Anaemia and Haemoglobinopathies Flashcards
What is a full blood count?
Assess number and size of cells found in blood eg RBC/WBC/platelets (baseline test)
What is haematopoeisis and where does it occur?
The production of all types of blood cells which occurs in the bone marrow in long bones. Maturation of immature bood cells occurs in bone marrow and mature cells then circulate within peripheral blood
What is EDTA?
Collating agent and it stops the blood from clotting so you are able to analyse cells properly. Used in FBC
What does FBC tell us?
Hb: conc of Haemoglobin (g/L)
Haematocrit: % of blood volume as RBC
MCV (mean cell vol): Average size of RBC
MCH: Average haemoglobin content of RBC
MCHC: calculated measure of haemoglobin concentration in given red blood cells
RDW (red cell distribution width): Range of deviation around RBC size, tells you if theres a great difference between shapes and sizes of cells
Can also request:
Reticulocyte count (immature RBC)
Blood film: microscopy
What are the features of red blood cells in a blood film regarding:
a) Size
b) Shape
c) Colour
d) Inclusions?
Size : big, small, normal
Shape : fragments, tear drop, spiculated, ovalocyte, spherocyte, eliptocyte
Colour: pale (hypochromic), normal, polychromasia
Inclusions: howell-jolly bodies, nuclear reminants, malarial parasites, basophilic stippling
What are some additional tests that can be done besides a FBC?
Bone Marrow Aspirate & Trephine
Haematinic levels: B12 & folate
Iron studies: ferritin, serum Fe, TIBC
High Performance Liquid Chromatography (for Hb variants eg in someone with anaemia)
What is the structure of Haemoglobin?
4 polypeptide chains, 2 alpha and 2 beta globin chains each with own haem group
What is the difference between Aspirate and Trephine?
Aspiration: thin needle used to remove fluid from bone marrow (aspirate) pelvic bone but sometimes chest
Trephine: bone marrow biopsy removes pieece of bone from bone marrow
What is present in normal adult blood?
HbA (a2b2)
Small amounts of HbF and HbA2
Where do the genes for globin chains occur?
In 2 clusters on chromosomes 11 and 16
Define haemoglobinopathies
Genetic conditions from either abnormal Hb variants eg HbA (sickle cell) or reduced rate of synthesis of alpha or beta chains (thalassemia)
Why do patients with sickle cell disease not exhibit symptoms from birth?
After the first three months of life, your body stops producing foetal haemoglobin and produces adult haemoglobin, in which the point mutation will be present
What is anaemia?
Hb below normal ranges
Classified as microcytic, normacytic and macrocytic
What are causes of microcytic anaemia?
Iron deficiency
Thalassemia
Anaemia of chronic disease
What are causes of normocytic anaemia?
Anaemia chronic disease
Aplasia
Chronic renal failure
What are causes of macrocytic anaemia?
B12 deficiency
Folate deficiency
Myelodysplasia
Reticulocytosis
Drug induced
Liver disease
Myxoedema
Why is a reticulocyte count important in investigations?
Clue into causes of anaemia like failure of RBC production or increased losses
What are the causes of failure of production in anaemia?
B12, folate deficiency, iron deficiency, erythropoeitin deficiency in CKD, bone marrow failure (eg aplastic anaemia)
What are the causes of failure of appropiate utilisation in anaemia?
anaemia of chronic disease
What are the causes of increased destruction in anaemia?
Blood loss, haemolysis, (autoimmune, sickle, hereditary spherocytosis, TTP) reticulocytosis
What is the length of life cycle of a red blood cell in sickle cell?
20 days (120 normal)
What would you see in the blood film of a patient with microcytic anaemia?
The blood film of a patient with iron deficiency anaemia showing anisocytosis, poikilocytosis (including elliptocytes), hypochromia and microcytosis.
How is iron transported in blood?
Iron (comes from diet) transported from enterocytes then either into plasma or stored as ferritin
Once attached to transferrin it is transported and binds to transferrin receptors on RBC precursors
(so low iron = low ferritin bc stores being used up)
What will you see happen to ferritin and transferrin in iron deficiency?
A state of iron deficiency will see reduced ferritin stores and then increased transferrin
What is observed in iron studies?
Serum Fe
Hugely variable during the day
Ferritin
Primary storage protein & providing reserve, Water soluble (if low = iron deficient, BUT can go up if infection so need other studies)
Transferrin Saturation
Ratio of serum iron and total iron binding capacity – revealing %age of transferrin binding sites that have been occupied by iron (if low = iron deficient)
TIBC (Total Iron Binding Capacity)
Measurement of the capacity of transferrin to bind iron
What are some causes of iron deficiency?
poor diet
malabsorption
increased physiological needs
What could happen if someone loses too much iron?
Blood loss
menstruation, GI tract loss, paraistes
Why would it still be anaemia if someone is iron deficient with low Hb, but a normal reticulocyte count?
Because Hb is low, would assume reticulocyte production would double (go to higher end of normal range to compensate) so if it remains in lower end of normal range, patient cannot keep up and not making enough RBCs FAILURE OF PRODUCTION
What happens to reticulocyte count in SCD?
RETICULOCYTOSIS
Higher than normal- suggests increased haemolysis, could become a sickle cell crisis
What kind of neutrophil would you see in macrocytic anaemia?
Hypersegmented or right shifted, having many lobes eg around 8 instead of 4-5
What happens to reticulocyte count in macrocytic anaemia?
Low numbers, failure of production
What effect does alcohol have in regards to macrocytic anaemia?
Lowers b12 and folate levels, reduces utilisation of folate and have toxic effect on bone marrow
Where does folate come from and where is it absorbed?
Folate comes from most foods with 60-90% lost in cooking. It is absorbed in the Jejunum and the body has enough stores usually for 3-5 months
Why is B12 deficiency a problem?
B12: Essential co-factor for methylation in DNA and cell metabolism
Intracellular conversion to 2 active coenzymes necessary for the homeostasis of methylmalonic acid (MMA) and homocysteine
Why would a patient with a gastrectomy be B12 deficient (pernicious anaemia)?
They cant absorb it because the site of instrinsic factor secretion is in the stomach, which is needed fot B12 to be absorbed, which is removed in a gastrectomy (no intrinsic factor = no B12 absorption = B12 supplements wont help)
What is pernicious anaemia?
Autoimmune disorder
Gastric Parietal cell antibodies
IF antibodies
Lack of IF
Lack of B12 absorption
Pernicious anemia occurs when your body can’t absorb enough vitamin B12 to function properly.
If someone is exposed to lots of nitric oxide what type of anaemia are they likely to develop?
Macrocytic anaemia due to B12 deficiency (NO - problems absorbing B12)
What is the structural difference between thalassaemia and sickle cell?
SCD: haemolysis (abnormal Hb structure)
Thalassaemia: disorder of globin chain synthesis
What happens to globin chains in thalassaemia?
Reduction in globin chain production:
Alpha thalassaemia: usually due to large deletions in alpha globin complex
Beta thalassaemia: usually due to point mutations in beta gene
What is the structure of Hb globin chains?
4 alpha genes: 2 on each chr 16
2 beta genes: 1 on each chr 11
What are the different severities of alpha thalassaemia?
Severity depends on number deleted genes
- α_/αα = Asymptomatic
- _ /αα and α/α_ = alpha thal trait (microcytic anaemia)
- α_/_ _ HbH disease (anaemia and haemolysis)
- –/– Hydrops Fetalis (No alpha chain production)
Why is beta thalassaemia not diagnosed at birth?
Beta chains not in foetal Hb so problems in beta chain only apparent once switching from foetal Hb to adult Hb in first 3 months of life
What are the different severities of beta thalassaemia?
TWO TYPES:
β0: absent globin production
β+: reduced production
B THAL TRAIT = abnormality in 1 inherited gene (Microcytic anaemia, high HbA2)
B THAL INTERMEDIA OR B THAL MAJOR (BTM) = Abnormality in both genes
Transfusion requirements and varying severity
How do we define beta thal patients?
Whether or not they are transfusion dependant or not (TDT)
TDT: usually beta zero, excess alpha chains and ineffective erythropoeisis and severe anaemia
Transfudion dependant life long: blood they get has too much iron leading to iron overload - liver and cardiac loading and failure
What are the different types of sickle cell?
Inherited chronic haemolytic anaemia due to formation fo abnormal Hb (sickling and unsickling)
Most common form = HbSS
Other sickle disorders are compound heterozygotes: HbSC, HbSD, HbSG, HbSBthal
What is an occlusive crisis?
In SCD, RBCs not as mallebale and increased viscosity because they don’t flow well and get stuck, causing vaso-occlusion of vessels so no O2 delivery = pain due to ischaemia
What are the triggers for an occlusive crisis?
Hypoxia
Dehydration
Fever
Acidosis
What is needed to diagnose SCD?
HAEMOLYTIC ANAEMIA
Low Hb
High reticulocyte count
High LDH and bilirubin
BLOOD FILM
sickle cells
polychromasia
target cells
What is the sickle solubility test?
Confirms diagnosis of SCD alongside blood film
Functional test
Lysed RBC released Hb
Presence of phosphate buffer
HbS insoluble and turbid solution
Will be positive for HbSS and HbAS
Useful for OOH, primary screening
What is HPLC?
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Separation technique
Under high pressure components will separate along a chromatographic column
Retention time: is the characteristic time taken for the haemoglobin species to elute from the column, and is printed above the peak on the chromatogram elution profile. The amount of each haemoglobin fraction present in percentage terms is represented by the area under the curve.
What emergency events can occur in SCD?
Acute Anaemia (Haemolysis, sequestration, Parvo B19 aplastic crisis)
Acute Painful Crises
Acute Chest Syndromes (Hypoxaemia, chest pain, fever, infiltration)
Stroke (Ischaemia or Haemorrhage)
Priapism
Abdominal/ Mesenteric crises
AKI / Papillary Necrosis
Overwhelming infection (Hyposplenism, osteomyellitis (salmonella))
What chronic problems do we see in SCD?
Chronic Haemolysis and anaemia
Pulmonary Hypertension
Nephropathy (Proteinuria
)Retinopathy
AVN
Chronic Pain
Iron Overload
(Memory, cognition, headaches, mood disorderspublic health and social needs)
How do we manage an emergency in SCD vs long term issues?
EMERGENCIES:
pain relief
oxygen
antibiotics
blood transfusions
LONG TERM:
clinic follow up AT LEAST ONCE A YEAR
echocardiographs
hip examinations
retinal screening
urinalysis: protein
BP monitoring
pain relief
HYDROXYCARBAMIDE (acts on bone marrow and increases FHb and reduces HbS, reduces sickling)
EXCHANGE BLOOD TRANSFUSIONS
Which lineage goes on to form RBCs?
Myeloid stem cells
What is haematopoiesis and where does it occur?
The differentiation processes that leads to the formation of all blood cells from haematopoietic stem cells.
At birth in marrow of almost every bone
In adults, in central skeleton and proximal ends of long bones
What is erythropoiesis?
Development of RBCs, mature from multipotent stem cell to reticulocyte to erythrocyte
How are RBCs removed from circulation?
By macrophages in spleen, liver or bone marrow
What are reticulocytes and what happens to them in circulation?
Reticulocytes: immature RBCs, no nucleus but contains RNA and some organelles
Develop into mature RBC within 1-2 days after release into circulation. Loss of RNA.
Reticulocyte count: can be used to represent marrow production of new RBCs. Useful
measure of erythropoiesis.
What is EPO?
Erythtropoietin, drives proliferation and maturation of RBC precusors
Mainly secreted in kidney by tubular and interstitial cells that sense hypoxia
Describe the structure and different types of haemoglobin
Cytosol of RBC contains Hb – synthesised before loss of nucleus
Each RBC contains ~280 million Hb molecules.
Hb Structure: Tetramer - 4 polypeptide globin chains
Each globin subunit contains a haem group.
Haem group: porphyrin ring + ferrous (Fe2+) atom
Fe2+ can bind reversibly to one oxygen molecule.
HbA -most common type of Hb in adult.
HbF is most dominant Hb 3-9months post conception
From 9 months post conception Y chain synthesis progressively reduces to a trace. HbF is replaced by adult Hb
By 1 year old – adult Hb ( a2, b2) is established
Where is the spleen located?
Left hypochondriac region
What are the two types of tissue in the spleen?
White pulp: lymphatic tissue (mostly lymphocytes and macrophages) arranged around
the branches of splenic artery - central arteries
Red pulp: blood filled venous sinuses and cords of splenic tissue called splenic cords (veins closely associated with red pulp)
What are splenic cords?
consist of RBCs, macrophages , lymphocytes, plasma cells and granulocytes.
What is the function of red pulp?
Removal of ruptured, worn out or defective blood cells and platelets by macrophages
Storage of platelets - up to 1/3rd of body’s supply
Production of blood cells (haemopoiesis ) during foetal life
What is the function of white pulp?
B cells and T cells carry out immune functions, similar to lymph nodes
Opsonisation of encapsulated bacteria
Spleen macrophages destroy blood borne pathogens by phagocytosis
Where does blood from the splenic artery enter?
Central arteries of white pulp
What does FISH stand for regarding spleen function?
F- Filtration of encapsulated organism and
blood cells
I- Immunological function
S-Storage of blood
H- Haematopoeisis – in foetus to neonate
What organ is important for opsonisation of encapsulated bacteria?
Spleen
What is whole blood composed of?
Plasma and formed elements
What is a useful measure of erythropoiesis?
Reticulocyte count
Adult Hb is normally composed of…
97% HbA
2% HbA2
less than 1% HbF
What is the
a) definition
b) signs
c) symptoms
of anaemia?
a) Low haemoglobin concentration:
men:<135 g/L , women: <115g/L
b) Pallor
• Tachycardia
• Systolic Flow murmur
• Cardiac Failure
• Specific
• Koilonychia: Spoon shaped nails - IDA
• Jaundice - Haemolytic anaemia
• Leg ulcers - SCA, Haemolytic anaemia
• Bone deformities - Thalassemia major
c) asymptomatic or specific:
fatigue
dyspnoea
palpitations
faintness
headache
What is the
a) genetic mutation
b) risk factors for polymerisation
c) consequences
of sickle cell anaemia?
a) Point mutation (adenine → thymine) in Hb-β gene (ch-11)
• Hence, amino acid change (glutamic acid → valine)
• This alters the properties of the β-chain where the AA R-group changes:
hydrophilic (Glu) → hydrophobic (Val)
• Under hypoxia the R-group changes shape exposing a ‘pocket’ where all
the Hb molecules join up in a stiff line (polymerisation)
• This alters the RBC structure → sickle-shape
b) • Hypoxia - increases deoxygenated HbS
○ e.g. tissues with ↓O2, intense exercise, high altitudes
• Acidosis - decreases Hb affinity for O2
• Dehydration - increases concentration of HbS
• Infection/fever
•Cold temperature
c) •Reduces RBC life span (120 days to 30 days) → premature destruction and
high turnover
•Sequester in liver and spleen
•Occlude small vessels → vaso-occlusive crisis
What are the clinical features of SCA?
Chronic pain
Anaemia (haemolytic)
○ Lethargy
○ Pallor
○ Weakness
○ Jaundice - from high levels of bilirubin
due to excessive RBC breakdown
Splenomegaly
Recurrent infections
Priapism (serious painful erections)
Delayed puberty and growth restriction
Periodic ‘attacks’ AKA sickle-cell crises
N.B. some patients remain asymptomatic until adulthood
What is a vaso-occlusive crisis?
• In peripheral vessels this causes extreme agonising pain
• In other areas: vision loss, strokes, bowel ischaemia, renal papillary
necrosis, congestive heart failure, cholelithiasis, ACS
• Risk factors: cold, smoking, pregnancy, dehydration, stress, exercise
What is acute chest syndrome?
• Vaso-occlusive crisis in lungs
• Causes chest pain, dyspnoea, fever
• Leading cause of death
What is sequestration?
Trapped RBC in spleen
• Acute: acute splenomegaly, acute drop in Hb, haemodynamic instability
• Chronic: fibrosis and autosplenectomy ➝ increased infections
What is an aplastic crisis?
• Transient cessation of haematopoiesis associated with parvovirus B19
• Presents similarly to heart failure
What common infections can badly affect sickle cell patients?
Encapsulated organisms especially
Osteomyelitis
Malaria
Pneumonia
Salmonella
Parvovirus B19
Meningitis
What investigations are done for sickle cell anaemia?
- Assess parental carrier status (if both parents carriers 25% risk of SCA in child)
- Offer antenatal diagnosis (Chorionic villus sampling, amniocentesis, non-invasive prenatal testing)
- Provide neonatal screening (heel prick test)
- Obtain bloods (FBC: Hb of 60-80g/l is anaemic, peripheral smear to see if reticulocyte is increased by 10-20% and sickled RBCs)
- Sickle solubility test (Normal Hb is negative, HbS is positive, precipitates and turbid)
- Hb electrophoresis (seperates different types of Hb eg HbSS and HbAS)
HPLC used for older screening to confirm diagnosis
How do we manage SCA via patient education and general care?
- Patient education
● Increase fluid intake
● Avoid triggers (e.g. excessive
exercise - General care
● Follow up regularly by secondary
care
● Folate supplementation - high folate
turnover due to haemolysis
● Immunisations - flu, HepB, PPV
● Antibiotic/malaria prophylaxis
How do we manage a vaso-occlusive crises?
Preventing vaso-occlusive crises
● Top up transfusions
● Hydroxycarbamide/hydroxyurea -
increases HbF ➝ reduces sickling
How do we manage acute complications in SCA?
Managing acute complications
● IV fluids, transfusions, O2,
● Analgesia (pain management)
○WHO pain ladder
○Usually opiates if in A&E - must
be given <30 mins and
reassessed every 30 mins
How do we manage chronic complications in SCA?
Iron chelators - reduce excess iron
in blood
Define thalassaemia
Thalassaemia: Inherited globin gene abnormalities
that result in underproduction or absence of either βglobin or α-globin chain.
• Classified according to which chain of globin molecule
is affected ( α or β):
• α genes – two genes, each on chromosome 16
• β genes – one gene, each on chromosome 11
Where is beta thalassaemia prevalent?
Mediterranean, Middle East, South and Southeast Asia and Southern China
Where is alpha thalassaemia prevalent?
Southeast Asia, Africa and Southern China
What is the management for beta thalassaemia?
• 4-6 weekly red cell transfusion – to keep Hb > 10g/dL –
to supress bone marrow hypertrophy and iron absorption
• Iron chelation
• Splenectomy – may be indicated if hypersplenism.
Predisposes to life-threatening infections.
• Allogenic bone marrow transplantation
• Counselling and antenatal diagnosis made available
What are the differences between beta thalassaemia intermedia and minor?
Beta Thalassaemia Intermedia:
• Less severe.
• Bone deformity and hepatosplenomegaly may occur.
• Haemoglobin maintained without transfusion.
• Blood film – similar to thalassaemia major.
• Iron overload – may occur. Increased iron absorption
Beta Thalassaemia Minor:
• Heterozygous state ( normal b-globin gene plus either a
b0 or b+ gene.
• Affected individuals are asymptomatic
• Carrier for thalassaemia major- reproductive advice and
antenatal diagnosis and foetal selection if partner is also
carrier.
• FBC: low Hb, low MCV. (Mimics iron deficiency)
• Hb electrophoresis: increased HbA2
• Iron status – normal
What is curative treatment for SCA?
Haemopoietic stem cell transplants
(SCT)
What are the purines and pyramidines?
PURINES: Adenine and Guanine (2 rings)
PYRAMIDINES: Cytosine and Thymine (1 ring)
What bonds bind purines and pyramidines together?
Hydrogen bonds
What is the central dogma?
DNA (replication) –> transcription –> RNA –> translation –> protein (‘phenotype’)
DNA to RNA in nucleus
RNA to protein in cytoplasm
Can DNA be transported out the nucleus?
No, therefore transcribed to RNA as it can be transported out
What is the structure of a gene?
Exons (coding)
Introns (non coding)
5’ end to 3’ end (prime)
What are exons?
Code for amino acids except for Untranslated Regions (5’ UTR and 3’ UTR)
What are UTRs?
Contain regulatory elements that are important for control of protein synthesis (don’t code for amino acids)
What are introns?
Non coding section of gene between exons, if a gene has 2 exons it needs an intron inbetween
What is the promotor region?
5’ end (upstream) of gene and contains important regulatory elements for transcription
What is transcription?
Going from double stranded DNA to single stranded RNA, transcript has same base sequence as gene
What base replaces thymine in RNA?
Uracil
What are the types of RNA?
mRNA = messenger RNA – carries genetic information from nucleus to cytoplasm
rRNA = ribosomal RNA – located in cytoplasm; important in directing translation
tRNA = transfer RNA – located in cytoplasm; transfers amino acids to ribosome to generate protein
What are post transcription modifications?
Capping, polydenylation and splicing = mature mRNA
What does mRNA contain?
mRNA contains cap, 5’UTR, exons, 3’UTR, polyA tail
What is translated?
Only RNA from coding exons
What is the triplet code?
RNA “read” in threes = codons
64 possible combinations of 4 bases
Codon →specific amino acid
20 amino acids
Degenerate = >1 codon can code for same amino acid
What are the types of mutation?
Substitution (point mutation)
Deletion
Insertion
Where are mutations found?
Coding and non-coding DNA like promotors/introns
What is a silent/synonymous base substitution?
Nucleotide change without amino acid change
What is the difference between a missense and nonsense mutation?
Missense: single nucleotide change = incorrect amino acid = possible malfunctioning protein
Nonsense: single nucleotide change = amino acid for stop codon = shortening of protein
What happens in an insertion or deletion?
If number of bases inserted divisible by 3 = IN FRAME
eg adds in one codon, other codons unaffected
If not = FRAMESHIFT (more likely to be pathogenic)
shifts all codons over by one base
Same for deletion, 3 bases deleted (in frame) or one base deleted causing frameshift
What is the difference in cell wall structure between gram positive and negative bacteria?
Gram positives (purple) – large peptidoglycan layer
(easier to treat due to porous wall)
Gram negatives (pink) – thin peptidoglycan layer
What colour does gram positive bacteria stain in iodine?
Purple
What colour does gram negative bacteria stain in iodine?
Pink
What allows encapsulated organisms to infect an organism?
● Prone to causing invasive infections due to
added virulence provided by their
polysaccharide capsule
● The capsule is antiphagocytic (hard to digest)
○ Allows evasion of the immune system
○ Conceals surface proteins that the
innate immune system would
recognise
● The body is dependent on splenic function for
clearing these organisms
Serum Fe+
Ferritin
Transferrin Saturation
Serum Fe*
Hugely variable during the day
Ferritin*
Primary storage protein & providing reserve, Water soluble
Transferrin Saturation*
Ratio of serum iron and total iron binding capacity – revealing %age of transferrin binding sites that have been occupied by iron
Transferrin/Transferrin receptors
Made by liver, Production inversely proportional to Fe stores. Vital for Fe transport. Uptake of Fe from protein needs transferrin to be attached to the cell via the transferrin receptor
What is total iron binding capacity (TIBC)?
Measurement of the capacity of transferrin to bind iron
It is an indirect measurement of transferrin – a transport protein that carries iron
TIBC is technically easier to measure in the laboratory that transferrin levels directly
What are TIBC levels in iron deficiency anaemia?
TIBC HIGH
more transferrin produced aiming to transport more iron to tissues in need
What are TIBC levels in anaemia of chronic disease?
TIBC LOW
less transferrin produced (but more ferritin), aim to reduce availability of iron for pathogens. This is mainly regulated by increased hepcidin production.
Pernicious anaemia
Autoimmune disorder
Gastric Parietal cell antibodies
IF antibodies
Lack of IF
Lack of B12 absorption
