ankyrin + spectrin
Describe how hereditary spherocytosis may arise
Most common RBC membrane defect
Common hereditary haemolytic anaemia
Autosomal dominant inheritance (75%)
Defects in proteins involved in vertical interactions between the membrane skeleton and the lipid bilayer - spectrin + ankyrin, protein 4.2
Decreased membrane deformability
Bone marrow produces biconcave RBC but as membrane is lost, RBC becomes spherical
Hereditary Spherocytosis Blood Film
Sphere shape
Deeply stained, no central pallor
Polychromatophilic - ⬆ RNA. membrane protein defects
These RBCs go through reticuloendothelial system, can’t retain biconcave shape = attain spherical shape = ⬇ deformability = haemolysis
Hereditary elliptocytosis blood film
Elongated RBCs but with no pointed ends (teardrop cells have 1 pointed end)
How is Haemolytic spherocytosis managed ?
Monitored
Folic Acid
Transfusion
Splenectomy
MCV in Hereditary Spherocytosis is ……….
Normal
Clinical Features of Hereditary Spherocytosis
Asymptomatic - severe haemolysis
Neonatal jaundice
Splenomegaly - Spleen constantly removing spherical RBCs=spleen enlarges
Pigment gall stones - Caused by constant RBC breakdown ➔ bilurubin (pigment)
Reduced eosin-5-maleimide (EMA) binding - EMA binds to RBC plasma membrane Band 3 protein= test for Hereditary Spherocytosis
Positive family history (autosomal dominant inheritance)
Negative direct antibody test
What are enzymopathies ?
These are enzymes which are responsible for producing energy in the glycolytic pathway.
This is used to maintain the RBC size and haemoglobin
What are the RBC metabolic pathways?
- Glycolysis
- HMS
- Rapoport Leubering shunt
What are the roles of HMP shunt ?
-Generates reduced glutathione
-Protects the cell from oxidative stress
So RBC that are G6P deficient look fine and live a normal amount of time but only when exposed to oxidizing precipitants e.g. drugs, fava beans etc. you get oxidation of membrane proteins and Hb.
How do RBC generate energy ?
They do this through Glycolysis (Emden-Meyerhof pathway),Hexose Monophosphate shunt , Rapoport Luebering Shunt.
What are the two most common enzyme abnormalities ?
Glucose -6-Phosphate deficiency
Pyruvate Kinase deficiency
G6PD in RBC HMP Shunt (Pentose Phosphate Pathway)
Glucose -> G6P (G6PD) -> 6PG.
G6PD enzyme converts G6P -> 6PG. This reaction produces NADPH. NADPH converts oxidised glutathione (GSSG) to reduced glutathione (GSH).
Reduced glutathione = antioxidant, protects RBC membrane + RBC Hb against oxidative stress
Absent reduced glutathione = RBC exposed to oxidative stress = haemolysis
Absent reduced glutathione = Hb oxidation = Hb denatures + aggregates + forms Heinz bodies within RBC
Absent reduced glutathione = membrane proteins oxidise = ⬇ RBC deformability
Describe G6PD deficiency
- Hereditary, X-linked disorder (if its in the X chromosome, men have it and it can also occur in women-In cells they randomly switch off 1 of the genes and so 50% of cells will be G6P Deficient)
- Common in African, Asian, Mediterranean and Middle Eastern populations
- Mild in African (type A), more severe in Mediterraneans (type B)
- Clinical features range from asymptomatic (except for neonatal jaundice)until they get exposed to things to acute episodes to chronic haemolysis (which is less common)
What are the effects of oxidative stress?
Oxidation of Hb by oxidant radicals
- Resulting in denatured Hb aggregates and forms Heinz bodies (bind to membrane)
- Oxidised membrane proteins-Reduced RBC deformability
What are some oxidative precipitants ?
-Infections
-Fava/broad beans
-Drugs :
Anti-biotics/Anti-Malarial
Dapsone
Nitroflurantoin
Ciprofloxacin
Primaquine - antimalarial drug = causes oxidative stress + Heinz bodies + impact RBC membrane
Patients with G6PD deficiency have protection against………
Severe malaria
What exactly does the HMP shunt do ?
The HMP shunt extends the life span of RBC by maintaining membrane proteins and lipids.
It diverts Glucose-6-phosphate to 6-phosphogluconate through Gluycose-6-phosphate dehydrogenase
What are the features of G6PD deficiency ?
- Haemolysis
- Blood Film - target cells, bite cells, blister cells, ghost cells, Heinz bodies with methylene blue)
How can you make a diagnosis of G6PD deficiency?
This can be done using an enzyme assay.
- May be falsely normal if reticlocytosis is occurring after a recent episode as reticulocytes usually have high enzyme levels .
- Wait for the reticlocytes to decrease or check ratios between enzyme levels .
What happens to patients who are having a acute Haemolytic episode ?
- They will be come acutely jaundiced.
- May have intravascular Haemolysis, dark urine from Hb in the urine.
- Anaemic
What is the morphology of oxidative haemolysis ?
The presence of Heinz bodies and bite cells
What are bite cells?
These are abnormally shaped mature RBC with one or more semicircular portions removed from the cell margin.
What are Heinz bodies?
These are inclusions within RBC composed of denatured Hb
Describe Pyruvate Kinase deficiency
-Pyruvate kinase is required to generate ATP.
-This is essential for membrane cation pumps (deformability)
-Autosomal recessive and much rarer.
-Can cause chronic haemolytic anaemia
Mild to transfusion dependent
(Depends on genetics do you make a tiny bit less or do you make no PK)
Improves with splenectomy (HS goes to normal Hb remember)
what you see characteristically on a blood film are prickle cells (small and spikey), polychromasia.
PK deficiency = ⬇ intracellular ATP
ATP maintains RBC shape + deformability
Na+/K+ ATPase pump = cell dehydrates + lyses
Blood film - prickle cells, large central pallor = ⬇ Hb
non-spherocytic haemolytic anaemia
What are Haemoglobinopathies ?
This is a group of disorders which are inherited. They cause a abnormal production or structure of Hb.
Example is SCD
What is the normal structure of Haemoglobin ?
Ferrous iron + Protoporphoryin IX =Haem
Globin protein
Haem + Globin = Haemoglobin
Describe normal Haemoglobin
HbA -Adult which consists of α2β2 (97%)with some α2 delta2 e (2-3.5%)
and alpha2 gamma 2 (foetal Hb)
Describe the globin gene expression during foetal development
Alpha chains are expressed from the beginning and if problems occur in utero , alpha chains are required.
For e.g. Alpha thalassemia zero
Beta chains are not expressed until a few weeks old during the neonatal period.
The most common haemoglobinopathies are Beta chains.
What are the 2 groups of globin disorders?
Quantitative =thalassaemias
(The production of increased/decreased amount of globin chain) - Reduced/absent alpha/beta globin chain synthesis, structurally normal
Qualitative =variant haemoglobins - Production of a structurally abnormal globin chain.
Describe the different Haemoglobin disorders
HbS =This decrease solubility and causes polymerisation
Hb Koln=Decreases stability and increases Heinz body formation
HbC=Decreases solubility and increases crystallisation
What are Thalassaemias?
This is an imbalanced alpha and beta chain production
Excess unpaired globin chains are unstable
-Precipitate and damage RBC and precursors
-Ineffective erythropoiesis in bone marrow
-Haemolytic anaemia
What are the 2 types of Beta thalassaemia that can be inherited?
Beta thalassaemia trait
Beta Thalassaemia major
How do we diagnose the thalassaemia trait ?
-Asymptomatic
-Microcytic hypochromic anaemia
-Low Hb, MCV, MCH
-Increased RBC
-Often confused with Fe deficiency
Because your making less β chains, HbA2 is increased in b-thal trait –(diagnostic test)
a-thal trait often by exclusion
-globin chain synthesis (rarely done now)
-DNA studies (expensive)
Beta thalassaemia major
Absence of both beta globin chains
Severe anaemia, 3-6 months after birth
Pallor
Progressive hepatosplenomegaly - erythroid hyperplasia
Bone marrow expansion – facial bone abnormalities
Mild jaundice
Transfusion = Iron overload, affects endocrine organs
Splenectomy = Intermittent infections
Microcytic hypochromic RBCs with ⬇ MCV, ⬇ MCH, ⬇ MCHC
Anisopoikilocytosis: target cells, nucleated RBC, tear drop cells
Reticulocytes ⬆ >2%
Describe Beta Thalassaemia major
- Transfusion dependent in 1st year of life
- When they stop making Foetal Hb they become progressively anaemic
-If not transfused:
Failure to thrive
Progressive hepatosplenomegaly (big liver and spleen and so have big tummies)
Bone marrow expansion – skeletal abnormalities (facial shape changes aswell)
Death in 1st 5 years of life from anaemia
Side effects of transfusion:
Iron overload from the excess iron in the transfused blood - usually treated through medication (iron chelators help iron be excreted in urine) if not can cause:
Endocrinopathies
Heart failure
Liver cirrhosis
And so Treated with transfusions and iron chelators
β-thalassaemia trait (minor)
Asymptomatic
Often confused with Fe deficiency
α-thal trait often by exclusion
HbA2 increased in b-thal trait – (diagnostic - electrophoresis) - both beta thalassaemia trait + beta thalassaemia major A2 bands are increased
What are the chronic complications of SCD?
Silent infarcts
Pulmonary hypertension
Chronic lung disease, bronchiectasis
Erectile dysfunction
Azoospermia
Chronic pain syndromes
Delayed puberty
Avascular necrosis
Moya-moya
Retinopathy, visual loss
Describe Sickle cell disease
Tested for in newborn babies in the UK
Clinically significant sickling syndromes:
HbSS
HbSC
HbS-D Punjab (started in india)
HbS- O Arab (started in arab peninsula)
HbS- β thalassaemia – ( the other gene can’t make beta chains and so you get SC)
And so as you can see you can get a sickle cell disease by having one S mutation on a beta gene matched up with other mutations on the other gene.
Point mutation in the β globin gene: glutamic acid → valine
Insoluble haemoglobin tetramer when deoxygenated → polymerisation
“Sickle” shaped cells
SC cause a huge spectrum of problems; intravascular haemolysis and so changes in NO, Abnormal shaped RBC have abnormal membranes which effect vasculature.
What are the acute complications of SCD?
Stroke-Ischaemia and Haemorrhagic
Cholecystitis
Hepatic sequestration
Dactylitis
Bone pain & infarcts
Osteomyelitis
Retinal detachment
Vitreous haemorrhage
Chest syndrome
Splenic sequestration
Haematuria: papillary necrosis
Priapism
Aplastic crisis
Leg ulcers
What are the clinical features of SCD
Painful crises
Aplastic crises
Infections
Acute sickling:
Chest syndrome
Splenic sequestration
Stroke
Chronic sickling effects:
- Renal failure
- Avascular necrosis bone
What are the laboratory features of SCD?
Anaemia (Hb often 65-85)
Reticulocytosis
Increased NRBC (nucleated RBC)
Raised Bilurubin - increased RBC breakdown
Low creatinine
How can we confirm the diagnosis of SCD ?
-Through a solubility test
Expose blood to reducing agent
HbS precipitated
Positive in trait and disease
-Using electrophoresis structure
A-C =SCT
D-E = HbC trait
F= SCD
-HPLC
The extra portion at the end of the trait machine result when compared to normal.
SS patient= no HbA.
To diagnose SCD, use HPLC/Hb electrophoresis
What are the two types of acquired Haemolytic anaemia ?
Autoimmune
Alloimmune
Describe Autoimmune Haemolytic Anaemia
Idiopathic
(Usually warm)
(IgG, IgM)
Drug mediated
Cancer associated
(LPDs)
Describe Alloimmune Haemolytic Anaemia
Transplacental transfer:
Haemolytic disease of the newborn:D, c, L
ABO incompatability
Transfusion related:
Acute haemolytic transfusion reaction
ABO
Delayed haemolytic transfusion reaction:
E.g Rh groups, Duffy
Describe Non-Immune acquired haemolysis
Paroxysmal nocturnal haemoglobinuria
RBC become vulnerable through a mutation in proteins which protects the cell normally from the complement mediated lysis. And so there is intravascular Haemolysis through the lysis of the RBC.
Fragmentation haemolysis:
Mechanical
Microangiopathic haemolysis
Disseminated intravascular coagulation – fibrin strands from clotting factors because of sepsis in the capillaries, damage the red cells
Thrombotic thrombocytopenic purpura
Other:
Severe burns
Some infections: e.g. malaria
Breakdown of RBCs in Haemolytic anaemia is often accompanied by……….
This may maintain……
⬆ RBC production
Normal Hb level (if it compensates for shortened RBC lifespan)
Anaemia can be classified based on:
Cause of anaemia:
- RBC loss
- Insufficient normal RBC production
- Excessive destruction of RBCs (haemolytic anaemia)
RBC morphology:
- Microcytic
- Macrocytic
- Normocytic
Haemolytic Anaemia - Intrinsic Classification
Membrane defects:
- Hereditary Spherocytosis
- Hereditary Elliptocytosis
- Hereditary Pyropoikilocytosis
Enzyme Defects:
- G6PD deficiency
- PK deficiency
Haemoglobin Defects:
- Sickle Cell Disease
- Thalassaemias
Haemolytic Anaemia - Extrinsic Classification
Immune-Mediated:
-Autoimmune:
- Warm - high temp. 37oC
- Cold - low temp. 4oC-37oC
- Drug-induced
-Alloimmune:
- Haemolytic Disease of Newborn
- Haemolytic Transfusion Reaction
Non-Immune-Mediated:
-RBC Fragmentation Syndrome:
-Mechanical damage - e.g. artificial valve destroys RBCs
-Microangiopathic Haemolytic Anaemia = destroy RBCs within vascular system, caused by fibrin deposited in vascular endothelium - e.g. Haemolytic Urinic Syndrome, Thrombotic thrombocytopenic purpura
(Blood disorder which causes clots forming in blood vessels -low RBC, platelets due to breakdown), Disseminated Intravascular Coagulation
- Drugs + Chemicals
- Infection - e.g. Malaria, Clostridium
- March Haemoglobinuria
- Hypersplenism
Intravascular Haemolytic Anaemia
- G6PD deficiency
- PK deficiency
- Drug-induced
- Haemolytic transfusion reaction
- RBC fragmentation syndrome
- Infection
- March haemoglobinuria
Poikilocytosis =
Anisocytosis =
Hypochromic =
Poikilocytosis = RBCs of diff. shapes
Anisocytosis = RBCs of diff. sizes
Hypochromic = RBCs with larger central pallor
