1BMS326 1-16 Combined & Compressed

Introduction to Anaemia
BMS326 Clinical Haematology I
Murray Adams
Learning Objectives
• After completing this lecture, you should be able to:
1. Describe and categorise causes of anaemia

2. Describe and differentiate laboratory tests used in the diagnosis of anaemia
3. Apply correct haematological terminology in describing the pathophysiology
and laboratory diagnosis of anaemia
2
Overview
• Definition
• Classification
• General aspects of anaemia
• Laboratory investigations (general)
• This lecture will provide a foundation for the first half of BMS326
3
Anaemia: Definition
Anaemia is defined as a significant decrease in the haemoglobin
concentration of blood, below the normal for the age and sex of the patient
RBC usually decreased

PCV usually decreased
Compare laboratory result of parameters to reference intervals

(Refer to BMS326 Clinical Haematology practical manual)
4
Anaemia: Introduction
• Anaemia is not a specific disease
• Reflects an underlying disorder or disease
• Important to diagnose exact cause of anaemia
• RCC and PCV are usually decreased
• Prevalence is higher in females
• Low socioeconomic groups
• Anaemia results from:
• RBC loss/destruction > BM RBC production
• Impaired BM RBC production
5
Anaemia: Diagnosis
• Patient History
• Physical Examination
• Laboratory Testing:
• FBC
• Blood films: peripheral blood and BM
• Specific tests: Haem & Biochem
• Systems of classification:
• Morphological
• Aetiological (functional)
6
Morphological Classification
• Based on the appearance of RBC's in blood film
• “- cytic” = size
• “- chromic” = colour
• “poikilocytosis” = shape
• Normocytic/normochromic anaemia (MCV 80 - 100 fL)
• Macrocytic anaemia (MCV > 100 fL)
• Microcytic anaemia (MCV < 80 fL)
7
Aetiological Classification
• Morphological assessment not sufficient
• Persistent anaemia may be due to one or more pathophysiological
mechanisms involving:
• Proliferation defects
• Maturation defects
• Survival defects
• Abnormal blood loss (acute vs chronic)
• Note: increased plasma volume i.e. haemodilution
8
Proliferation Defects
• ↓proliferation, maturation & release rates of RBCs
• Insufficient erythropoietin production
• Bone marrow damage
• Stem cell damage
• Liver disease, renal disease
• Normocytic/normochromic anaemia (usually)
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Maturation Defects
Abnormal nuclear or cytoplasmic development
Nuclear Defects Cytoplasmic Defects
Affect all cell lines Abnormal Hb production
e.g. Megaloblastic Anaemia e.g. Iron Deficiency Anaemia

10
Survival Defects
• Premature loss/destruction of RBC's
• Haemolysis
• Schistocytes, spherocytes
• Normocytic/normochromic anaemia (usually)
• For example: haemolytic anaemias
11
Acute and Chronic Blood Loss
• Due to:
• Haemorrhage
• Accidents/injuries
• Underlying disorders
• Internal bleeding
• External bleeding
NB Causes of anaemia may be multi-factorial
12
Laboratory Investigation
• Laboratory investigation of anaemia occurs after:
• Evaluation of patient history
• Physical examination
• Incidence higher:
• Females
• Age (> 65 years)
• Children susceptible via diet (malnutrition)
• Genetic and regional factors (Mediterranean/SE Asia)
• Pregnancy (increased requirement for Fe/Folate)
13
Laboratory Tests
• Full Blood Count (FBC)
• Peripheral Blood Examination
• Bone Marrow Examination https://www.sysmex.com/us/en/Company/News/XN-SpecialEdition2012/Pages/XN.aspx
• Reticulocyte count
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Full Blood Count
• Hb, RCC, PCV
• Red cell indices i.e. MCV, MCH, MCHC
• RDW (red cell distribution width):
• %CV of RBC volume distribution
• Normal range: 11.5 - 14.5%
• Reticulocyte count:
• Reflects effective BM activity
• Normal range: 0.2 - 2.0%
15
Peripheral Blood Examination
• Morphological classification of anaemia
• Review BMS218 Haematology
• Examples in disease states:
• Anisocytosis
• Poikilocytosis
• Polychromasia
• RBC inclusions
Rodak 5th Ed. Page 287 16

Peripheral Blood Examination
• What type of anaemia is present?
• Normocytic normochromic
• Microcytic normochromic
• Microcytic hypochromic
• Macrocytic normochromic
17
Rodak 5th Ed. Page 292 18
Bone Marrow Examination
• Fragments
• Cellularity:
• Represents proliferation & maturation rates of cells
• Normal bone marrow (50% cells : 50% adipose/fat)
• Hypercellular (>70% cellularity e.g. leukaemia)
• Hypocellular (<30% cellularity e.g. myelodysplasia)
• M:E Ratio
• Cytoplasmic and nuclear maturation
• PBR stain - iron stores
19
Other Tests
• Iron Studies
• Serum bilirubin
• Vitamin B12 assay
• Folate assay
• Direct antiglobulin test
• What you will need to know:
• Principles and applications of a range of laboratory tests used for the
investigation and diagnosis of anaemia
20
Summary
• Anaemia is the significant decrease in the haemoglobin concentration
of blood
• Diagnosis of anaemia is based on clinical history and laboratory
investigation
• Classification of anaemia is based on morphological and functional
characteristics
21
References
• Rodak et al. Hematology: Clinical Principles and Applications (5th Ed).
• Chapter 19, pp 284 - 296
• Chapter 21, pp 283 - 298
• Bain et al. Dacie and Lewis Practical Haematology (12th Ed).
• Chapter 5, pp 61 - 80
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Describe the abnormalities….
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Normocytic Anaemia
Murray Adams
Learning Objectives
1. Describe and categorise causes of normocytic anaemia

2. Describe and differentiate laboratory tests used in the diagnosis of
normocytic anaemia
3. Apply correct haematological terminology in describing the pathophysiology
and laboratory diagnosis of normocytic anaemia
2
Normocytic Anaemia: Introduction
• Note (for diagnosis): • MCV = normal (also MCHC)
• Patient history / physical exam • Reticulocyte count:
• Laboratory testing: • Reflects bone marrow activity
• FBC (capacity to compensate
• Blood film features anaemia?)
• Bone marrow features (?)
• Normal/Low:
• Reticulocytes
• ? Bone marrow failure
• Specialised laboratory testing
• High:
• Apply morphological classification • ? Blood loss
of anaemia (Lecture 1 BMS326) • ? Haemolysis
3
Normocytic Anaemia: Classification
• Recent blood loss
• Haemolysis
• Aplastic anaemia***
• Pure red cell aplasia***
• Myelophthisis***
• Myelodysplastic syndromes
• Others e.g. CRF, ACD***
4
Aplastic Anaemia
• Characterised by failure of blood cell production:
• Pancytopaenia:
• Leukopaenia
• Anaemia
• Thrombocytopaenia
• Reticulocytopaenia
• Bone marrow hypocellularity:
• Depletion of haemopoietic stem cells
• Incidence: ~ 5 in 1,000,000
• Higher incidence in oriental populations
5
Aplastic Anaemia: Aetiology
• Acquired (~85% of cases):
• Idiopathic (~70%): unknown cause
• Secondary (~10 – 15%): known cause
• Drugs and chemicals (wide range of rare associations; benzene, chloramphenicol, others)
• Radiation
• Post-viral
• Others, including paroxysmal nocturnal haemoglobinuria (PNH)
• Hereditary (~15% of cases):
• Fanconi’s Anaemia (see later)
• Dyskeratosis congenita (very rare <600 cases ever reported)
• Shwachman-Bodian-Diamond syndrome (multi-system disorder)
6
Aplastic Anaemia: Pathogenesis / Mechanisms
• Quantitative/qualitative deficiency in CD34+ stem cells:
• Intrinsic stem cell defect (damage to DNA in cells)
• Increased expression of apoptosis-related genes
• Failure of stromal microenvironment:
• Increased levels of growth factors (erythropoietin, thrombopoietin, G-CSF)
• Can’t correct cytopaenias
• Autoimmune component?
• Immune suppression of BM improves AA
7
Aplastic Anaemia: Clinical Features
• Pallor, fatigue, weakness
• Mucosal bleeding, purpura, ecchymoses
• URT and LRT infections
• Bacterial and fungal infections
• Anaemic symptoms less severe – chronic onset
• Splenomegaly and hepatomegaly typically absent
• Treatment:
• Bone marrow transplant (BMT), and/or
• Immunosuppression
8
Aplastic Anaemia: Peripheral Blood
• Pancytopaenia:
• Normocytic/normochromic anaemia (Hb < 90 g/L)
• Leukopaenia (< 2.0 x 109/L)
• Thrombocytopaenia (< 70 x 109/L)
• Reverse differential:
• Lymphocytes > neutrophils
• Decreased reticulocytes
• Absence of “abnormal” cells in BM
Rodak. p335
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Aplastic Anaemia: Bone Marrow
• Aspiration and biopsy:
• Several biopsies may be required
• Foci of normal cellularity
• Cellularity:
• Decreased
• Especially granulocytes and megakaryocytes
• Increased adipose (fat) tissue
• Focal hypercellularity
• Relative lymphocytosis (60% - 100%)
• Usually increased iron in RE cells
Rodak. p335
10
Aplastic Anaemia: Bone Marrow
Biopsies from patients with Bone Marrow Aspirate

normal (L) and aplastic anaemia (R)
bone marrows
http://www.ashimagebank.org/cgi/content/full/2001/1205/100241 http://www.ashimagebank.org/cgi/content/full/ashimgbank;2004/0519/101115
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Aplastic Anaemia: Other Lab Tests
• Iron Studies:
• Serum iron:
• Increased due to impaired marrow utilisation of iron
• Total iron-binding capacity (TIBC): decreased
• Transferrin saturation:
• Increased (may be almost 100%)
• Often first sign of reduced marrow utilisation of iron
• Increased serum and urine erythropoietin
• Prolonged bleeding time and poor clot retraction
12
Aplastic Anaemia: Treatment / Prognosis
• Treatment:
• Remove causative agent (acquired aplastic anaemia)
• Stem cell transplantation (HLA-identical sibling)
• Prognosis (outcomes improved significantly in last 20 years):
• 10 year overall survival:
• 91% (children); 74% (adults) (HLA-identical sibling)
• 75% (children); 63% (adults) (HLA-matched unrelated donor)
• Neutrophils most important prognostic feature:
• < 0.5 x 109/L: increased risk of infection
• < 0.2 x 109/L: very poor prognosis
• Death mainly due to infection and haemorrhage
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Fanconi’s Anaemia
• Chromosome instability disorder characterised by:
• Aplastic anaemia (laboratory results same as for aplastic anaemia)
• Physical abnormalities (skeletal abnormalities, skin pigmentation, short stature)
• Susceptibility to cancer (leukaemia and solid tumours)
• Incidence ~ 1-5 per million (higher in Ashkenazi Jews, Afrikaners)
• Pancytopaenia usually develops in childhood
• Genetic association (15 genes associated with Fanconi’s anaemia):
• All are autosomal recessive (except 1 is X-linked recessive):
• Usually biallelic mutations or deletions of gene
• Relationship between mutations and disease pathology is not clear
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Pure Red Cell Aplasia (PRCA)
• Rare disorder of erythropoiesis:
• Isolated loss of erythroid precursors in BM
• Reticulocytopaenia, but normal WBC and platelet counts
• Also:
• Red cell agenesis
• Erythroblastic hypoplasia
• Erythroid hypoplasia
• Diamond-Blackfan Syndrome
• Acquired and congenital forms
15
Acquired PRCA
• Incidence (rare):
• Acute or chronic
• Children or adults
• Causes:
• Idiopathic
• Autoimmune related
• Secondary:
• Tumours, infection, chronic haemolytic anaemia, infections, drugs, chemicals
• Transient erythroblastopaenia of childhood:
• Association with viral infections
• Treatment with RBC transfusion (normalisation of erythropoiesis)
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Congenital PRCA
• Diamond-Blackfan Syndrome
• Inherited stem cell defect
• Mutations identified in 9 genes:
• Encode structural ribosome proteins (RPS19; ~25%)
• Anaemia diagnosed in first year
• Normocytic/normochromic anaemia (often macrocytic)
• Decreased reticulocytes
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Myelophthisis (Myelophthisic Anaemia)
• Infiltration of abnormal cells into bone marrow:
• Destruction / replacement of normal bone marrow haemopoietic cells
• Tumour cells
• Leukaemic cells
• Fibroblasts
• Laboratory features:
• Normocytic/normochromic anaemia
• Reticulocytopaenia
• Leukoerythroblastic reaction
• Anisopoikilocytosis
• Dacrocytes (if fibrotic – myelofibrosis)
• Platelet anisocytosis and variable count Rodak. p342
• Bone marrow biopsy demonstrates infiltrating cells

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Anaemia of Chronic Kidney Disease
• Inadequate renal production of erythropoietin
• Haemolysis (extravascular)
• Bleeding – abnormal platelet function
• Peripheral blood:
• Poikilocytosis +++
• Burr cells, fragments, spherocytes
• Treatment:
• Dialysis
• Erythropoietin (expensive)
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Anaemia of Chronic Kidney Disease
Burr cells
Spherocytes
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Anaemia of Chronic Disease (ACD)
• 2nd most common cause of anaemia
• Mild-moderate normocytic/normochromic anaemia
• Associated with:
• Malignancy
• Normal aging
• Trauma
• Inflammation
• Defective iron absorption
• Inefficient iron recycling
• Iron retained in macrophages
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ACD: Pathogenesis Anaemia
“Chronic Disease”
Interference to iron
absorption/metabolism
Inflammation
Inhibits iron absorption (GIT)
Inhibits iron release (macrophages)
IL-6
(Hepatocytes)
↑ Hepcidin
22
Summary
• Normocytic anaemias are caused by a variety of abnormalities e.g.
blood loss, haemolysis, malignancy, chronic disease, etc.
• Aplastic anaemia is a rare disorder characterised by pancytopaenia
and decreased CD34+ stem cells
• Anaemia of chronic kidney disease occurs due to a lack of
erythropoietin production
• Anaemia of chronic disease occurs due to interference to iron
absorption or release
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Case Study 1 WBC (x109/L) 6.8 (4.0 – 11.0 x 109/L)
Hb (g/L) 68 (115 – 155 g/L)
• 25 y.o. female PCV (L/L) 0.34 (0.37 – 0.47)
• Tired 6/12 MCV (fL) 72 (80 – 98 fL)
• S.O.B when exercising Platelets (x109/L) 220 (150 – 400 x 109/L)
• GP requested;
• FBC
• U&E’s
• LFT’s
• Thyroid function tests
• What is the most likely diagnosis?
• What further tests should be
performed?
Case Study 2
WBC (x109/L) 13.5 (4.0 – 11.0 x 109/L)
Hb (g/L) 57 (115 – 155 g/L)
• 29 y.o. female
PCV (L/L) 0.30 (0.37 – 0.47)
• Received 4 units RBCs transfusion
following bleeding during delivery of MCV (fL) 101 (80 – 98 fL)
2nd child Platelets (x109/L) 435 (150 – 400 x 109/L)
• What may have caused the
anaemia?
• What is the most likely diagnosis?
• What further tests should be
performed?
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References
• Rodak. Hematology: Clinical Principles and Applications (5th Edition).
• Chapter 22, pp 332 - 339
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Abnormal Haemoglobins I:
Thalassaemia
Murray Adams
Learning Objectives
1. Describe and categorise genetic mutations that result in thalassaemia
2. Explain the pathophysiologic effects caused by the imbalance of globin chain
synthesis in α- and β-thalassaemia
3. Describe and differentiate laboratory tests used in the diagnosis of α- and β-
thalassaemia
4. Recognize the pattern of laboratory findings in heterozygous and homozygous
β-thalassemias, including hereditary persistence of foetal Hb (HPFH)
5. Correlate the clinical syndromes of α-thalassemia with the number of α genes
present
2
Introduction
• Abnormal haemoglobins are formed in two ways
• Thalassaemias:
• Mutations in globin chain gene
• Decreased globin chain production
• Disordered globin synthesis
• Haemoglobinopathies
• Disordered haemoglobin structure
3
Abnormal Haemoglobins
• ~5% of world’s population are carriers
• β-thal: Sardinia, Cyprus, Greece
• α-thal: Cyprus, Saudi Arabia, Thailand
• Coincides with prevalence of malaria:
• Selective resistance to infection
• Infected cell:
• Defective growth of parasite
• Increased phagocytosis
4
Thalassaemia
• First described in 1925 (‘Cooley’s anaemia’)
• Mediterranean (-thal) and Asian races (-thal)
• Common genetic disorder:
• Mild forms: most common genetic disorder
• Severe forms: significant morbidity/mortality
• Genetic mutations that decrease the rate of synthesis of one of the
two constituent globin chains of normal haemoglobin (usually  or )
5
Thalassaemia
• Mutations result in:
• Unbalanced synthesis of the  and  chains
• Decreased total haemoglobin
• Decreased haemoglobin synthesis may be due to:

• Structural gene deletions
• Mutations preventing gene expression
6
Thalassaemia
• Globin Chains:
• Hb A  /  (97%)
• Hb A2  /  ( 2%)
• Hb F  /  ( 1%)
• The 2 main variants of classical thalassaemia are:

•  - Thalassaemia ( - globin chain affected)
•  - Thalassaemia ( - globin chain affected)
7
Genes for Globin Chains
Chromosome 16 Chromosome 11
    G A  
Globin
Chain     G A  
Globin
Tetrad  2 2 22 22 22 22 22 4 4
Hb Portland Hb Gower I Hb Gower II HbF HbA2 HbA Hb Barts HbH

Hb
Embryonic/Foetal Adult Abnormal Hb:
thalassaemias
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-Thalassaemia: Overview
• Imbalance of  and  globin chains leads to:
• Decreased rate of synthesis of  globin chains
• Unpaired  globin chains (precipitate to form Heinz bodies)
• Causes oxidative stress and damage to cell membranes
• Apoptosis of RBC precursors in BM:
• Phagocytosed by macrophages
• Ineffective erythropoiesis
• BM attempts to produce RBCs:
• Full of inclusion bodies that are rapidly sequestered and phagocytosed (spleen)
9
-Thalassaemia: Overview
• > 250 mutations in β-globin gene cluster (β, δ, γ-globin genes):
• Partial to complete absence of  gene expression
• Mutations affect any step in the pathway of globin gene expression:
• ↓/absent transcription of mRNA:
• Mutations in promoter region
• Mutations in initiation codon
• mRNA processing errors (splicing errors)
• Translation errors:
• Frame shift, missense, and nonsense mutations
• Removed stop codon
• Results in a wide range of clinical expression
10
-Thalassaemia: Nomenclature & Genotypes
• Nomenclature:
 Normal  globin chain synthesis
˚ Denotes severe reduction in  globin chain synthesis (no β-globin chains)
+ Denotes mild reduction in  globin chain synthesis (5-30% of normal)
silent Denotes small reduction in  globin chain synthesis (near normal)
• Genotype permutations result in 4 catergories of clinical manifestion:

 - Thalassaemia Major: homozygous / compound heterozygous (˚/˚)
 - Thalassaemia Intermedia: (˚/+ or ˚/)
 - Thalassaemia Minor: heterozygous (+/ or ˚/)
 - Thalassaemia Silent Carrier: heterozygous (silent/)
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-Thalassaemia: Clinical Features
• Symptoms dependant on severity of mutations
• Increased synthesis of  and  globin chains:
• Ineffective erythropoiesis in BM due to the presence of unpaired  globin chains
• Increases in HbA2 and HbF (NB reduced HbA)
• Haemolytic anaemia
• Compensatory increased erythropoiesis in BM:
• Expansion of bones & deformities
• Hepatosplenomegaly
• Iron overload if repeated transfusions
12
β-Thalassaemia: Bone Deformities
13
-Thalassaemia: Laboratory Testing
• FBC:
• Haemoglobin reduced (~ 25 g/L in  Thal Major)
• RBC indices decreased (MCV, MCH, MCHC)
• Peripheral blood film: see next slide
• Other Laboratory Testing:
• Demonstration of Heinz bodies (supravital staining)
• Haemoglobin electrophoresis:
• HbA Normal to Decreased
• HbA2 Increased
• HbF Increased
• Other indicators of haemolytic anaemias
• Bilirubin, reticulocytes, haptoglobin
• Molecular testing (DNA analysis) - detection of thalassaemia mutations
14
• Peripheral Blood:
• Microcytic/hypochromic anaemia (MCV < 75 fL)
• Anisocytosis (Mild to Marked)
• Poikilocytosis (Mild to Marked)
• Codocytes, NRBCs, dacrocytes
• Basophilic stippling
• Patient history:
• Family history
• Previous transfusion(s)?
Rodak. p461 15
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Hb Electrophoresis (Cellulose Acetate pH 8.4)
“Slow” Hbs “Fast” Hbs
Direction of Migration
CA A2 D F A Portland Bart’s
C S
E G
O Arab Lepore
Origin C Harlem
Cathode (-) Anode (+)
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Carbonic A2 S F
Anhydrase
Control Lysate
HPFH
β - Thal Trait/Intermedia
Normal Adult
origin A
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Carbonic A2 F
Anhydrase
Control Lysate
HPFH
Sickle Cell Trait (AS)
Normal Adult
origin S A
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Hb Electrophoresis (Agarose pH 8.4)
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-Thalassaemia: Therapy
• Blood transfusions:
• Life threatening -thal major
• Helps maintain normal Hb levels
• May develop iron overload:
• Iron chelation e.g. desferrioxamine
• Splenectomy
• Bone marrow / stem cell transplant
• Gene therapy - correction of molecular defect?
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-Thalassaemia
• Group of disorders:
• Decreased synthesis of  chains
• Can be difficult to diagnose (usually milder than  – Thalassaemia)
• Chinese, Thai and Middle Eastern persons
• Most commonly a deletion of one or more  genes
• Excess  chains: Tetramer of  chains (Hb Barts):
• Stable, no precipitation
• High oxygen affinity
• Excess  chains: Tetramer of  chains (HbH):
• Initially stable, but precipitates as RBC ages in circulation (extravascular haemolysis)
• High oxygen affinity
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-Thalassaemia: Genotypes
- -/- - Thalassaemia Major (Hb Barts)
-/- -  Thalassaemia Intermedia (HbH Disease)
/- -  Thalassaemia Minor (-Thal Trait)
-/-  Thalassaemia Minor (-Thal Trait)
-/  Thalassaemia Minor (Carrier)
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-Thalassaemia: Clinical Features
•  Thal Major (Hb Barts):
• No  globin chains synthesised
• “Hydrops fetalis”
• Foetus dies 28 - 34 weeks into pregnancy
•  Thal Intermedia (HbH):
• Moderate problems
• Patient will live a normal life course
•  Thal Minor:
• Mild to no problems
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-Thalassaemia: Laboratory Testing
• FBC:
• Haemoglobin reduced (~90 g/L)
• RBC indices decreased (MCV, MCH, MCHC)
• Microcytic Hypochromic anaemia (MCV < 75 fL)
• Poikilocytosis (Mild to Moderate)
• Anisocytosis (Mild to Moderate)
• Bone Marrow:
• Erythroid hyperplasia in bone marrow Schrier, S. ASH Image Bank 2002;2002:100327
Copyright ©2002 American Society of Hematology.
Copyright restrictions may apply.
25
-Thalassaemia: Laboratory Testing
• Further laboratory testing:
• HbH inclusions (supravital stain, e.g., methylene blue)
• Molecular testing (DNA analysis) - detection of thalassaemia mutations
• Other indicators of haemolysis e.g.  bilirubin
• Haemoglobin Electrophoresis:
• HbA Reduced
• HbA2 Normal
• HbF Normal
• Hb Barts ( chains): 4, 3 and 2 gene deletions
• HbH bodies ( chains): 3 and 2 gene deletions
Rodak. p466 26
-Thalassaemia: Therapy
• 1 or 2 gene deletion:
• None required
• 3 gene deletion:
• Transfusions
• Vitamin supplements (Fe/B12)
• Note: iron overload (chelators)
• 4 gene deletion:
• Fatal in third trimester or shortly after birth
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Other Thalassaemias
• Hereditary Persistence of Foetal Haemoglobin (HPFH):
• β-globin gene contains a deletion in the δβ region
• Increased production of HbF
• Usually asymptomatic (unless with other form of thalassaemia / structural variant)
• Hb Lepore Thalassaemia:
• Structural variant (fusion of the δβ-globin genes: δβLepore)
• Clinically similar to β-thalassemia minor
• Others:
• Hb Constant – Spring
• β0 – Thalassaemia
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Summary
• Abnormal haemoglobins are formed by:
• Reduced globin chain synthesis (thalassaemias)
• Disordered haemoglobin structure (haemoglobinopathies)
• β-thalassemia is due to a wide variety of genetic mutations resulting
in decreased β-globin chain production
• α-thalassaemia is due to structural gene deletions resulting in
decreased α-globin chain production
• Thalassaemias present as microcytic/hypochromic anaemias and have
variable phenotypic expression (particularly β-thalassemia)
• Diagnosis is based on FBC, peripheral blood examination and
specialised testing such as haemoglobin electrophoresis
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References
• American Society of Hematology Image Database
• Hoffbrand and Pettit (3rd Ed). Essential Haematology. pp 94 - 120.
• Rodak. Hematology: Clinical Principles and Applications (5th Edition).
• Chapter 28, pp 455 - 471
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Genotype Hb A Hb A2 Hb F Hb Lepore
Normal (Normal Hematologic Parameters)
β/β N N N 0
Silent Carrier State (Asymptomatic; Normal Hematologic Parameters)
silent
β /β N N N 0
Thalassemia Minor (Asymptomatic; Mild Hemolytic Anemia; Microcytic, Hypochromic)
β+/β N to Sl ↑ 0
β0/β N to Sl ↑ 0
δβ0/β N to ↓ 5%–20% 0
Lepore
δβ /β 5%–15%
Thalassemia Major (Severe Hemolytic Anemia; Transfusion-Dependent; Microcytic, Hypochromic)
+
β /β+ ↓ V ↑ 0
β+/β0 ↓↓ V ↑ 0
β0/β0 0 V ↑ 0
Lepore Lepore
δβ /δβ 0 0 80% 20%
Thalassemia Intermedia (Mild to Moderate Hemolytic Anemia; Transfusion-Independent; Microcytic, Hypochromic)
silent silent
β /β 0
silent silent
β+/β or β0/β
δβ0/δβ0 0 0 100% 0
β0/δβ0 0 N ↑ 0
Abnormal Haemoglobins 2:
Haemoglobinopathies
Murray Adams
Learning Objectives
1. Explain the difference between thalassemias and haemoglobinopathies
2. Describe the types of mutations found in the haemoglobinopathies
3. Differentiate between homozygous and heterozygous states and the terms
“disease” and “trait” as they relate to the hemoglobinopathies
4. Describe the genetic mutations, the effect of the mutations on hemoglobin,
pathophysiology, clinical findings, peripheral blood findings and laboratory
diagnosis of the haemoglobinopathies.
2
Haemoglobinopathies
• Genetically determined abnormality of the structure or synthesis of the
haemoglobin molecule
• Affects ~7% of the world’s population
• 300,000 children born each year with a haemoglobinopathy
• Africa, Mediterranean and South East Asia
• > 1000 structural variants described:
• Single (or multiple) amino acid substitutions; deletions; insertions; gene fusion
• External site on globin chains (less severe) (except: HbS)
• Internal site on globin chains (more severe)
• Produce structural or qualitative defects of the haemoglobin molecule
• May occur in association with thalassaemia
3
Haemoglobinopathies
• Sickle Cell Disease (Syndromes):
• Sickle cell trait (AS)
• Sickle cell anaemia (SS)
• Double heterozygotes:
• Sickle trait + b-thal, HbC, HbE, etc
• Common variants e.g. HbC, HbE
• Less common variants e.g. HbD, HbO, HbG, etc
• Unstable haemoglobin variants
• Haemoglobin variants affecting oxygen affinity
4
Sickle Cell (HbS) Disease
• Most common haemoglobinopathy worldwide:
• Africa, India, Middle East and Mediterranean
• Two forms of disease:

• Sickle cell trait (Heterozygous - AS)
• Carriers are asymptomatic
• FBC is normal
• HbS = 50%
• Sickle cell anaemia (SCA) (Homozygous - SS)
• Glutamic acid to valine substitution at position 6 in b chain
5
Sickle Cell Disease Inheritance
A. Normal + Carrier
B. Normal + Sickle Cell Anaemia
C. Sickle Cell Trait + Sickle Cell Trait
D. Sickle Cell Trait + Sickle Cell Anaemia
E. Heterozygous HbC + Sickle Cell Trait
Rodak. p431 6
Sickle Cell Anaemia
• HbS polymerises (low O2 + pH) to form sickle cells:
• Increases blood viscosity - slows blood flow/circulation
• Promotes further sickling of RBCs
• Sickling causes blockage of microcirculation
• Painful crises (episodes) and organ infarction
• Reduced RBC lifespan ~ 10-12 days
• Identification of HbS:
• Hb electrophoresis
• HPLC
• ? Resistance to P. falciparum infection
7
Normal flexible RBC
Normal
RBC with HbS (inflexible)

RBC that has undergone repeated
sickling becomes inflexible and is
caught in microvasculature
O2
RBC’s with HbS behind the inflexible
RBC give up oxygen to surrounding
hypoxic tissue causing them to sickle
O2
As more RBCs with HbS give up oxygen

a blockage forms, with tissue necrosis
eventually forming due to lack of oxygen
O2
8
Sickle Cell Anaemia: Clinical Features
• Highly variable
• SCA diagnosed in first year of life
• High morbidity and mortality due to infections
• Anaemic symptoms mild (HbS = low O2 affinity)
• Sickle ‘crises’:
• Severe anaemia (Hb = 60 – 90 g/L)
• Sickle “crises” triggered by:
• Infections, deoxygenation, cold temperatures
• Others: dehydration, alcohol, menstruation, idiopathic
9
Sickle Cell (HbS) Disease: Blood Film
• Sickle cell trait:
• Unremarkable (occ. sickle cell)
• Sickle cell anaemia:
• Microcytosis = coinheritance of thalassaemia or HbC
• Polychromasia
• Anisopoikilocytosis
• Sickle cells
• Target cells
• Basophilic stippling
• NRBCs
10
Sickle Cell (HbS) Disease: Blood Film
Rodak. p436 11
Sickle Cell (HbS) Disease: Further Tests
Alkaline Electrophoresis High-performance liquid chromatography (HPLC)
1. Normal adult.
2, 3. 17-year-old patient with sickle cell anaemia (Hb SS).
5, 6. Patient with sickle cell anaemia, recently transfused (note the presence of
Hb A from the transfused red blood cells).
4, 7. Hbs A/F/S/C standard Rodak. p438 12
Sickle Cell Trait vs Sickle Cell Anaemia
Sickle Cell Trait Sickle Cell Anaemia
Hb Normal 60 – 90 g/L
Reticulocytes Normal Up to 20%
Sickle cells Rare Prominent
HbS (%) 50 80 – 99
HbA (%) 50 0
HbF (%) Normal Up to 15
13
Haemoglobin C Disease
• Frequency (17-28% of West Africans; 2-3% African Americans)
• Glutamic acid to lysine substitution (pos 6, β-globin chain)
• HbCC – homozygous (No HbA)
• HbAC – heterozygous (HbA ~ 60%; HbC ~ 30%)
• Haemolytic anaemia (milder than HbSS):
• Usually compensated
• Microcytic/hypochromic; codocytes, polychromasia
• HbC polymerises under low oxygen tension:
• Occasionally HbC crystals seen on blood film (hexagonal)
• HbC polymer structure (hexagonal) different to HbS polymers (long/thin)
14
Haemoglobin C Disease: Blood Film
Lazarchick, J. ASH Image Bank 2005;2005:101384 Rodak. p440

15
Haemoglobin E Disease
• Frequency (SE Asia up to 30%; second most common Hb variant)
• Lysine to glutamic acid substitution at position 26 (β-globin chain)
• Mild microcytic/hypochromic anaemia:
• Usually well compensated
• Few symptoms
• Codocytes +++
• AE heterozygotes MCV ~ 70 fL (Hb normal)
• Often seen with a and b thalassaemia
• Can resemble thalassaemia trait (differentiation is important)
• Clinically severe when combined with β-thal (resembles β-thal major)
16
Haemoglobin E Disease: Blood Film
Rodak. p444 17
HbE – b Thalassaemia
Schrier, S. ASH Image Bank 2001;2001:100208

Copyright ©2001 American Society of Hematology. Copyright restrictions may apply.
18
HbS – b Thalassaemia
• Most common sickle cell syndrome in Mediterranean patients
• Both β chains affected (compound heterozygosity):
• Glutamic acid to valine substitution (HbS)
• Variable mutations in other b chain (b-Thal)
• Variable clinical manifestation
• Moderate - severe haemolytic anaemia
• Splenomegaly in 70% of patients
• Microcytic/hypochromic
• Polychromasia, codocytes, stippling
19
HbS – HbC (SC) Disease
• As common as sickle cell anaemia (25% in West Africa)
• Both β chains affected (compound heterozygosity):
• Glutamic acid to valine substitution at position 6 on one β-chain (HbS)
• Gluatmic acid to lysine substitution at position 6 on the other β-chain (HbC)
• Blood film:
• Mild to moderate normocytic anaemia
• Codocytes
• Occasional sickle cells and HbSC crystals:
• Crystalline aggregate hybrids – protrude from membrane
• Alkaline electrophoresis:
• ~ equal HbS and HbC
20
HbS – HbC (SC) Disease: Blood Film
21
Other Haemoglobinopathies
• Hb SC-Harlem
• Hb O-Arab
• Hb D
• Hb G
• Hb SG
• Hb SO-Arab
• Hb SG-Philadelphia
• Hb SD-Punjab
• etc….
22
Unstable Haemoglobin Variants
• > 200 variants (mainly β - chain variants)
• Unstable haemoglobin oxidises to methaemoglobin
• Heinz bodies:
• Oxidised haemoglobin
• Precipitated and denatured
• Irreversible
• Not demonstrated using Romanowsky stains
• Demonstrated using supravital stains e.g. methylene blue
• ↓MCV, ↓/N Hb, heinz bodies, electrophoresis (?)
• 25% will produce a haemolytic anaemia (mild to severe)
23
Haemoglobin Variants Affecting Oxygen Affinity
• Increased oxygen affinity:
• >90 variants
• Haemoglobins fail to release oxygen leading to hypoxia
• Kidneys release erythropoietin leading to compensatory erythrocytosis
• No precipitation; normal RBC morphology
• Most patients asymptomatic (diagnosis following investigation of erythrocytosis)
• Decreased oxygen affinity:
• Quickly release oxygen to tissues
• Hb Kansas
24
Summary
• Haemoglobinopathies are genetic disorders of globin genes:
• Produce structurally abnormal haemoglobins
• Altered amino acid sequences affect hemoglobin function and stability
• HbS is the most common haemoglobinopathy (African descent)
• HbS polymerises under various ‘stressors’ to generate sickle cells
• HbC and HbE are common haemoglobinopathies, but milder than HbS
• HbC disease is characterised by RBC crystals
• HbE disease is characterised by marked numbers of codocytes
• Unstable haemoglobin variants are characterised by Heinz bodies
25
References
• American Society of Hematology Image Database
• Hoffbrand and Pettit (3rd Ed). Essential Haematology. pp 94 - 120
• Rodak. Hematology: Clinical Principles and Applications (5th Edition)
• Chapter 27, pp 426 - 453
• Thien et al 2017. Pathology. 49, 1 – 9. (Review)
26
Disorders of Iron 1:
Iron Deficiency
Murray Adams
Learning Objectives
1. Compare and contrast storage forms of iron in the body
2. Discuss the factors that influence iron absorption/metabolism, including
‘iron recycling’
3. Describe the pathogenesis of iron deficiency anaemia
4. Differentiate between the different stages of iron deficiency
5. Describe the morphological changes to the blood in iron deficiency
anaemia
2
Overview
• Iron Metabolism:
• Distribution
• Storage
• Absorption
• Transport
• Iron Deficiency Anaemia
• Clinical features
• Laboratory testing
• Is Guinness a good source of iron???
3
Microcytic Anaemia
• A group of disorders where there is a quantitative decrease in Hb
synthesis leading to the production of microcytic red blood cells
• MCV < 80 fL (also ↓ MCHC)
• DNA synthesis and cellular division not impaired
• Causes:
• Impaired globin synthesis (Thalassaemias)
• Disordered haemoglobin structure (Haemoglobinopathies)
• Impaired heme synthesis:
• Unavailability of iron e.g. Fe ↓, sideroblastic anaemia
• Defective iron re-utilisation e.g. (ACD)
4
Iron Metabolism
• Fe 2+ (ferrous)
• Fe 3+ (ferric)
• Free iron
• Any iron stored must be bound to proteins
• Iron is required by every cell in the body
5
Iron Metabolism
• Total iron = 2 - 4 grams
• Body has no active mechanism for excreting iron
• Increased demand:
• Pregnancy
• Growing children
• Average daily intake:
• Males: 15 - 18 mg
• Females: 12 mg
• Only 10% of daily iron intake is absorbed
6
Iron Distribution
• There are three major compartments of iron
• Storage:
• Ferritin
• Haemosiderin
• Transport:
• Transferrin
• Functional (metabolic or enzymatic functions):
• Haemoglobin
• Myoglobin
• Cytochromes
7
Storages Forms of Iron
Ferritin Haemosiderin
• Major storage form of iron (65%) • 35% storage
• Found mainly in BM, spleen, liver • Long term storage for iron
• Water soluble • Stains with Prussian blue stain
• In equilibrium with serum iron • Not readily available
• Not stainable
• Not visible by microscopy
• Readily available
8
Iron Absorption
• The amount of iron absorbed is dependent on:
• Condition of mucosal cells in GI tract
• Intraluminal factors
• Dietary iron intake
• Tissue iron stores
• Haematopoietic activity of bone marrow
9
Dietary Iron
• 2 forms of dietary iron:
• Non - heme iron
• Heme iron
• Ferric iron is reduced to ferrous iron, bound to HMW chelators e.g.

lactic acid, ascorbic acid
• Ferrous iron is more stable and easily absorbed
10
Iron Transport
• When iron is absorbed into the mucosal cell:
• Combines with apoferritin to form ferritin (storage)
• Binds in ferric form to transferrin (transport)
• Transferrin:
• Plasma transport protein (2 binding sites for ferric iron)
• Delivers iron to marrow for heme synthesis (incorporation into erythroblast)
• High expression of transferrin receptors on Fe requiring cells
• After releasing Fe to the cell transferrin and transferrin receptor are recycled
• Laboratory measures:
• % Transferrin saturation
• Total iron binding capacity (TIBC)
11
Total Iron Binding Capacity (TIBC)
• Ability of transferrin to bind iron
• TIBC: 3.0 - 3.6 mg/L
• Serum iron: 0.6 - 1.6 mg/L (1/3 saturation)
•  TIBC: < 1/3 saturation
•  TIBC: > 1/3 saturation
TIBC ↓TIBC ↑TIBC
Serum
Iron
12
85% of Fe derived from Bone Marrow
Hb catabolism is recycled Transferrin
to plasma transferrin Fe3+ Hb
Fe2+ Heme
65% of total Fe
Stomach
Other Body Cells
Fe2+
Blood Vessel Fe3+

Fe3+
3 - 4% of total Fe
Fe2+ Fe2+ Fe3+ Fe2+

Liver/Spleen
Duodenum Ferritin
Mucosal Fe3+ Ferritin

1mg/day
Cell
Haemosiderin
Faeces TOTAL BODY: Fe
2 - 4g
30% of total Fe
Storage forms
13
Iron Recycling
• Erythroblast matures into RBC Circulating RBC
• RBC life span 120 days

• Removed from circulation Erythroid
Hepatocytes Macrophage
• Degradation of RBC
marrow
• 85% of iron is recycled to plasma

by transferrin Transferrin
• Transferrin delivers iron back to

Mucosal cells
1 mg in duodenum
Loss Other body 1 mg (Absorption)
marrow for heme synthesis cells
14
Causes of Iron Deficiency Anaemia
• Inadequate dietary intake
• Increased requirements:
• Pregnancy
• Rapid growth in infancy and adolescence
• Menstruation
• Impaired absorption:
• Malabsorption e.g., coeliac disease
• Inherited e.g., mutations in iron regulatory proteins (matriptase 2 protein)
• Loss of gastric acidity e.g., aging, gastrectomy
• Medications
• Chronic blood loss:
• Haemorrhage
• Haemolysis 15
Foods Rich in Iron
• Grains (rice, oatmeal)
• Dried fruit (prunes, figs, peaches)
• Meat (steak, liver)
• Nuts (almonds, cashews, sunflower seeds)
• Potatoes
• Curry Powder
16
Stages of Iron Deficiency
• Iron deficiency anaemia
develops slowly (3 stages)
• Storage iron depletion:
• Serum ferritin decreases
• No morphological changes to RBCs
• Transport iron depletion:
• Storage pool depleted
• TIBC increases
• Functional iron depletion:
• Iron deficiency anaemia
• Morphological changes
17
Clinical Features
• Slow onset (months to years)
• No clinical signs early
• Symptoms are generally non-specific:
• Weakness
• Lethargy
• Others
• Clinical history useful to identify higher risk groups:
• Increased demand; age (adolescents), female, pregnancy
• Underlying bleeding disorder
• Hookworm and other parasitic infections
18
Laboratory Diagnosis: Full Blood Count
• Haemoglobin decreased (< lower limit of reference interval)
• MCV decreased (< 75 fL) (decrease associated with severity)
• MCH and MCHC progressively decrease
• RCC and PCV progressively decrease
• RDW increased (>15%)
• Reticulocytes decreased
• Increases in response to treatment
• WBC usually normal
• Platelets may have thrombocytosis if underlying chronic bleeding
19
Laboratory Diagnosis: Peripheral Blood Film
• Microcytic hypochromic
• Anisocytosis
• Poikilocytosis:
• Codocytes occasional
• Elliptocytes occasional
• No single change is characteristic of
iron deficiency anaemia
• WBCs and platelets typically normal
in number and morphology
Rodak, page 300
20
Laboratory Diagnosis: Bone Marrow
• Not routinely conducted
• Cellularity is normal or increased
• Decreased M:E ratio
• Mild to moderate erythroid hyperplasia
• Scanty "moth-eaten" normoblasts
• Prussian Blue Reaction (PBR):
• Iron stain Rodak, page 302
• Demonstrate absence of haemosiderin in macrophages
• Sideroblasts markedly reduced
21
Laboratory Diagnosis: Iron Studies
Iron Increased Iron
Overload Iron Stores Reduced Iron Iron Deficient Deficiency
Normal Iron Stores Depletion Erythropoiesis Anaemia
Storage Iron
Transport Iron
Erythron Iron
Serum iron decreased

Marrow Iron Stores 4+ 3-4+ 2-3+ 1+ 0 - Trace 0 0
TIBC (µg/dL)
Serum ferritin decreased
< 300 < 300 330 ± 30 330 - 360 360 390 410
Plasma Ferritin (µg/L)
Plasma Iron (µg/dL)

% Transferrin saturation decreased
> 250
200
> 250
> 150
100 ± 60
115 ± 50
< 25
< 115
< 20
< 115
10
<60
<10
<40
Transferrin Saturation (%)

TIBC increased
> 60 > 50 35 ± 15 30 < 30 <15 <10
Sideroblasts (%) 40 - 60 40 - 60 40 - 60 40 - 60 40-60 <10 <10
RBC Protoporphyrin (µg/dL RBC) 30 30 30 30 30 100 200
Erythrocytes Normal Normal Normal Normal Normal Normal Microcytic

Hypochromic
22
Treatment
• Treat underlying cause: dietary, hookworm, stomach ulcers, etc
• Nutritional deficiency:
• Ferrous sulfate (oral supplement)
• Without food + vitamin C (aid absorption)
• Reticulocyte response:
• Within 48 - 72 hours
• Reticulocytes rise to 4 - 10%
• Haemoglobin levels normal within 6 - 10 weeks
• If no response, perform BM
23
Iron Deficiency (Treatment)
24
Summary
• The most common nutritional deficiency in the world is iron
deficiency anaemia
• The major compartments of iron in the body are:
• Storage (ferritin, haemosiderin)
• Transport (transferrin)
• Functional (haemoglobin, myoglobin, cytochromes)
• Diagnosis of iron deficiency anaemia is based on a combination of:
• Clinical history
• FBC + blood film
• Iron studies
25
References
• Beutler, Hoffbrand and Cook. Iron Deficiency and Overload. American
Journal of Hematology. 2003, 40-61.
• Chapter 20, pp 298 – 302.
26
Disorders of Iron 2:
Iron Overload
Murray Adams
Learning Objectives
1. Describe the pathogenesis of sideroblastic anaemia
2. Recognize predisposing factors for sideroblastic anemias or conditions in which
sideroblastic anemias may develop
3. Describe the pathogenesis of hereditary haemochromotosis
4. Identify the morphological changes to the blood in sideroblastic anaemia
5. Describe the bone marrow features in sideroblastic anaemia
6. Differentiate between the different stages of iron deficiency
7. Describe the morphological changes to the blood in iron deficiency anaemia
2
Overview
• Disorders of iron excess/overload
• Sideroblastic anaemia
• Hereditary haemochromotosis
• Pathophysiology
• Laboratory diagnosis
• Case studies of iron disorders
3
Sideroblastic Anaemia
• An enzyme defect disorder where there is sufficient iron but it is not
incorporated into Hb
• An arbitrary classification of patients with:

• Increase in total body iron
• Hypochromic anaemia
• Ringed sideroblasts in bone marrow
4
Sideroblastic Anaemia: Cause
• Iron enters developing RBC and accumulates:
• Not incorporated into haemoglobin
• Due to defects:
• Within initial stages of heme synthesis
• With the incorporation of heme into protoporphyrin ring
• Iron accumulates inside the cristae of mitochondria:

• Mitochondria overloaded with iron (ringed sideroblast)
• Iron granules (haemosiderin) present in sideroblasts
• Ringed sideroblasts do not develop into mature RBC’s
5
Sideroblastic Anaemia: Classification
• Hereditary:
• X - linked
• Autosomal recessive
• Acquired:
• Primary sideroblastic anemia (refractory)
• Secondary sideroblastic anemias caused by drugs and bone marrow toxins
• Anti-tubercular drugs
• Chloramphenicol
• Alcohol
• Lead
• Chemotherapeutic agents
6
Sideroblastic Anaemia: Morphological Features
• Blood film:
• Dimorphic blood picture:
• Often microcytic
• Normochromic and hypochromic RBC populations
• WBC's and platelets normal
• Inclusion bodies:
• Pappenheimer bodies, basophilic stippling
• Iron Studies:
• Serum Iron: Increased
• Serum Ferritin: Increased
• % Transferrin Sat.: Increased
• TIBC: Decreased
7
Bone Marrow: Ringed Sideroblasts
• Bone marrow:
• Erythroid hyperplasia
• "Moth eaten" late normoblasts
• Ringed sideroblasts:
• Occasional megaloblastic changes (?folate deficiency)
Rodak, page 305 8

Sideroblastic Anaemia: Treatment
• Depends on cause
• Acquired:
• Remove offending agent
• Hereditary:
• Pyridoxine
• Desferrioxamine
9
Other Causes of Iron Overload
• Increased intake:
• Excess medicinal iron
• Dietary
• Increased transfusion load:
• Treatment of BM failure
• Treatment of thalassaemia major and sickle cell anaemia (ongoing)
• Inappropriate increase in intestinal absorption:
• Hereditary haemochromotosis (HH)
• Various iron-loading anaemias
10
Aetiology of Iron Overload
• Body’s rate of iron absorption > rate of iron loss (> 1 mg/day)
• Excess iron initially stored (ferritin, then haemosiderin):
• Ferrous iron accumulates intracellularly
• Ferrous iron generates superoxide, free radicals and peroxidation of
membrane lipids
• Once storage is overwhelmed cell death / organ damage occurs:
• Liver
• Heart
• Pancreas
11
Hereditary Haemochromotosis: General
• Hereditary disorder of abnormal iron metabolism
• Then:
• Initial discovery of two genetic mutations (1996)
• HFE gene (chromosome 6) encodes for 343 a.a. protein
• 2 mutations:
• C282Y (cysteine to tyrosine) and H63D (histidine to aspartate)
• Homozygotes clinical symptoms; Heterozygotes associated with iron loading
• Now:
• Multiple genotypes of iron regulatory proteins produce a general phenotype
• Commonly misdiagnosed (FBC usually normal)
12
Hereditary Haemochromotosis: Genes & Proteins
Feature Hemochromatosis Hemochromatosis, Hemochromatosis, Hemochromatosis, Hemachromatosis, Hemachromatosis,
Type 1 Type 2A Juvenile Type 2B Juvenile Type 3 Type 4 Type 5
HFE-Associated HFE2-Related HAMP-Related TFR2-Related Ferroportin-Related

Hereditary Juvenile Juvenile Hereditary Iron Overload
Hemochromatosis Hemochromatosis Hemochromatosis Hemochromatosis
Affected gene HFE HFE2 (HJV) HAMP TFR2 SLC40A1 FTH1
Mutated protein Hereditary Hemojuvelin Hepcidin Transferrin receptor Solute carrier family Ferritin
hemochromatosis protein 2 40 member 1 heavy chain
protein (Ferroportin-1)
Normal function Inhibits TfR1-mediated Regulates hepcidin Downregulates Provides hepatocyte Transports iron out Iron storage
of affected iron uptake; expression ferroportin-mediated iron uptake; of enterocytes and
protein regulates hepcidin iron transport in regulates hepcidin macrophages
expression macrophages and expression
enterocytes
Age of onset of 30–40 Teens–20 Teens–20 20–40 (mild) 30–40 Found in 3 members
symptoms (yr) of a Japanese family
Rodak, page 307, Table 20.3 13

Hereditary Haemochromotosis: HFE Gene/Protein
• Incidence: ~5 in 1000 northern Europeans
• Common genetic disorder of Caucasians
• HFE gene (chromosome 6) encodes for 343 a.a. protein
• Normal HFE protein regulates iron absorption:
• Binds β2-microglobulin intracellularly
• Allows HFE to appear on cell surface; HFE binds transferrin receptor 1 (TfR1)
• HFE binds to TfR1 until TfR1 binds transferrin; HFE then released
• HFE binds to transferrin receptor 2 (TfR2):
• Initiates a signal for hepcidin production
• Reduces iron absorption
14
3. Interaction of HFE with transferrin receptor-1 reduces transferrin binding
to receptor via hepcidin signalling (↓ iron absorption)
2. HFE protein interacts with

↓ Transferrin
transferrin receptor-1 on cell surface
Transferrin HFE β2-microglobulin

Receptor
HFE β2-microglobulin
1. Normal HFE protein binds β–microglobulin intracellularly
15
3. Transferrin receptor-1 available for transferring binding (↑ iron absorption)
2. HFE protein doesn’t interact with

Transferrin
transferrin receptor-1 on cell surface
Transferrin
Receptor
HFE β2-microglobulin
1. Abnormal HFE protein doesn’t bind

β–microglobulin intracellularly
16
Hereditary Haemochromotosis: Symptoms
• ‘Golden skin’:
• Also ‘bronzed complexion’
• Accumulation of haemosiderin
• Chronic fatigue
• Arthritic pain in joints
• Changes in skin colour
• Abdominal pain
• Weight loss
• Hypothyroidism
17
Haemochromotosis: Laboratory Diagnosis
• Laboratory testing required: • Iron studies:
• Screening • Serum iron increased*
• Diagnose cause of organ damage • Serum ferritin increased*
• Identify mutation for genetic counselling • TIBC normal/decreased
• Monitor treatment • Transferrin saturation increased
• Investigation of: • Massive iron deposits in BM
• Organ function problems (LFT’s) • Molecular analysis:
• Non-specific symtoms • Detection of mutations in HFE gene
• Not detected in routine lab tests/films • Other mutations
• Usually normal FBC
• Anaemia not usually detected
18
Haemochromotosis: Treatment
• Phlebotomy:
• Inexpensive
• Regular venesection, removal of ~500mL of blood weekly (initially)
• Maintenance for life every three months
• Monitor haemoglobin levels (maintain mild anaemia)
• Iron chelators:
• Desferrioxamine
• Injected subcutaneously
• Excreted through urine
• Monitor iron status
19
Iron Anaemia of Thalassaemia Sideroblastic
Deficiency Chronic Disease Trait ( or ) Anaemia
Anaemia / Inflammation
M.C.V. all reduced in low normal all reduced very low in congenital
M.C.H. relation to or mild reduction very low for type but MCV often
M.C.H.C. severity degree of anaemia raised in acquired type
Serum Iron reduced reduced normal raised
TIBC raised reduced normal normal
Serum ferritin reduced normal normal raised

Bone Marrow
absent present present present
Iron Stores
Erythroblast
absent absent present ring forms
Iron
Haemoglobin Hb A2 raised in
normal normal normal
Electrophoresis Thal.
20
Case Study 1
RCC (x1012/L) 3.57 (3.50 – 5.50 x 1012/L)
• 76 year old female Hb (g/L) 94 (115 – 155 g/L)
• History: Fatigue 2/12 WCC (x109L) 5.2 (4.0 – 11.0 x 109/L)
• Doctor requested: MCV (fL) 79 (80 – 98 fL)

• FBC Platelets (x109/L) 220 (150 – 400 x 109/L)
• Coag screen (INR/aPTT/fbg)
• U&E’s
• Liver enzymes
• Coags and biochem normal
Case Study 1
• Film Comment:
• Anisocytosis +
• Poikilocytosis +
• Hypochromasia +
• Occ. Codocytes and Dacrocytes
• Iron Studies:
• Serum Iron 58 µg/dL [65 – 165 µg/dL]
• TIBC 445 µg/dL [300 – 360 µg/dL]
• Iron Saturation 27% [20 – 50 %]
• Ferritin 25 µg/L [50 – 150 µg/L]
22
Case Study 2
• 45 year old female
• Post hysterectomy 4/12
• Fatigued during this period
• Doctor requested:
• FBC
• Iron studies
RCC (x1012/L) 3.12 (3.50 – 5.50 x 1012/L)
Hb (g/L) 87 (115 – 155 g/L)
WCC (x109L) 8.8 (4.0 – 11.0 x 109/L)
MCV (fL) 71 (80 – 98 fL)
Platelets (x109/L) 358 (150 – 400 x 109/L)

Case Study 2
• Film Comment:
• Anisocytosis ++
• Poikilocytosis +
• Hypochromasia ++
• Target and teardrop cells seen
• Iron Studies:
• Serum Iron 32 µg/dL [65 – 165 µg/dL]
• TIBC 515 µg/dL [300 – 360 µg/dL]
• Iron Saturation 7% [20 – 50 %]
• Ferritin 12 µg/L [50 – 150 µg/L]
24
Summary
• Sideroblastic anaemia:
• Develops when synthesis of protoporphyrin / incorporation of iron into
protoporphyrin ring is blocked
• Iron accumulates in the mitochondria of developing RBCs and are detected as
ringed sideroblasts in the laboratory
• Iron studies show elevated levels of serum iron and serum ferritin, decreased
or normal TIBC, and increased transferrin saturation
• Hereditary haemochromotosis:
• Results from mutations of proteins involved in regulation of iron absorption
• Free iron accumulates and can cause organ damage
• No single test can diagnose hereditary haemochromotosis (iron studies useful)
25
References
• Beutler, Hoffbrand and Cook. Iron Deficiency and Overload. American
Journal of Hematology 2003, 40-61.
• Chapter 20, pp 305 - 309.
26
Macrocytic Anaemia 1
Murray Adams
Learning Objectives
1. Discuss the classification of macrocytic anaemias
2. Describe the major haematological features of liver disease
3. Discuss the physiological roles of vitamin B12 and folate in DNA synthesis
4. Describe the absorption and distribution of vitamin B12 and folate
5. Describe how megaloblastic erythropoiesis occurs in vitamin B12 / folate
deficiency
2
Overview
• Introduction to macrocytic anaemias
• Non-megaloblastic macrocytic anaemias:
• Liver disease
• Other causes
• The roles of vitamin B12 and folate in DNA synthesis
• Vitamin B12 and folate:
• Absorption
• Transport
• Function
Introduction to Macrocytic Anaemias
• Macrocytic anaemias - MCV > 100 fL
• Causes of macrocytosis:
• Alcoholism
• Drugs
• Liver Disease
• Folate and cobalamin deficiency
• Classification:
• Megaloblastic
• Non-megaloblastic
Non-megaloblastic Macrocytic Anaemia
• DNA synthesis not impaired
• Macrocytic circulating RBC’s:
• Normoblastic erythropoiesis in BM
• Different mechanisms vs megaloblastic anaemia
• Macrocytes are round shaped
• MCV usually < 110 fL
• Mild anaemia
• Exclude B12/folate deficiency
• Clinical history is important
Non-megaloblastic Macrocytic Anaemia
• Liver disease***
• Alcohol
• Reticulocytosis***
• Cytotoxic drugs
• Aplastic anaemia
• Pregnancy
• Myelodysplastic syndromes
• Myeloma
Liver Disease
• Most common (non-megaloblastic)
• Contributing factors to anaemia:
• Blood loss
• Iron/folate deficiency
• Suppression of haemopoiesis by alcohol
• MCV 100 – 110 fL
• Codocytes (↑cholesterol in membrane)
• WBCs, platelets normal
• Coagulation abnormalities:
• FII, FVII, FIX, FX ’s (prolonged PT / aPTT)
Reticulocytosis
• Stimulated erythropoiesis
• Variety of causes:
• Blood loss
• Destruction of RBC’s
• Variable (↑) MCV
• Anisocytosis (↑RDW)
• Polychromasia
• NRBCs
Rodak, page 412
DNA Synthesis
• Rapidly dividing cells require high levels of DNA synthesis
Aberrant DNA synthesis
Abnormal nuclear : cytoplasmic maturation
Vitamin B12 & folate required for normal DNA synthesis

Megaloblastic Anaemia: Introduction
• Causes include:
• Vitamin B12 deficiency
• Folic acid deficiency
• B12 and folate metabolism enzyme abnormalities
• Other defects of DNA synthesis
• Nuclear maturation defect
• Anaemia due to ineffective erythropoiesis
• MCV usually > 110 fL
• Macrocytes are oval shaped
• Megaloblastic changes occur in the bone marrow
Vitamin B12
• Generic term for family of cobalamin vitamins
• The cobalamins have the same basic structure:
• Corrinoid:
• Central cobalt
• Corrin ring with four pyrrole groups
• Nucleotide attached to ring and cobalt
• Methylcobalamin required for methionine synthesis
• Main dietary intake; milk, eggs, fish (3 - 30 µg/day):
• ~70% absorption
Vitamin B12: Absorption
• Released from food by peptic digestion
• Cobalamin binds to R-binding protein (haptocorrin)
• Pancreatic proteases (trypsin) degrade haptocorrin
• Cobalamin binds to intrinsic factor
• Intrinsic factor - cobalamin complex binds to receptors for IF on
mucosal cells in ileum
Vitamin B12: Transport
Cobalamin leaves mucosal cell
Transcobalamin II
Taken to cells of liver, bone marrow (others)
Vitamin B12 stored mainly in liver (1 - 3 mg)

Vitamin B12: Absorption and Transport
Food
Gastric Parietal Cells Intake
B12
HCL Haptocorrin
Saliva
IF
IF Stomach
HCl
Trypsin Haptocorrin-B12 TCII
Small B12
Intestine IF TCII B12 TCII
IF-/-B12
IF-B12 B12-TCII
IF-B12 B12-TCII B12-TCII
Illeal Cell BM Cell

Vitamin B12: Functions
• Conversion of methyl malonyl CoA to succinyl CoA
• Succinyl CoA required for myelin synthesis (CNS)
Propionyl CoA
• ↓ vitamin B12 leads to:
• ↓ succinyl CoA and
• ↓ myelin synthesis Methylmalonyl CoA
• ↑ methylmalonyl CoA (urine)
adenyslcobalamin
• Lack of myelin – neurological symptoms
• Conversion of homocysteine to methionine Succinyl CoA
• Central reaction in DNA synthesis

• Vitamin B12 required for generation of tetrahydrofolate (THF):
• Active form of folate
dTTP dTMP dUMP
DNA DHF 5,10-methylene-THF
dATP dCTP THF Methionine
dGTP Vitamin B12

Homocysteine
Methyl-THF
Cell
Plasma Methyl-THF
Small Intestine Dietary Folates

dTTP dTMP dUMP
DNA DHF 5,10-methylene-THF
dATP dCTP THF
dGTP FOLATE TRAP

Homocysteine
Methyl-THF
Cell
Plasma Methyl-THF
Small Intestine Dietary Folates

Causes of Vitamin B12 Deficiency
• Vitamin B12 deficiency is reflected by:
• Impaired DNA synthesis (↓methionine; ↓THF)
• Defective fatty acid degradation and disruption of neural membranes
(↓succinyl CoA)
• Causes may be due to:

• Nutritional deficiency
• Malabsorption
• Impaired utilisation
• Increased demand
Folate
• Folate is the general term used for any form of folic acid
• Folates consist of:
• Pteridine ring
• p-amino-benzoic acid
• Glutamic acid
• Inactive form: Folic Acid

• Active form: Tetrahydrofolate (THF)
Folate: Functions
• Transfer carbon (methyl groups)
Deoxyuridylate Thymidylate DNA
(dUMP) (dTMP)
from donor to acceptor:
• Homocysteine → methionine 5,10-methylene THF
Dihydrofolate
(DHF) (inactive)
• ‘Central’ reaction DNA synthesis
Folic acid
DHF reductase (inactive)
• Metabolism of nucleotides and Glycine
amino acids: Tetrahydrofolate

(THF)
N5-formimino-
• Histidine → glutamic acid Serine
(active)
THF tetrahydrofolate
• Serine → glycine
Formiminglutamic acid Glutamic
Histidine Urocanic acid
(FIGLU) acid
Folate: Absorption and Transport
• Folate present in most foods
• Intake: 500 µg/day (50% absorbed) Folate deficiency
• Require: 100 µg/day
• Stored: 5 - 10 mg (liver)
• Absorption: ↓ Synthesis of 5,10-methylene THF
• Small intestine (jejunum)
• Reduced to 5-methyl tetrahydrofolate
• Plasma transport: Marked slowing of DNA synthesis
• Cells with folate receptors
Causes of Folate Deficiency
• Inadequate intake: • Impaired use:
• Poor diet • Drug interference
• Overcooking food (heat labile)
• Excessive loss:
• Increased demand: • Renal dialysis
• Pregancy
• Infants and children
• Impaired absorption:
• Intestinal disease
Summary
• Macrocytic anaemias (MCV > 100 fL) classified according to the
development of RBC’s in BM:
• Megaloblastic:
• Folate and cobalamin deficiency
• Non-megaloblastic:
• Alcoholism, drugs and liver disease
24
References
• Chapter 21, pp 314 - 330.
• Thien et al 2017. Pathology. 49, 1 – 9. (Review).
Macrocytic Anaemia 2
Murray Adams
Learning Objectives
1. Describe the clinical features of megaloblastic anaemia
2. Describe how neural tube defects develop in utero
3. Compare and contrast the features of pernicious anaemia and
megaloblastic anaemia
4. Describe the FBC, peripheral blood and bone marrow features found in
megaloblastic anaemia
2
Overview
• Clinical features of megaloblastic anaemia:
• Differences between vitamin B12 and folate deficiencies
• Neural tube defects
• Laboratory investigations of megaloblastic anaemia:
• FBC
• Peripheral blood
• Bone marrow
• Further tests
• Pernicious anaemia
• Treatment
3
Megaloblastic Anaemia: Clinical Features
• Typical symptoms of anaemia:
• Asymptomatic initially (?)
• Slow developing
• Jaundice (coupled with anaemia)
• Neurological complaints (B12 deficiency)
• In early stages treat with both B12 and

folate prior to knowing vitamin levels Hoffbrand & Pettit. Essential Haematology (3rd Ed.) p63.
4
Megaloblastic Anaemia: Clinical Features
• Folate Deficiency:
• More rapid onset
• Excess demand or poor diet
• Vitamin B12 Deficiency:

• Slow onset
• GIT problems
• Intrinsic factor
Hoffbrand & Pettit. Essential Haematology (3rd Ed.) p63.
5
Neural Tube Defects
• Neural tube is the part of an embryo that becomes the brain or
spinal cord (closure occurs ~ 3-4 weeks of gestation)
• Neural tube defects (NTDs):
• Anencephaly (brain - lethal)
• Spina bifida (spine)
• Incidence of NTDs reduced with folate:
• Dietary supplementation prior to and during pregnancy (since 1995)
• Food fortification; flour for bread (since 2009)
• Previously common (10/10,000 live births) (Lancaster et al 1993)
• Now uncommon (0.22/10,000 live births) (AIHW National Perinatal Statistics Unit 2011)
6
Neural Tube Defects
http://www.anencephalie-info.org/pdf/anen-info_2006-USA.pdf http://www.getfolic.com/professionals/birth-defects.html#1
7
Neural Tube Defects
8
Laboratory Results: Haematology / Biochemistry
• FBC:
• MCV >110 fL
• Haemoglobin and RCC vary (usually decreased)
• Pancytopaenia:
• WCC reduced
• Platelet count reduced
• Clinical chemistry:
• Increased bilirubin
• Increased LDH
9
Laboratory Results: Blood Film
• Anisocytosis and poikilocytosis (++ to +++)
• Macrocytes (ovalocytes)
• HJ bodies
• Hypersegmented neutrophils
• occ. NRBC's
10
Rodak, page 322
Blood Film
11
Laboratory Results: Bone Marrow
• Hypercellular (ineffective erythropoiesis)
• Megaloblastic changes (asynchrony):
• Large erythroblasts
• Open chromatin pattern
• Normal haemoglobinisation
• Giant metamyelocytes
• Hypersegmented megakaryocytes
12
Development of Megaloblastic Anaemia
1. Decrease in vitamin levels
2. Hypersegmentation of neutrophils in peripheral blood
3. Oval macrocytes in peripheral blood
4. Megaloblastosis in bone marrow
5. Anaemia
13
William Castle: “The Disgusting Experiment”
• Patients with pernicious anaemia lacked an “intrinsic factor”

in their gastric juices to release an “extrinsic factor” in food
• Tested the hypothesis:
• Ate raw liver
• Regurgitated (his own) gastric juices
• Gave to patients with pernicious anaemia
• Patients generated a reticulocyte response
14
Pernicious Anaemia
• Absence of Intrinsic Factor (IF) Food
Gastric Parietal Cells
• Absence of IF leads to B12 deficiency Intake
• [note: IF required for B12 absorption] HCL

IF
B12
Haptocorrin
Saliva
• ? Autoimmune disease
• Diagnosis:
IF Stomach
HCl
• Previously; gastric analysis (Schilling Test)

Trypsin Haptocorrin-B12 TCII
• Now; ELISA (antibodies): Small

Intestine
B12
IF TCII B12 TCII
• Blocking (anti-IF antibodies) IF-B12
IF-/-B12
B12-TCII
• Binding (anti-parietal cell antibodies) IF-B12 B12-TCII B12-TCII
• > 40 years Illeal Cell BM Cell
• Women > Men

15
Comparison of Macrocytic Anaemias
Megaloblastic Liver Disease Reticulocytosis
MCV (fL) 120 +/- 30 105 +/- 10 110 +/- 20
Anisocytosis Increased Normal Increased
Poikilocytosis Increased Normal/Increased Normal
RBC Oval Macrocytes Codocytes, Shift Reticulocytes,

Acanthocytes Polychromasia
WBC/Platelets Decreased Variable Increased or

Hypersegmented Normal
Bone Marrow Megaloblastic Nonmegaloblastic; Nonmegaloblastic

Erythroid Variable Erythroid
Hyperplasia Erythropoiesis Hyperplasia
16
Other Laboratory Tests
• Serum vitamin B12:
• Microbiological (historical)
• Immunoassays
• Chemiluminescence
• Vitamin B12 absorption tests:
• Schilling Test (rarely used)
• Other tests for vitamin B12:
• Antibody assays (anti-IF; anti-parietal cell antibodies)
• Methyl malonic acid (MMA) (serum / urine)
• Deoxyuridine suppression test (dUT): requires BM sample
17
Other Laboratory Tests
• Tests for folate deficiency (immunoassay):
• Serum folate:
• Levels fluctuate with diet
• Red cell folate:
• Levels more stable that serum levels assay
• Assays have lower sensitivity and specificity
• Tests for vitamin B12 and folate are influenced by an array of factors:
• Concurrent disease
• Diet, alcohol, pregnancy
• Homocysteine levels:
• Increased in folate or vitamin B12 deficiencies
18
Some Useful Journal Reviews
• Review of laboratory investigation of B12 and folate
• Laboratory methods:
• Peripheral blood
• Bone marrow
• Vitamin and metabolite assays
• Zittoun and Zittoun, 1999. Seminars in Haematology. 36, 35 - 46.

• Wickramasinghe, 2006. Blood Reviews. 20, 299-318.
• Kaferle and Strzoda, 2009. Am. Acad. Fam. Physicians. 79, 203-308.
19
Treatment
• Appropriate vitamin / folate
• Severe anaemia - both
• Avoid transfusion
• 24 - 48 hour response
• RBC abnormalities disappear quickly
• Reticulocyte response after 2 - 3 days
• Hypersegmented neutrophils
20
Treatment
B12 Deficiency Folate Deficiency
Drug Hydroxocobalamin (I/M) Folic Acid (Oral)
Dose 6 x 1000 μg over 2-3 weeks 5 mg daily for 4 months

1000 μg every 3 months
Prophylactic Total gastrectomy Pregnancy
Ileal resection Severe haemolytic anaemia
Dialysis
Prematurity
21
Rodak, page 327 22
Summary
• Vitamin B12 and folate deficiency can result in neural tube detects
• Pernicious anaemia is caused by a deficiency in intrinsic factor
resulting in decreased absorption of vitamin B12
• The major haematological features of megaloblastic anaemia include:
• FBC: MCV > 110 fL, pancytopaenia, anaemia
• Peripheral blood film: macrocytes, anisocytosis, hypersegmented neutrophils
• BM: megaloblastic erythropoiesis
• Causes of megaloblastic anaemia can be determined using:
• Clinical history
• Assays for vitamin B12 (serum B12, serum folate, RBC folate assays)
• Auto-antibody assays
23
References
• Hoffbrand and Pettit. Essential Haematology (3rd Ed.). pp 53-73.
• Chapter 21, pp 314 - 330.
• Zittoun and Zittoun, 1999. Seminars in Haematology. 36, 35 - 46.
• Wickramasinghe, 2006. Blood Reviews. 20, 299-318.
• Kaferle and Strzoda, 2009. Am. Acad. Fam. Physicians. 79, 203-308.
• AIHW National Perinatal Statistics Unit 2011. Neural tube defects in
Australia: prevalence before mandatory folic acid fortification. Cat.
no. PER 53. Canberra: AIHW.
24
Haemolytic Anaemia 1
Murray Adams
Learning Objectives
1. Define haemolysis and describe compensated erythropoiesis
2. Discuss the processes of intravascular and extravascular haemolysis
3. Compare and contrast intravascular and extravascular haemolysis
4. Discuss the classification and causes of haemolytic anaemia
5. Describe the types of laboratory methods used for the investigation of
haemolytic anaemia
2
Overview
• Terminology
• Sites of haemolysis (RBC destruction):
• Intravascular and extravascular
• Classification:
• Hereditary and acquired
• Examples:
• Hereditary spherocytosis, G6PD deficiency
• Laboratory investigation
3
Haemolytic Anaemia
Hereditary Acquired
Membrane Enzyme Abnormal

Disorders Deficiencies Hb
HS G6PD Thals
HE PK Hb Variants
Immune
Non-Immune
MAHA Drug Induced Allo Auto
TTP/HUS
March Haemoglobinuria HDN Warm AIHA
Hypersplenism Incompatible CHAD
Sepsis transfusion Mycoplasma
Malaria EBV
PNH PCH
LPD’s
4
Terminology
• Haemolysis is the premature destruction of RBC's i.e. lysis of RBC’s
+/- anaemia
• Haemolytic anaemia refers to the normocytic normochromic
anaemia caused by the increased and premature destruction of RBC's
• Compensated erythropoiesis occurs when increased BM stimulation
balances increased haemolysis
• No anaemia with compensated (balanced) erythropoiesis
5
Sites of Haemolysis
• Haemolysis (RBC Destruction) may be:
• Intravascular:
• Within circulation
• Rarer
• More severe
• Extravascular:
• Outside circulation
• More common
• Less severe
Normal Catabolism of Haemoglobin

Rodak, page 350 6
Intravascular Haemolysis
• Occurs within the circulation (normally ~ 10%)
• Associated with more severe problems
• Examples of diseases with increased intravascular haemolysis:
• Activation of complement on RBC membrane
• Physical or mechanical trauma to RBC
• Toxic microenvironment
7
Intravascular Haemoglobin Degradation
Free Hb in blood
haptoglobin
Hb - haptoglobin Liver (catabolism)
ab dimers Hb in excess of haptoglobin
Methaemoglobin
kidney
globin amino acid
pool
heme
haemopexin haemopexin - heme
urine urine
Hb haemosiderin
albumin methaemalbumin
albumin
RE cells in liver heme 8

Intravascular Haemoglobin Degradation
Free Hb in blood (haemoglobinaemia)
haptoglobin
Hb – haptoglobin () Liver (catabolism)
ab dimers Hb in excess of haptoglobin
Methaemoglobin
kidney
globin amino acid
pool
heme
Haemopexin () haemopexin - heme
urine urine
Hb haemosiderin
albumin methaemalbumin
(haemoglobinuria)
albumin
(haemosiderinuria) RE cells in liver heme 9
Extravascular Haemolysis
• Occurs within macrophages (normally ~ 90%):
• Spleen, liver and bone marrow
• Examples of disease with increased extravascular haemolysis:
• Inherited RBC Defects e.g. Thalassaemias
• Acquired RBC Defects e.g. Spur cell anaemia
• Autoimmune haemolytic anaemia (AIHA)
10
Extravascular Haemoglobin Degradation
Haemoglobin Heme + globin amino acid pool
biliverdin + CO + Fe transferrin + Fe BM
lungs
bilirubin
plasma albumin
bilirubin-albumin (unconjugated)
liver
bilirubin diblucuronide (conjugated)
bile duct to duodenum

kidney
urobilinogen blood urobilinogen (urine)
stool
urobilinogen + stercobilinogen
11
Extravascular Haemoglobin Degradation
Haemoglobin Heme + globin amino acid pool
biliverdin + CO + Fe transferrin + Fe BM
lungs
bilirubin
plasma albumin No release of Hb into plasma

bilirubin-albumin (unconjugated) No haemoglobinaemia
No haemoglobinuria
liver
No haemosiderinuria
bilirubin diblucuronide (conjugated)
bile duct to duodenum

kidney
urobilinogen blood urobilinogen (urine)
stool
urobilinogen + stercobilinogen
12
Classification of Haemolytic Anaemia
The classification of haemolytic anaemias is based on aetiology of RBC
haemolysis
Hereditary/Genetic
or
Acquired
13
Aetiology
• Genetic/Hereditary:
• Intrinsic abnormalities (within RBC)
• Cell membrane defects - H.S/H.E
• Enzyme deficiencies
• [Abnormal haemoglobin – haemoglobinopathies]
• [Defects in globin synthesis – thalassaemias]
• Acquired:
• Extrinsic abnormalities (external to RBC)
• RBC antibodies
14
Hereditary RBC Abnormalities
• Membrane Defects:
• Abnormalities in membrane proteins or lipids
• Changes in stability, shape, permeability
• Enzyme Disorders:
• Deficiency causes changes to membrane or Hb, resulting in haemolysis
• Pyruvate kinase, Glucose-6-phosphate-dehydrogenase
• Haemoglobinopathies: (see also Microcytic anaemia)
• Structurally abnormal haemoglobins (HbS, HbC)
• Defects in globin synthesis (thalassaemia)
15
Acquired RBC Abnormalities
• Immune:
• Due to immune mediated process
• Complement and/or antibody
• AIHA and HDN
• Abnormal Plasma Lipids:

• Excess membrane lipid
• Spur cell anaemia (acanthocytes)
AIHA
Rodak, page 397
16
Acquired RBC Abnormalities
• Physical or Mechanical Trauma:
• Trauma to RBC in vascular circulation
• Microangiopathic Haemolytic Anaemias (MAHA):
• HUS: Haemolytic Uraemic Syndrome
• DIC: Disseminated Intravascular Coagulation
• TTP: Thrombotic Thrombocytopaenic Purpura
• Antagonists: TTP
• Drugs, chemicals, infections, toxins Rodak, page 397
17
Clinical Features
• Primary symptoms associated with
anaemia (Hb):
• Fatigue, pallor http://www.med.umich.edu/lrc/coursepages/m1/anatom
y2010/html/clinicalcases/cholelithiasis/cholelithiasis.html
• Other symptoms associated with

↑RBC destruction & erythropoiesis:
• Jaundice, gallstones (pigmented),
darkened urine
• Other changes:
• Thinning of cortical bone
• Extramedullary haemopoietic masses
• Splenomegaly https://askhematologist.com/2016/02/22/hemolytic-anemias/
18
Laboratory Investigations
• Wide range of aetiology (causes)
• Lab tests will help identify the cause of haemolysis
• Haematological findings reflect:
• Increased destruction of RBC's
• Increased activity of bone marrow (erythropoiesis)
• Investigation:
• Full blood count
• Peripheral blood film
• Bone marrow
• Other laboratory tests
19
• FBC:
• Decreased Hb (anaemia)
• Variable MCV (macrocytic if increased retics)
• Anisocytosis
• Poikilocytosis e.g. spherocytes, schistocytes
• Polychromasia (+++)
• Bone Marrow:
20
• Other Laboratory Tests:
• Increased RBC Breakdown Products:
• Unconjugated bilirubin (< 20 µmol/L) increased
• Haptoglobin (1 - 3 g/L) decreased
• Faecal and urine urobilinogen increased http://healthsaline.com/bilirubin-in-urine.html
• Increased RBC Production:

• Reticulocytes (% and absolute numbers)
• Bone marrow activity (M:E ratio)
• Bone marrow changes
21
Summary
• Haemolytic anaemias are a group of disorders charactersised by
reduced RBC lifespan
• Haemolysis occurs at both intravascular and extravascular sites
• Hereditary haemolytic anaemias are due to intrinsic abnormalities:
• Cell membrane defects
• Enzyme deficiencies
• Acquired haemolytic anaemias are due to extrinsic abnormalities:
• RBC antibodies
22
References
• Chapter 23, pp 349 - 365.
• Hoffbrand and Pettit (3rd Ed). Essential Haematology.
23
Haemolytic Anaemia 1
Murray Adams
Learning Objectives
1. Describe intrinsic causes of haemolytic anaemia
2. Describe extrinsic causes of haemolytic anaemia
3. Describe the pathophysiology and laboratory investigations of the following
haemolytic anaemias:
• Hereditary spherocytosis and hereditary elliptocytosis
• Paroxysmal nocturnal haemoglobinuria
• G6PD and pyruvate kinase deficiencies
• Non-Immune cause, including the microangiopathic haemolytic anaemias
2
Haemolytic Anaemia
Hereditary Acquired

HS G6PD Thals
HE PK Hb Variants
PNH
Immune
Non-Immune
TTP/HUS/DIC Drug Induced Allo Auto
HELLP Syndrome
Malaria EBV
PCH
LPD’s
3
Hereditary Spherocytosis
• Incidence 1:5000 (~1:2500 of northern Europeans)
• Autosomal dominant (75%)
• Genetically encoded membrane defect of main structural proteins:
• 130 different mutations: disrupt vertical membrane linkages
• ~10% of patients have no identifiable mutation
• Loss of RBC shape: biconcave to spherical
• Severity:
• Mild (20%),
• Moderate/Typical (70%),
• Severe (~10%)
4
Hereditary Spherocytosis: Gene Mutations
• Cytoskeleton proteins:
• ANK1: ankyrin
• SPTA1: α-spectrin
• SPTB: β-spectrin
• EPB42: protein 4.2
• Transmembrane proteins:
• SLC4A1: band 3
5
• Mild HS:
• Compensated haemolysis
• Diagnosis later in life (anaemia, infections, bleeding)
• Typical HS:
• Moderately severe anaemia
• Splenomegaly
• Diagnosis early childhood/neonatal period
• Severe HS:
• Marked anaemia
• Transfusion dependent survival (iron overload)
• Splenomegaly, skeletal abnormalities
6
• FBC:
• Anaemia (Hb variable)
• MCHC >360 g/L
• Reticulocytes > 5%
• Blood film:
• Spherocytosis +++ (microspherocytes)
• NRBC’s
• Other Tests:
• Bilirubin increased
• Osmotic fragility increased
• DAT negative
• Decreased haptoglobins
Rodak, page 368 7
Rodak, page 370 8

Osmotic Fragility
9
Hereditary Elliptocytosis
• Heterogenous disorder:
• Autosomal dominant
• ~1 in 3000 persons affected (many undiagnosed due to mild/no symptoms)
• Genetically encoded mutations of cytoskeleton proteins:
• Weakens stability of membrane
• Gene mutations (46 mutations described):
• SPTA1: α-spectrin (65%): affect dimer-dimer association
• SPTB: β-spectrin (30%): affect dimer-dimer association
• EPB41: protein 4.1 (5%): weakened spectrin–actin–protein 4.1 junction complexes
• Characterised by elliptocytosis on peripheral blood film
10
Hereditary Elliptocytosis
• FBC:
• Anaemia (Hb variable)
• Reticulocytes > 5%
• Blood film:
• Elliptocytosis +++
• Other Tests:
• Osmotic fragility increased
• DAT negative
• (Sensitivity to heat)
11
Hereditary Elliptocytosis: ‘Subtypes’
Subtype Clinical Severity Laboratory Features
Common HE Nil – Mild 30 – 90% Elliptocytes
Hb usually normal
Spherocytic HE Mild – Moderate Elliptocytes
Spherocytes
Polychromasia
Stomatocytic HE Nil Oval RBCs
(SE Asian Ovalocytosis)
(Melanesian Ovalocytosis)
Hb = normal
Hereditary Severe Poikilocytosis +++

Pyropoikilocytosis (Homozygous HE) Fragmentation
Microspherocytes
Elliptocytes
Rodak, page 376 12
Paroxysmal Noctural Haemoglobinuria
• Rare acquired haematopoietic stem cell mutation disorder (< 1 in 100,000)
• Lack of glycosylphosphatidylinositol (GPI) linked proteins
• GPI anchors surface proteins to membrane
• RBCs have abnormal sensitivity to complement (lack CD55 + CD59)
• Haemolysis occurs while sleeping (haemoglobinuria)
• Laboratory Tests:
• DAT (negative) and OF (normal)
• Haemosiderinuria
• Ham’s Test - RBC lysis at low pH
• Flow cytometry - CD55 and CD59 deficient
13
Haemolytic Anaemia
Hereditary Acquired

HS G6PD Thals
HE PK Hb Variants
PNH
Immune
Non-Immune
HELLP Syndrome
Malaria EBV
PCH
LPD’s
14
G6PD Deficiency
• Glucose-6-phosphate dehydrogenase (G6PD):
• Deficiency or functional abnormality
• G6PD required for maintaining glutathione levels
• Glutathione an important buffer to oxidants in RBC
• Sex linked inheritance
• >400 variants affecting 400 million people
• RBCs vulnerable to oxidative stress
• G6PD null (-/-) is lethal
• G6PD deficiency confers resistance to malaria
15
G6PD Deficiency
• WHO Classification for G6PD Deficiency:
• Class I: Severe symptoms (<1% G6PD activity)
• Class II: Mild clinical expression (<10% activity)
• Class III: Mild clinical expression (10-60% activity)
• Class IV: No clinical expression (60-90% activity)
• Class V: No clinical expression (>100% activity)
• Many asymptomatic until exposure to oxidative agent:

• Variable responses in different patient groups
• Acute haemolytic anaemia after exposure to drugs, infections, fava beans (favism)
• Pallor, jaundice, increased bilirubin (2-3 days)
• Heinz bodies
16
G6PD Deficiency
• Blood Film:
• Normal film (not in crisis)
• Normocytic/normochromic anaemia (in crisis)
• Blister cells
• Bite cells
• Reticulocytes increased (up to 30%)
• Fragments (crisis)
• Heinz bodies (in response to certain drugs)
• Enzyme assay (quantitative)
• Fluorescent Test (screening)
17
Detection of G6PD Deficiency
Glucose-6-Phosphate + NADP [No fluorescence]
G6PD (Patient’s sample)
6-Phosphogluconate + NADPH [Fluorescence]
Note: can also use a substrate (dye) for NADPH

e.g. Brilliant cresyl blue [blue to colourless colour change]
18
Pyruvate Kinase Deficiency
• Autosomal recessive
• Results in decreased ATP in RBC
• Variable anaemia (Hb 60 – 120 g/L)
• Severe neonatal haemolysis
• Fully compensated subclinical haemolysis
• Reticulocytes 5 – 20%
• Poikilocytosis +++
• Increased bilirubin
• Decreased haptoglobins
19
Haemolytic Anaemia
Hereditary Acquired

HS G6PD Thals
HE PK Hb Variants
PNH
Immune
Non-Immune
HELLP Syndrome
Malaria EBV
PCH
LPD’s
20
Microangiopathic Haemolytic Anaemia
• Group of potentially life-threatening disorders characterised by:
• RBC fragmentation (mechanical shearing of RBCs in circulation)
• Thrombocytopaenia (consumption due to clotting)
• Disorders:
• Thrombotic Thrombocytopaenic Purpura (TTP)
• Haemolytic Uraemic Syndrome (HUS)
• Disseminated Intravascular Coagulation (DIC)
• HELLP Syndrome (hemolysis, elevated liver enzymes, low platelet count)
21
Microangiopathic Haemolytic Anaemia
• Laboratory results:
• Haemoglobin decreased
• Reticulocytes decreased
• Blood film:
• Fragmentation
• Helmet cells
• Microspherocytes
22
Infections
• Malaria (see later lecture in BMS326)
• Babesiosis:
• Tick-transmitted disease
• Intraerythrocytic protozoa (Babesia microti)
• Clostridial Sepsis: Rodak, page 376
• Clostridium perfringens (anaerobic GPB)

• Produces α-toxin that hydrolyses RBC membrane phospholipids
• Development of spherocytes and susceptibility to osmotic lysis
• Triggers DIC and renal failure
23
Other Causes
• Burns:
• Fragmentation, microspherocytes
• Venoms:
• Snakes, spiders, bees
• Australian snakes can induce DIC (death)
• Drugs and chemicals:
• Oxidative denaturation of haemoglobin
• Dapsone (leprosy); naphthalene flakes
• March (Mechanical) Haemoglobinuria: Burns, Rodak, page 406
• Direct damage to RBCs in circulation
• Military personnel, hikers
24
Summary
• HS and HE are caused by mutations of membrane structural proteins
that result in loss of membrane stability
• HS is characterised by spherocytosis, polychromasia and increased OF
• HE is characterised by elliptocytosis (depending on variant)
• G6PD deficient RBCs are susceptible to oxidative stress
• Patients with PNH lack inhibitors of complement (CD55/59) and are
more susceptible to complement-mediated haemolysis
• MAHA is characterised by thrombocytopaenia and RBC fragmentation
25
References
• Chapter 23, pp 349 - 365.
• Chapter 25, pp 394 – 410.
26
Malaria
Murray Adams
Learning objectives
1. Discuss the transmission and global distribution of malaria
2. Describe the lifecycle and disease process of malaria
3. Discuss prophylaxis and treatment of malaria
4. Explain laboratory procedures used to detect and diagnose malaria
5. Differentiate the five clinically significant species of malaria morphologically
What is Malaria?
• Febrile protozoan infection
• Major haematological features:
• Haemolytic anaemia
• Intra-erythrocytic parasite
• 5 species of Plasmodium infect
humans:
• P. falciparum
• P. malariae
• P. ovale
• P. vivax Tropical Medicine & International Health
Volume 14, Issue 7, pages 802-809, 26 MAY 2009 DOI: 10.1111/j.1365-3156.2009.02287.x
•
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3156.2009.02287.x/full#f1
P. knowlesi (simian/monkey malaria)
Global Impact of Malaria
• Most common cause of haemolytic anaemia
• 2015*
• 214 million new cases of malaria reported
• 10% in South East Asia region
• 2% in Eastern Mediterranean region
• 88% in WHO Africa region
• 438,000 deaths estimated (90% in WHO Africa region)
• 306,000 children <5 years globally (292,000 in Africa)
• 2000 – 2015
• World mortality ↓60% (66% in Africa)
• Children <5 years ↓65% (71% in Africa)
*WHO World malaria report 2015 http://www.who.int/malaria/media/world-malaria-report-2015/en/
Malaria in Australia
• Not endemic to Australia at present
• 400 - 1000 cases reported per year since 1991
• Global travel (‘Airport Malaria’)
• Global warming
• Humanitarian migrants
• 48% P. vivax
• 43% P. falciparum
Transmission
• Bite of the Anopheles mosquito (female)
• Africa: A. gambesi
• Australia: A. farauti
http://saurashtrapestcontrol.com/MosquitoContro.html
• Blood transfusion
• Sharing of needles/needle-stick injury
https://ucrtoday.ucr.edu/36057/anopheles_minimus
Enter salivary glands Enter blood
Sporozoites Sporozoites
Exoerythrocytic Stages
Enter liver cells

Rupture
of Oocyst Merozoites
RBCRBC
Invasion
Invasion
Oocyst Merozoites
Uninfected RBC
RBC
Invasion
RBC Cell Rupture
Ookinete Invasion
Early Trophozoite
Zygote Mature Schizont
Macrogamete
Microgamete Macrogametocyte Mature Trophozoite
Exflagellation Microgametocyte
Mosquito Phase Human Phase

Disease Process: Differs with Species
P.falciparum P.malarie P.ovale P.vivax P.knowlesi
Incubation
7 - 27 23 - 69 16 - 18 13 - 17 5-7
period (days)
Length of life
36 - 48 72 48 48 24
cycle (hours)
RBC invaded Retics & Retics &

All ages Older All ages
(parasite load) young young
Malignant (Benign) Benign Benign

Severity of illness Malignant
tertian quaternary tertian tertian
Clinical Features
• Early symptoms resemble influenza
• Chills, fever, headache, dizziness, myalgia
• Most common symptoms:
• Recurrent fever & sweating
• Abdominal pain
• Altered mental state
• Jaundice, splenomegaly, hepatomegaly (less common)
• Haemolytic anaemia in 25% of cases
• Haemolysis  parasitisation
• Bleeding gums, epistaxis, needle sites (less common)
• DIC
Laboratory Diagnosis
• Patient history:
• Overseas travel, prior infection, fever
• FBC:
• Thrombocytopaenia (< 120 x 109/L)
• Haemolytic anaemia (25% of cases; splenic conditioning of erythrocytes)
• Normocytic anaemia (not always)
• Leukocytosis or neutropaenia (not always)
• Blood Film:
• Demonstration of parasites in blood film
• Screening kits for malaria (RDTs)
• Detection of malarial antigens (P. falciparum V the rest)
• Polymerase chain reaction (PCR)
RTDs for P. falciparum / P. vivax
• Detection of HRP-II • Detection of pLDH
• Parasight F (Becton-Dickinson) • OptiMAL-IT (TCS Biosciences)
• ICT Malaria P.f. (AMRAD Diagnostics)
• Kat Quick Test (Kat Medical)

• Travellers kit
When to Collect Blood Specimens
• There should be parasites in the peripheral blood at all times:
• P. falciparum usually develops with overlapping broods reaching maturity at
different times (asynchronous)
• Seldom develop as a single generation (synchronous)
• With rare exceptions the blood is continually positive
• Re- collections (negative films + strong suspicion):

• Repeat blood films every 12 hours for 24-48 hours
• Three consecutive negative blood films
Laboratory Diagnosis: Microscopy (Blood Films)
• Blood sample
• Venous, capillary or arterial
• Direct from finger-prick or syringe preferable
• More practical from anti-coagulated blood
• Make films as soon as possible after collection
• 3x thick
• 2x thin
• Use x40 or x100 (oil immersion) lenses for examination
Laboratory Diagnosis: Microscopy (Thick Films)
• From finger or EDTA
• Unfixed
• 3% Giemsa, pH 7.2
• Just able to read print underneath
• Too thick:
• Flake off slide
• Blue background
Left: Unstained thick film

Right: Thick film - too much blood
Laboratory Diagnosis: Microscopy (Thick Films)
• Background made up of remnants of
lysed and dehaemoglobinised red cells
• Ideally pale, clean and faintly mottled
• Ranges from light blue to mottled grey-
blue to pale pink
• Look for dark stained chromatin dots
against paler background
Laboratory Diagnosis: Microscopy (Thin Films)
• Regular blood film
• Evenly spread
• No holes
• Smooth tail
• Not too thick
• Fixed in methanol
• 10% Giemsa, pH 7.2
Left: Unstained thin film
Right: Thin film - too much blood
Microscopic Examination of Structural Features
• For each species there is a: • Common features (all species)
• (Merozoite) • Chromatin (red-purple dot)
• Trophozoite • Cytoplasm (blue-purple ring)
• Ring form – early trophozoite • Vacuole (in the middle)
• Amoeboid form – later trophozoite • Pigment (black/brown haemazoin)
• Schizont
• Differential features:
• Gametocyte
• Schuffner’s dots (P. vivax & P. ovale)
• Maurer’s clefts (P. falciparum)
• Most important species is • Ziemann’s stippling
P. falciparum • P. malariae
• P. knowlesi
Effect of Parasite on RBC: Key Points
Cell Infected Cell Size Cell Shape Inclusions
Crescent
P. falciparum Older RBCs Retics Normal Maurer’s clefts
(gametocyte)
Schuffner’s
P.vivax Retics ↑↑ Amoeboid
dots
Ziemann’s
P. malariae Older RBCs N-↓ Normal
stippling
Irregular
P. knowlesi n/a N-↓ Normal dots/stippling
in later forms
Schuffner’s
P. ovale Retics ↑ Comet
dots
Differentiation of Parasite Forms in Blood
P.falciparum P.vivax P.malariae P.ovale P.knowlesi
Ring form
Parasites/cell Numerous Single Single Single Multiple
Shape Multiple Rough; single Compact; Rough; single Delicate
Delicate chromatin dot inverted chromatin dot
chromatin dot
Trophozoite
Size Rarely seen >1/3 of RBC >1/3 of RBC >1/3 of RBC >1/3 of RBC
Shape Amoeboid Bandforms Compact Bandforms
Schizont
Frequency Very rare Common Common Common Common
Configuration Random Random Daisy-head Daisy-head Grape cluster
# Merozoites 8 – 24 12 – 24 8 – 12 8-12 ≤ 16
Gamete Crescent forms Large & round Round-oval Small & round Round
Central Fills RBC Fills ⅔ to all of Fills 1/3–1/2 of Fills ⅔ to all of
chromatin RBC RBC RBC
Differentiation: Plasmodium falciparum
• World wide distribution (2nd most common to P. vivax)
• Invades young and older red cells
• Most pathogenic form:
• High parasitic load
• Massive IV haemolysis & haemoglobinuria (Blackwater fever)
• ARF
• DIC
• Cerebral malaria
• Mainly ring forms and gametocytes in circulation:
• Multiple ring forms per cell
• Hard to treat (drug resistance)
Differentiation: Plasmodium vivax
• Most widespread & most common:
• Not as common in Africa (esp W. Africa)
• Fya- Fyb- confers resistance
• Invades young RBC’s & reticulocytes
• Schizonts (Hypnozites) lie dormant in liver:
• Relapses may occur
• Difficult to eradicate
• Drug resistant strains developing
Differentiation: Plasmodium malariae
• Africa, India, Burma, Malaysia, Indonesia
• Infection usually mild:
• Nephropathy due to deposition of immune
complexes in renal tubules
• Invades older red cells:
• Cells not enlarged
• Squarish ring forms
• ‘Band form’ trophozoites
• ‘Daisy head’ shizonts
Differentiation: Plasmodium ovale
• Tropical Africa, PNG, Philipines
• Invades young red cells and reticulocytes
• Schizonts lie dormant in the liver:
• Relapses
• Hard to eradicate
• Developmental stages similar to P. vivax
• Red cells become enlarged, oval and fringed:
• ‘Comet’ formation
• Schuffner’s dots
Differentiation: Plasmodium knowlesi
• Geographical distribution as for P. malariae
• Infection mostly uncomplicated
• 10% cases are fatal
• Morphological identification extremely difficult:
• Early trophozoites resemble P. falciparum:
• Double chromatin dots
• 2-3 rings per red cell
• More mature forms resemble P. malariae:
• Schizonts with 16 merozoites (cf 12)
Prophylaxis/Treatment: Control the Vector
• Removal of stagnant water
• Indoor residual spraying (IRS)
• DDT
• Vector resistance to DDT
• Larvicide
• Methoprene
• Bacillus sphaericus
• Genetic engineering of fungus
• Metarhizium anisophilae
Prophylaxis/Treatment: Avoid the Vector
• Residents: • Travellers:
• Long-lasting insecticidal nets (LLIN): • Breeding times
• Permathrin • Protective clothing
• 145,000,000 delivered in Sub Saharan Africa • Insect screens
in 2010
• Room sprays
• 50% households have one net (96% usage)
• Bed nets
• Infants, young children, pregnant women
• Insecticide spray
• Avoiding insect ‘habitat’: • DEET
• Jungle clearing – Asia, South America • Eucalyptus oil
• Avoid exposure at dawn and dusk • Insecticide treated bed nets
• Mosquito coils
Prophylaxis/Treatment: Avoid the Vector
https://www.smallchange.ngo/fundraiser/fund-mosquito-nets-to-stop-disease/
Prophylaxis/Treatment: Chemoprophylaxis
• Rapid response to infection required
• Quinine:
• Used > 300 years until 20th century (1930’s)
• Still largely effective against P. falciparum
• Undesirable side effects
• Many other drugs subsequently developed and trialed
• Issues:
• Cost – malaria is endemic in poorer parts of world
• Drug resistance
Prophylaxis/Treatment: Chemoprophylaxis
1. Suppressive 2. Causal
• Erythrocytic phase • Ex-erythrocytic phase (liver)
• (Chloroqine) • Atovaquone/proguanil
• Mefloquine • Malarone
• Doxycyclin • Primaquine
• Hypnozoites
• ACT: Artemisinin-based Combination Therapy

• Anti-malarial drugs will clear the blood cycle of the parasite (not gametocytes)
• Treatment is usually successful given early detection
• Treatment for P. ovale, P. malariae & P. knowlesi is the same for P. vivax
• P. knowlesi responds very well to treatment
Prophylaxis/Treatment: Vaccines
• Attack particular lifecycle stages:
• Sporozoite/exerythrocytic vaccines
• Blood-stage vaccines
• Subunit proteins vs whole parasite:
• Circumsporozoite protein (CSP) of P. falciparum
• RTS,S the only one to reduce infections (2006)
• Attenuated whole parasites at low dosage
• Stimulate T cells rather than destroying them
• >40 vaccine projects at clinical trial stage
• 1 vaccine approved by European regulators
Summary
• Malaria is a protozoan infection (vector: Anopheles mosquito):
• Causes haemolytic anaemia
• Associated with significant morbidity and mortality (particularly in Africa)
• 5 species of Plasmodium infect humans:
• Variations in lifecycle & pathogenesis
• Increasing emergence of drug resistant strains
• Clinical signs include chills, fever, jaundice, hepatosplenomegaly
• Laboratory diagnosis is based on FBC, blood film and rapid detection test
• Demonstration of intra-erythrocytic parasite is diagnostic for infection
• There are preventative measures to avoid and treat infection, but a
successful vaccine remains the holy grail….
References
• Jane Pittaway, University of Tasmania.
• Makhija et al. The utility of serial blood film testing for the diagnosis
of malaria. Pathology 47, 68-70. 2015.
• Chapter 25, pp 399 – 403.
Acute Leukaemia
Murray Adams
Learning Objectives
• Compare and contrast the morphological features of myeloblasts and
lymphoblasts
• Describe the major features of the WHO Classification for acute leukamia
• Describe the major clinical features of acute leukaemia and link these with
laboratory results
• Summarise the laboratory tests involved and typical results of these in the
investigation of acute myeloid and acute lymphoid leukaemias
2
Overview
• Acute Leukaemia:
• Acute Myeloblastic Leukaemia (AML)
• Acute Lymphoblastic Leukaemia (ALL)
• Classification:
• French American British (FAB) (no longer used clinically)
• World Health Organisation (WHO) (2001 / 2008 / 2016)
• Laboratory Diagnosis:
• Blood film and bone marrow morphology
• (Cytochemistry)
• Immunophenotyping
• Cytogenetics
3
Introduction
• Refer to lectures from BMS218 (Introduction to Leukaemia)
• Acute leukaemia is characterised by the accumulation of abnormal,
malignant WBCs that replaces normal BM:
• Dissemination into tissues occurs if left untreated
• Patients die from complications of cytopaenias and organ infiltration
• Main morphological feature are blast cells in the peripheral blood:
• Inability of these cells to differentiate into normal functional WBCs
Diagnosis (Treatment and Prognosis)

Clinical -> FBC -> Peripheral Blood Film -> Immunophenotyping -> Cytogenetics
4
Introduction Blasts
Leukaemia Immature Precursors
Mature Cells
Acute Chronic
Normal Acute Leukaemia
Leukaemia Leukaemia Bone Marrow Bone Marrow
Acute Acute Chronic Chronic

Myeloblastic Lymphoblastic Myelogenous Lymphocytic
Leukaemia Leukaemia Leukaemia Leukaemia
AML ALL CML CLL https://www.slideshare.net/NCSLS/aml-vs-all

5
Acute Myeloblastic Leukaemia
• Introduction: • Clinical Features:
• Incidence ~ 1 in 10,000 • Acute presentation; often critically unwell
• Highest < 1 year & ~ 40 years • Symptoms of anaemia (tiredness, lethargy)
• Predisposing factors: • Symptoms of thrombocytopaenia (bleeding)
• Radiation • Organomegaly (hepatomegaly, splenomegaly)
• Benzene exposure • Recurrent infections
• Alkylating agents
• Viral symptoms (children)
• Hereditary chromosomal abnormalities
• Existing haematopoietic stem cell injury
• Bone pain
• Most patients idiopathic • Weight loss
• Skin lesions
6
AML: Laboratory Features
• FBC:
• Anaemia +++
• Thrombocytopaenia +++
• Neutropenia +++
• Leukocytosis (often)
• Peripheral blood film:
• Reflects FBC results
• WCC typically elevated with blast cells present: http://slideplayer.com/slide/4494569/ (Aldosari)
• 50% of cases have WCC < 5.0 x 109/L

• May not demonstrate circulating blast cells
• Cytogenetic abnormalities have been demonstrated in ~ 70% of cases
7
FAB Classification (no longer used clinically)
Note morphology of myeloblasts and blasts of the myeloid cell line
Type Description % Bone Marrow Features

M0 Undifferentiated <5 > 90% blasts*, <3% MPO or SBB positive
M1 Myeloblastic without 10 >90% myeloblasts*, < 4% promyelocytes
maturation
M2 Myeloblastic with 40 30-60% myeloid cells mature beyond blast stage
maturation
M3 Promyelocytic 5-8 30-90% promyelocytes
M4 Myelo-Monoblastic 15 > 30% myeloblasts, >20% monoblasts + promonocytes
M5 Monoblastic 10 30-90% monoblasts

M6 Erythroleukaemia 5 50% or more erythroid cells
M7 Megakaryoblastic 5 >10% megakaryoblasts
* of non-erythroid cells
8
Acute Myeloblastic Leukaemia
(AML FAB M0) (AML FAB M1) (AML FAB M2 )
9
Acute Promyeloctic Leukaemia
(AML FAB M3) (AML FAB M3v)

10
Acute Myelomonoblastic Leukaemia
(AML FAB M4) (AML FAB M4Eo)

11
Acute Monoblastic Leukaemia
(AML FAB M5a) (AML FAB M5b)

12
Acute Erythroblastic Leukaemia
Acute Megakaryoblastic Leukaemia
(AML FAB M6) (AML FAB M7)

13
World Health Organisation (WHO) Classification
• WHO Classification System for AML combines:
• Existing features of the FAB Classification System
• Cytogenetic classification
• Molecular characterisation
• Diagnosis of AML using WHO Classification helps determine approach

to treatment
• Four categories of AML
14
WHO Classification
• AML with Recurrent Abnormal Karyotypes:
• AML with t(8;21)(q33;q22)
• AML with inv(16)(p13;q22) or t(16;16)(p13;q22)
• AML with t(15;17)(q22;q12)
• Others
• AML with Dysplasia:
• AML occurring after a diagnosis of a myeloproliferative or myelodysplastic
disease
• AML as a Result of Previous Therapy-Related Myelodysplasias:
• AML with history of alkylating agent exposure
• AML with topoisomerase exposure
• AML with other exposures such as radiation 15
WHO Classification
• AML Not Otherwise Categorised:
• AML minimally differentiated
• AML without maturation
• AML with maturation
Previous ‘FAB’
• Acute myelomonocytic leukaemia Classification
• Acute monoblastic/monocytic leukaemia
• Acute erythroid leukaemia
• Acute megakaryocytic leukaemia
• Acute basophilic leukaemia
16
17
18
Acute Lymphoblastic Leukaemia
• Introduction:
• Commonest malignancy of childhood
• Peak incidence; 2 – 10 years
• Incidence 1 in 70,000 in adults
• Aetiology same as for AML
• Clinical features (similar to AML):
• > involvement of CNS than AML (headache, vomiting, cranial nerve palsies)
• Testicular and ovary involvement
• Lymphadenopathy, splenomegaly & hepatomegaly
• Bone pain (BM infiltration)
19
ALL: Laboratory Features
• FBC:
• WCC variable (only 20% WCC > 50 x 109/L)
• Anaemia + to +++
• Thrombocytopenia + to +++
• Peripheral blood film:
• Reflects FBC results (+ lymphoblasts)
• Bone marrow:
• >20% lymphoblasts
• Hypercellular http://slideplayer.com/slide/4494569/ (Aldosari)
• Further tests:
• Refer to immunophenotyping, cytogenetics (+/- cytochemistry) 20
Acute Lymphoblastic Leukaemia
Type Description %C %A
L1 Small, homogenous blasts, single indistinct nucleoli 85 31

L2 Larger blasts, heterogenous, multiple nucleoli 14 60
L3 Larger blasts, deeply basophilic, numerous vacuoles 1 9
(ALL FAB L1) (ALL FAB L2) (ALL FAB L3) 21

WHO Classification
• Two major groups of ALL recognised by WHO;
• Precursor B cell ALL
• Precursor T cell ALL
Early Precursor
Antigen B Cell Pre-B Cell Pre-B Cell T Cell
CD2/3/5/7 - - - +
CD19 + + + -
CD20 + + - -
CD34 - - +
% of ALL 5 80 15-20
22
Cytogenetics
Abnormality Prognostic significance Association %C %A
Numerical change
High hyperploidy (> 50) Favourable 20-25 4-8
Hyperploidy (47-50) Intermediate
Pseudodiploidy Intermediate
(46 with structural change)
Hypoploidy Poor 1-2 4-5
Structural abnormality
Philadelphia [t(9,22)] Very poor 4 20-30
t(1,19) Poor Pre-B 5 3
t(4.11) Poor 2 5
t(11,14) Intermediate T cell 1 1
t(12,21) 25 1-2
t(8,14) Very Poor L3 2-5 2-5 23
Prognosis
• Childhood ALL:
• 90% complete remission rate
• 60% cure rate
• Adult ALL:
• 68% - 91% complete remission rate
• 25% - 41% cure rate
• Poor prognosis is predicted by:
• Increasing age (and children <1); male sex
• High WCC at presentation
• Certain cytogenetic abnormalities (see table above)
• Poor response to treatment
• L3 (and L2 to some degree)
• T-ALL
24
Cytochemistry
Reaction ALL AML AML M4 AML M5 AML M6 AML M7
Myeloperoxidase - + + - * -
Sudan Black B - + + +/- * -
AS-D - + + - * -
chloroacetate
esterase
α-naphthyl acetate +/- - + + - +
esterase
PAS Varied Varied Varied Varied + +/-
*Positive in myeloblasts; negative in normoblasts
25
Immunophenotyping
B-Lymphoid T-Lymphoid Myeloid
CD10 CD2 CD13
CD19 CD3 CD117
CD20 CD7 CD33
CD22 CD41
CD79a CD42
CD61
26
Summary
• Acute leukaemia is characterised by uncontrolled proliferation of
immature blast cells in the BM
• Acute leukaemia has two classification systems;

• FAB: morphology and cytochemistry (no longer used clinically)
• WHO: morphology, cytochemistry + cytogenetics (continues to evolve)
• Diagnosis of acute leukaemia is dependent on clinical presentation,

FBC, cell morphology, cytogenetics & immunophenotyping
27
References
• McKenzie. Clinical Laboratory Haematology.
• Rodak (4th Ed). Hematology: Clinical Principles and Applications.
Chapters 29 and 36.
• Leukaemia Foundation of Australia
28
Myeloproliferative Disorders 1
Murray Adams
Learning objectives
• Define myeloproliferative disorders, listing the most common diseases
• For each of the most common myeloproliferative disorders:
• Describe the pathophysiology
• Describe the clinical phases and manifestations
• Describe the cell lines involved, peripheral blood and bone marrow findings
• Describe the diagnostic criteria (including molecular testing)
• Discuss the significance of the Philadelphia Chromosome and JAK2 mutation
in the development of myeloproliferative disorders
2
Overview: Myeloproliferative Disorders
• Classification:
• Myeloproliferative/myelodysplastic disorders
• Laboratory diagnosis:
• FBC
• Blood film morphology
• Bone marrow morphology
• Further testing
• Treatment strategies:
• Relate to pathophysiology
3
Neoplastic Disorders of the Bone Marrow
BENIGN MALIGNANT
Myelodysplastic Syndromes
RA
Reactive Leukocytosis
RARS
Leukamoid Reaction
RAEB
RAEBT
MPD/MDS
CMML
Atyp CML Acute Leukaemia
Myeloproliferative Disorders
CML, 1°MF Juvenile CMML
ET, PRV
CNL, CEL
4
• Clonal haemopoietic stem cell diseases with expansion, excessive
production, and over-accumulation of RBCs, granulocytes, platelets
• Four main disorders:
• Chronic Myeloid Leukaemia (CML)
• Polycythemia Rubra Vera (PRV)
• Essential Thrombocythemia (ET)
• Primary Myelofibrosis (PMF)
• Others (WHO Classification), including:
• Chronic Neutrophilic Leukaemia
• Chronic Eosinophilic Leukaemia
• Pathogenic similarities; common clinical & laboratory features
5
Chronic Myeloid Leukaemia
• Also: chronic myelogenous leukaemia
• CML is a myeloproliferative disorder characterised by a proliferation
of myeloid cells without loss of their capacity to differentiate
• If untreated develops into acute leukaemia
• 15 – 20 % of adult leukaemias (~3% children)
• Median age at diagnosis = 55 years
• M:F ratio ~ 1.5:1
• Associated with:
• Radiation, drugs and chemicals
• Philadelphia chromosome
6
Philadelphia (Ph’) Chromosome
• > 95% of CML is due to the translocation of Chr 22
Chr 22
genetic material from chromosome 22 to 9
• Some ALL patients also have Ph’ Chromosome:
• 20% of adults
• 5% of children
• Molecular analysis may detect Ph’ Chr. at any
stage of CML (prior to symptoms)
• Cause of translocation unknown:
• Ionising radiation? Chr 9
Chr 9
7
Philadelphia (Ph’) Chromosome
• BCR1 gene (chromosome 22)
• ABL proto-oncogene (chromosome 9)
• BCR-ABL1 chimeric gene:
• Three chimeric proteins:
• 210 kDa BCR/ABL fusion protein (p210)
• p190
• p230
• Expresses ↑ TK activity
• ↑ Cell division
8
• Chronic stable phase:
• May last for years (3 - 5)
• Weakness, malaise, abdominal discomfort, infections
• Splenomegaly (other organs) and bleeding complications
• Accelerated phase:
• Increasing blast numbers
• Blast crisis (transformation into acute leukaemia):
• Poor response to therapy
• Remission difficult to achieve
• Survival < 6 months
9
• FBC:
• Leukocytosis (WCC: 50 - 300 x 109/L)
• Haematology analysers flag “IG”; left shift (+++)
• Anaemia and normal or elevated platelet count
• Blood Film:
• Features reflect expansion of granulocyte pool
• Left shift; metamyelocytes, myelocytes (+++)
• Occasional promyelocytes and blasts (<5%)
• Eosinophilia and basophilia
• Mild-moderate normocytic/normochromic anaemia
• Blast crisis associated with thrombocytopaenia
10
Rodak p565
11
• Bone Marrow:
• Hypercellular
• Increased M:E ratio (up to 50:1)
• < 10% blasts (chronic phase)
• Marked proliferation of
granulocytic elements
• Increased megakaryocytes
(often smaller in size)
12
Treatment
• Early treatment regimes CML aimed to:
• Reduce WCC and regain normal bone marrow function
• Reduce splenomegaly and other symptoms
• Options:
• Chemotherapy:
• Alkylating agents: myelosuppression
• For example: busulphan (Inhibits DNA synthesis)
• Others:
• BMT / SCT: potentially curative (patients < 55 years)
• α-interferon: stimulates cell-mediated anti-tumour response
• Good response to treatment during chronic phase
• Poor response during accelerated and blast crises
13
Treatment: Tyrosine Kinase Inhibitors (Gleevec)
14
Tyrosine Kinase Inhibitors
• Synthetic proteins that bind the
abnormal BCR/ABL protein:
• Blocks constitutive tyrosine kinase
activity (binds ATP binding site)
• Reduces signal transduction activation
• Example:
• Imatinimb mesylate (Gleevec)
• Resistance (other mutations)
• 2nd, 3rd generation inhibitors
Rodak p568 15
Polycythemia
• Polycythemia Rubra Vera is a myeloproliferative disorder characterised
by varying degrees of erythroid, myeloid, megakaryocytic proliferation
• Predominantly increased RBC production
• Primary:
• Polycythemia Rubra Vera (PRV)
• Secondary:
• Physiological or inappropriate EPO production
• Most patients with PRV possess a mutation in the JAK2 gene:
• Probable that other mutations are also implicated
16
JAK2
• Janus kinase 2 (JAK2)
• Valine to phenylanaline (V617F) mutation (2005)
• Acquired, clonal mutation
• Can activate erythropoietic signal transduction pathway in absence of erythropoietin
• Present in:
• ~ 95% of patients with PRV
• ~ 50% patients with Essential Thrombocytosis (see next lecture)
• ~ 50% patients with Primary Myelofibrosis (see next lecture)
• Cells more susceptible to growth factors:
• Erythropoietin
• Thrombopoietin
• JAK2 inhibitors in development
Polycythemia Rubra Vera
• Clinical features:
• Slow insidious onset followed by dramatic complications
• Thrombosis, circulatory disturbances, hypertension
• Proliferative phase:
• Proliferation of RBC mass
• “Spent” phase:
• Progressive splenomegaly or hypersplenism
• Pancytopaenia
• Transformation:
• Acute leukaemia (5%)
• Myelofibrosis (5%)
18
• FBC:
• Hb > 185 g/L (males) and > 165 g/L (females)
• PCV increased
• Leukocytosis (12.0 – 25.0 x 109/L)
• Thrombocytosis (450 – 800 x 109/L)
• NRBCs occ.
19
• Blood Film:
• Normocytic normochromic RBCs
• “Thick” blood
• Poikilocytosis if transformation to
myelofibrosis
20
• Bone Marrow:
• Hypercellular
• Increased normoblasts
• Increased granulocytes
• Increased megakaryocytes
• Increased reticulin
21
• Treatment:
• Correct hyperviscosity - regular phlebotomy
• Myelosuppression:
• Complications:
• Thrombosis
• Acute leukaemia
• Haemorrhage
• Iron deficiency
22
WHO Criteria for PRV (Blood 114:937-951, 2009)
• Major criteria:
• Hemoglobin > 185 g/L in men, > 165 g/L in women or other evidence of
increased red blood cell volume
• Presence of JAK2 V617F or other functionally similar mutation such as JAK2
exon 12 mutation
• Minor criteria:
• BM biopsy with hypercellularity for age with trilineage growth (panmyelosis)
with prominent erythroid, granulocytic, and megakaryocytic proliferation
• Serum erythropoietin level below the reference range for normal
• Endogenous erythroid colony formation in vitro
Diagnosis: 2 Major + 1 minor OR 1 major + 2 minor

23
Secondary Polycythemia
• Erythrocytosis due to enhanced RBC production:
• Not a myeloproliferative disorder (by WHO criteria)
• Physiological:
• Contracted plasma volume (dehydration)
• Conditions causing reduced arterial oxygen:
• Prolonged high altitude
• Lung disease
• Cardiovascular disease
• Inappropriate EPO production:
• Some tumours and endocrine diseases
24
Summary
• CML is characterised by:
• Peripheral blood leukocytosis with left shift, eosinophilia and basophilia
• Bone marrow hypercellularity
• Philadelphia chromosome
• PRV is characterised by:
• Varying degrees of erythroid, myeloid, megakaryocytic proliferation
• JAKS V617F mutation (in the majority of patients)
25
References
• Chapter 33, pp 561 - 575.
• Vardiman JW et al. 2009. The 2008 revision of the World Health
Organization (WHO) classification of myeloid neoplasms and acute
leukemia: Rationale and important changes. Blood 114:937-951.
26
Myeloproliferative Disorders 2
Murray Adams
Learning objectives
• Define myeloproliferative disorders, listing the most common diseases
• For each of the most common myeloproliferative disorders:
• Describe the pathophysiology
• Describe the clinical phases and manifestations
• Describe the cell lines involved, peripheral blood and bone marrow findings
• Describe the diagnostic criteria (including molecular testing)
• Discuss the significance of the JAK2 mutation in the development of
myeloproliferative disorders
2
Overview: Myeloproliferative Disorders
• Classification:
• Myeloproliferative/myelodysplastic disorders
• Laboratory diagnosis:
• FBC
• Blood film morphology
• Bone marrow morphology
• Further testing
• Treatment strategies:
• Relate to pathophysiology
3
BENIGN MALIGNANT
RA
RARS
Leukamoid Reaction
RAEB
RAEBT
MPD/MDS
CMML
ET, PRV
CNL, CEL
4
Thrombocytosis
• Primary Thrombocytosis:
• Essential Thrombocythaemia (ET)
• Platelets > 600 x 109/L (no known cause)
• Secondary Thrombocytosis:
• Reactive thrombocytosis present in a range of conditions
• Chronic inflammatory disease
• Exercise
• Splenectomy
5
Essential Thrombocythaemia
• ET is a clonal myeloproliferative disorder with:
• Increased megakaryopoiesis
• Increased thrombocytosis:
• Sustained thrombocytosis > 450 x 109/L
• Usually >600 x 109/L
• Higher incidence > 50 years of age
• Must be differentiated from secondary thrombocytosis
• Often incidental finding
• JAK2 V617F ~ 50% of patients
• Other mutations identified (TET2, ASXL1, LNK, MPL genes)
6
• Exclusion criteria:
• No evidence of PRV
• No evidence of CML
• No Philadelphia chromosome or BCR/ABL gene
• No evidence of chronic idiopathic myelofibrosis
• No evidence of myelodysplastic syndrome
• No evidence of secondary (reactive) thrombocytosis
7
• Clinical Features:
• Thromboembolic complications:
• Erythromelalgia
• TIAs (transient ischemic attacks)
• Seizures
• MI
• Bleeding from mucous membranes in GIT / URT (abnormal platelet function)
• Splenomegaly (~50% of patients)
8
• Treatment:
• Prevention of thrombosis (low dose aspirin)
• BM suppression of megakaryocyte production (alkylating agents)
• Cytoreduction; interferon-α (younger), busulfan (older)
• Prognosis:
• Relatively long survival rates
• Median survival > 10 years from diagnosis
• Transformation to acute leukaemia or myelofibrosis
9
• FBC:
• Thrombocytosis (often > 600 x 109/L)
• Mild decreases in Hb and PCV
• Mild increases in WCC and neutrophils
• Blood and bone marrow films:
• Platelet anisocytosis (giant platelets)
• Platelet aggregates (tail of blood film)
• Megakaryocytic hyperplasia, hypersegmentation and clustering
• Abnormal platelet function (decreased)
10
11
Essential Thrombocythaemia: BM Biopsy
12
Primary Myelofibrosis
• A clonal myeloproliferative disorder with:
• Splenomegaly
• Ineffective haemopoiesis
• Areas of bone marrow hypercellularity & fibrosis
• Megakaryocytic (enlarged) hyperplasia
• Dacrocytes
• Previously known as:
• Chronic idiopathic myelofibrosis
• Myelofibrosis with myeloid metaplasia (MMM)
• “Myelofibrosis”
13
• Patients:
• > 60 years
• May be asymptomatic
• Fatigue, shortness of breath
• Splenomegaly, hepatomegaly (extramedullary haemopoiesis)
• Increases in collagen types I, III and IV
• Increased circulating CD34+ cells
• JAK2 V617F mutation in ~50% of patients
• Other mutations (MPL, CBL, TET2, ASKL1 genes)
14
• FBC:
• Hb normal or low
• RDW increased
• WCC and platelets variable
• Blood Film:
• Anisopoikilocytosis +++ (RBCs and platelets)
• Polychromasia ++
• NRBCs +
• Dacrocytes +++
• Micromegakaryocytes occ.
15
• Bone Marrow:
• Hypercellular
• Dysgranulopoiesis
• Dysmegakaryopoiesis:
• Increased numbers
• Hypersegmentation
• Often “dry-tap”
• Sheets of fibrosis throughout BM
16
Rodak p579
17
• A wide range of treatment options (usually supportive/palliative):
• Transfusions
• Splenectomy
• Interferon-α
• Prognosis:
• Median survival ~ 5 years from diagnosis
• ↑ megakaryocytes & blasts lead to BM failure
• Poor prognosis: anaemia and thrombocytopaenia
• Mortality: infections and haemorrhage
• 10% transform into acute leukaemia
18
Chronic Neutrophilic Leukaemia
• A myeloproliferative disease characterised by:
• Sustained neutrophilia
• BM hypercellularity:
• Neutrophilic granulocyte proliferation
• Hepatosplenomegaly
• Rare (~ 150 cases reported)
• Affects elderly individuals (M:F ratio 1:1)
• Aetiology unknown
19
Chronic Neutrophilic Leukaemia
• Neutrophilia > 25 x 109/L
• Left shift + (< 10%)
• Bone marrow hypercellularity
• M:E ratio increased
• Chromosomal abnormalities:
• Not reported in ~90% of patients
• Philadelphia chromosome negative
• Survival 6 months to 20 years
20
Chronic Eosinophilic Leukaemia
• Chronic eosinophilic leukaemia (CEL)
• Clonal disorder of eosinophilic precursors:
• Results in persistent eosinophilia
• Eosinophilia > 1.5 x 109/L
• Philadelphia chromosome negative
• < 20% blasts in blood or bone marrow
• CEL if clonal involvement or increased blasts
• Idiopathic hypereosinophilic syndrome (HES):
• If no clonal abnormality
21
Chronic Eosinophilic Leukaemia
• CEL diagnosis based on exclusion of:
• Reactive eosinophilia
• Neoplastic disorders with 2°eosinophilia
• Other neoplastic disorders with eosinophil involvement
• T cell popn with aberrant phenotype + cytokine production
• All of the above conditions + chromosomal abnormality of myeloid cells or
blasts >2% in blood or 5% < blasts < 19% in BM
• HES diagnosis based on exclusion of:
• Any demonstrable disease causing eosinophilia
22
BENIGN MALIGNANT
RA
RARS
Leukamoid Reaction
RAEB
RAEBT
MPD/MDS
CMML
ET, PRV
CNL, CEL
23
Summary
• ET is characterised by:
• Peripheral blood thrombocytosis
• Increased megakaryopoiesis in the bone marrow
• Primary myelofibrosis is characterised by:
• Splenomegaly, ineffective haemopoiesis & areas of BM fibrosis
• Megakaryocytic (enlarged) hyperplasia
• Dacrocytes in the peripheral blood
• JAKS V617F mutation (in ~50% of patients)
24
References
• Chapter 33, pp 575 - 583.
• Vardiman JW et al. 2009. The 2008 revision of the World Health
Organization (WHO) classification of myeloid neoplasms and acute
leukemia: Rationale and important changes. Blood 114:937-951.
25
Lymphoproliferative Disorders 1
Murray Adams
Learning objectives
1. Describe the approach to the diagnosis of mature lymphoid neoplasms as
outlined by the World Health Organization classification
2. Describe the epidemiology, clinical presentation, pathophysiology,
peripheral blood, bone marrow findings, and diagnostic test results of
common lymphoproliferative disorders, especially:
• Chronic lymphocytic leukaemia
• Hairy cell leukaemia
• Multiple myeloma
• Hodgkin lymphoma
3. Interpret diagnostic test results to identify lymphoproliferative disorders
2
Lymphoproliferative Disorders (Simple Outline) Hodgkin Lymphoma
Lymphocytic Leukaemias Plasma Cell Neoplasms Lymphomas
Multiple Myeloma
Waldenstroms
Macroglobulinaemia
MGUS
Plasma cell leukaemia Non-Hodgkin Lymphoma
Chronic Lymphocytic Leukaemia (CLL) Small Lymphocytic Lymphoma

Prolymphocytic Leukaemia (PCL) Follicular Small Cleaved Cell Lymphoma
Hairy Cell Leukaemia (HCL) Diffuse Small Cleaved Cell Lymphoma
Diffuse Large Cell Lymphoma
Immunoblastic Lymphoma
Lymphoblastic Lymphoma
3
Introduction
• The lymphoproliferative disorders are a large group of neoplastic
lesions of the lymphoid system:
• Prolymphocytic leukaemia
• Plasma cell neoplasms
• Hodgkin’s lymphoma
• Non-Hodgkin’s lymphoma
• Variable clinical presentation / organ involvement
• WHO Classification of Mature Lymphoid Neoplasms:
• Third Edition 2008
• Updated 2016
4
5
Chronic Lymphocytic Leukaemia
• Most common leukaemia of western world
• 30% of all leukaemias
• 2.7/100,000 in US (50/100,000 age > 70 years)
• 90% of patients > 50 years of age
• Rare in Asian populations
• M:F ratio 2:1
• Slow growing tumour of lymphocytes (>98% are B-cell lineage)
• No known environmental risk factors
• Also: small lymphocytic lymphoma (‘one entity, different clinically’)
6
Chronic Lymphocytic Leukaemia: Symptoms
• Many patients are asymptomatic at diagnosis
• Anaemia, lymphadenopathy and infections
• Splenomegaly
• Weight loss
• Progression:
• Severe immunodeficiency
• Autoimmune phenomena
• Accumulation of lymphocytes leads to BM failure
7
Chronic Lymphocytic Leukaemia: FBC + Film
• FBC:
• Lymphocytosis > 5.0 x 109/L (persistent)
• Leukocytosis may reach 500 x 109/L
• Anaemia (later in course of disease)
• Thrombocytopaenia (later)
• Blood Film:
• Lymphocytosis:
• Small, mature appearing lymphocytes
• Small amount of cytoplasm + course chromatin
• “Smudge” cells
http://rebeccanelson.com/leukemia/cll.jpg
• ~10 – 25% with AIHA
8
Chronic Lymphocytic Leukaemia: Bone Marrow
• Bone Marrow:
• Hypercellular
• “Sheets” or layers of cells
• Suppression of normal haemopoiesis
• M:E ratio increased
• >30% lymphoid cells
• Immunophenotyping:
• B-cell: CD5, CD19, CD20, CD22 positive
• T-cell: CD2, CD3, CD5, CD6 positive
Maslak, P. ASH Image Bank 2004;2004:101114
9
Chronic Lymphocytic Leukaemia: Other Tests
• ~50% produce monoclonal Ig (paraprotein)
• ~25% hypogammaglobulinaemia
• Patients susceptible to infections
• DAT positive with AIHA
• Immunoglobulin heavy chain (IGVH) mutation ass. with variable prognosis:
• Mutated = indolent disease; Not mutated = aggressive disease
• Chromosomal abnormalities:
• Trisomy 12
• Deletions; del13q14.3, del11q22-23, del17p13
• Lymph node biopsy:
• Infiltration of small well differentiated lymphoid cells
10
RAI Staging System Binet Classification
Stage Presentation Stage Presentation
0 Lymphocytosis in blood and bone marrow A Hb > 100 g/L

Platelets > 100 x 109/L
I Lymphocytosis and enlarged lymph nodes
< 3 lymph node areas enlarged
II Lymphocytosis with organomegaly
B Hb > 100 g/L
III Lymphocytosis and anaemia Platelets > 100 x 109/L
(Hb < 110 g/L) > 3 lymph node areas enlarged
IV Lymphocytosis and thrombocytopaenia
C Hb < 100 g/L or platelets < 100 x 109/L
(< 100 x 109/L)
11
Chronic Lymphocytic Leukaemia: Treatment
• If asymptomatic: no treatment (e.g. Stage A Binet)
• Indications for treatment:
• BM failure, lymphadenopathy, hypersplenism, AIHA
• Options for treatment:
• Chemotherapy, radiation, splenectomy, BM transplantation, intravenous Ig
• Apheresis (to reduce WCC)
• May transform into:
• Prolymphocytic leukaemia (less responsive to treatment)
• Large cell lymphoma (Richter Syndrome; rapidly lethal)
• Acute leukaemia (rare < 1% of CLL)
12
Prolymphocytic Leukaemia
• Rare variant of CLL
• Median age of presentation ~ 70 years
• Characterised by:
• High incidence in elderly males
• Massive splenomegaly
• No lymphadenopathy
• Poor prognosis
• Median survival ~ 3 years (B-cell PLL; TP53 tumour suppressor gene); ~ 1 year (T-cell PLL)
• Treatment using low dose or combination therapy
13
Prolymphocytic Leukaemia
• Lymphocytosis > 100 x 109/L
• Large lymphoid cells:
• >55% of circulating lymphoid cells are
prolymphocytes
• Nucleolus is distinct / prominent
• Abundant basophilic cytoplasm
• Can be B- or T-cell PLL:
• CD5 (some), CD19, CD20, CD22 (B-cell)
• CD5, CD2, CD3 (T-cell)
• Strong IgM expression Maslak, P. ASH Image Bank 2004;2004:101116
• Bone marrow infiltration variable

14
Hairy Cell Leukaemia
• Rare malignant disorder (< 2% of leukaemias):
• Middle aged persons (median = 50 years); males > females
• Insidious onset: weakness + lethargy + splenomegaly
• FBC: Pancytopaenia (WCC: 2 – 3 x 109/L)
• Hairy cells:
• Predominate in BM & spleen (red pulp)
• Some found in peripheral blood
• Bone Marrow:
• “Dry tap” bone marrow aspirates
• Infiltration of hairy cells
• Immunophenotyping:
• CD19, CD20, CD22 positive
• CD25 + CD11c expression Maslak, P. ASH Image Bank 2001;2001:100209
15
Summary
• Chronic Lymphocytic Leukaemia (CLL)
• Prolymphocytic Leukaemia
• Hairy Cell Leukaemia (HCL)
• Chronic Lymphocytic Leukaemia:
• Characterised by proliferation of lymphocytes in the bone marrow and
peripheral blood (exclude other causes of lymphocytosis)
• The vast majority of cases are B-cell CLL
• Diagnosis based on clinical presentation, FBC, blood film (bone marrow)
16
References
• Chapter 36, pp 619 – 640.
17
Lymphoproliferative Disorders 2
Murray Adams
Learning objectives
1. Describe the approach to the diagnosis of mature lymphoid neoplasms as
outlined by the World Health Organization classification
2. Describe the epidemiology, clinical presentation, pathophysiology,
peripheral blood, bone marrow findings, and diagnostic test results of
common lymphoproliferative disorders, especially:
• Multiple myeloma
• Hodgkin lymphoma
3. Interpret diagnostic test results to identify lymphoproliferative disorders
2
Lymphoproliferative Disorders (Simple Outline) Hodgkin Lymphoma
Lymphocytic Leukaemias Plasma Cell Neoplasms Lymphomas
Multiple Myeloma
Waldenstroms
Macroglobulinaemia
MGUS
Plasma cell leukaemia Non-Hodgkin Lymphoma
Chronic Lymphocytic Leukaemia (CLL) Small Lymphocytic Lymphoma

Prolymphocytic Leukaemia (PCL) Follicular Small Cleaved Cell Lymphoma
Hairy Cell Leukaemia (HCL) Diffuse Small Cleaved Cell Lymphoma
Diffuse Large Cell Lymphoma
Immunoblastic Lymphoma
Lymphoblastic Lymphoma
3
Introduction
• Prolymphocytic leukaemia
• Plasma cell neoplasms
• Hodgkin’s lymphoma
• Non-Hodgkin’s lymphoma
Variable clinical presentation / organ involvement 4

Plasma Cell Neoplasms
• B cell lymphoproliferative disorders characterised by:
• Clonal expansion of plasma cells
• Excess production of Ig’s or their chain components
• Fully differentiated B cells:

• Multiple Myeloma (MM)
• Waldenstroms Macroglobulinaemia
• Monoclonal Gammopathy of Uncertain Significance (MGUS)
• Plasma cell leukaemia (PCL)
5
Multiple Myeloma
• Most common plasma cell neoplasm
• Uncontrolled proliferation of malignant plasma cells (BM) leading to
bone marrow failure
• Tumour cells synthesise abnormal amounts of monoclonal
immunoglobulin:
• IgG, IgA, IgM, IgD, IgE, or
• kappa or lambda light chains
• Rarely no detectable secretion of Ig (serum/urine)
• ~3 per 100,000 persons affected (↑ with age)
• M:F ratio 1:1
6
Multiple Myeloma: Clinical Features
• Bone pain:
• Bone marrow infiltration by plasma cells
• Bone destruction by direct infiltration (fractures)
• Symptoms of anaemia due to BM infiltration
• Bleeding, bruising
• Infections
• Hypercalcaemia (↑Ca2+; 2° to bone lesions)
• Renal disease (renal tubular damage by Ig)
• Hyperviscosity syndrome (due to ↑Ig) https://minajamyelooma.fi/en/multiple-myeloma/symptoms/
7
Multiple Myeloma: FBC + Film
• FBC:
• Pancytopaenia
• Hb ~ 100 g/L, WCC ~ 3 – 4 x 109/L,
thrombocytopaenia
• Blood Film:
• Normochromic normocytic anaemia
• MCV 95 – 100 fL
• Small numbers of plasma cells common
• If plasma cells > 2.0 x 109/L or 20% of diff:
plasma cell leukaemia
• Rouleaux (increased ESR; > 100 mm/hr) Maslak, P. ASH Image Bank 2005;2005:101392
• “Blue” background (↑ protein) Copyright ©2005 American Society of Hematology. Copyright restrictions may apply.
8
Multiple Myeloma: Bone Marrow
• > 10% plasma (myeloma) cells:
• Highly basophilic cytoplasm
• Eccentric nuclei (can be multi-nucleated)
• “Flaming” (red) cytoplasm: classically IgA
myeloma
• Plasma cell inclusions (Russell bodies in
“Mott” cells)
• Often patchy or focal infiltrate
• Trephine biopsy
Maslak, P. ASH Image Bank 2003;2003:100672
9
IgA Myeloma Plasma Cell Inclusions
Braza, J. ASH Image Bank 2006;2006:6-00026 Lazarchick, J. ASH Image Bank 2004;2004:100984
Copyright ©2006 American Society of Hematology. Copyright restrictions may apply. Copyright ©2004 American Society of Hematology. Copyright restrictions may apply.
10
Multiple Myeloma: Other Lab Tests
• Biochemistry:
• Hypercalcaemia
• Hyperglobulinaemia
• Proteinuria
• Paraprotein (“M” protein) (95% of cases)
• Amount proportional to tumour mass
• Monoclonal band in serum electrophoresis:
• IgG (50%); IgA (25%)
• Bence-Jones protein: IgG light chains (20% of cases)
• Increased β2-microglobulin (marker of MM)
• Chromosomal abnormalities described (not constant)
11
Multiple Myeloma: Treatment
• Chemotherapy
• High dose chemotherapy
• Interferon
• Bone marrow transplant
• Radiotherapy
• Supportive therapy
• Pain relief, transfusions, G-CSF, antibiotics
• Complications
12
Waldenstroms Macroglobulinaemia
• Chronic lymphoproliferative disorder
• Uncommon; elderly males
• Infiltration of bone marrow by:
• Clonal lymphocytes
• Plasmacytoid lymphocytes
• Plasma cells
• Usually secrete IgM kappa paraprotein
• Circulating monoclonal IgM (macroglobulin)
13
Monoclonal Gammopathy of Uncertain Significance
(MGUS)
• Monoclonal Ig present in peripheral blood

• Usually IgG paraprotein
• Levels remain stable for years
• Bone marrow plasma cells < 20%
• Increasing incidence with age
• More common than myeloma
• No infiltration of bone marrow
• No lymph node, liver or spleen involvement
• Myeloma develops in 10% of cases
14
Plasma Cell Neoplasms
MM WM MGUS
Plasma cells PB plasma cells (+) Lymphocytes + plasma cells PB plasma cells absent
BM plasma cells (>10%) in PB and BM <20% plasma cells in BM
Immunoglobulin “M” paraprotein (>30 g/L) kappa paraprotein Usually IgG (<30 g/L)
IgG (50%) IgA (25%) IgM BJP absent
Urinary BJP (20%)
Symptoms Bone pain (osteolytic lesions) Visual disturbances None
Anaemia Anaemia
Bleeding/Bruising Bleeding/Bruising
Complications Hypercalcaemia Hyperviscosity No hyperviscosity,

Renal disease No hypercalcaemia or renal hypercalcaemia or renal
Hyperviscosity disease disease
Plasma cell leukaemia Multiple myeloma (10%)
15
Plasma Cell Leukaemia
• Aggressive form of multiple myeloma (~3% of MM progress to PCL)
• Characterised by high levels of plasma cells in peripheral blood:
• MM has few plasma cells in peripheral blood
• Diagnosis: >20% plasma cells in peripheral blood
• Rare:
• 1 in 1,000,000
• Increased incidence in elderly patients
• Symptoms: similar to multiple myeloma
• Treatment: similar to multiple myeloma
• Prognosis: poorer than multiple myeloma
http://imagebank.hematology.org/image/2518/plasma-cell-leukemia--1
16
Lymphomas
• Malignancies arising in:
• Lymph nodes
• Other lymphoid sites
• Hodgkin’s Lymphoma: Rodak, p630
• Characteristic clinical and pathological features
• Also:
• Hodgkin’s disease
• Non Hodgkin’s Lymphoma (NHL):
• All other lymphomas
• Also:
• Non-Hodgkin’s disease or malignant lymphoma
Rodak, p635
17
Clinical Staging for HL and NHL
Stage Extent of Disease
I Disease confined to one lymph node site
II Disease confined to lymphatic tissue in more than one site but on only
one side of the diaphragm
III Disease confined to lymphatic tissue or spleen but on both sides of
diaphragm
IV Bone marrow involvement, liver involvement, and widespread
lymphoma at any other site of extranodal tissue
18
Hodgkin Lymphoma
• Heterogenous neoplastic disorder of unknown aetiology
• Two broad categories:
• Modular lymphocyte-predominant Hodgkin lymphoma
• Classical Hodgkin lymphoma
• Bimodal peak incidence (15-40 & >60 years)
• ~ 1/3 of all malignant lymphoproliferative disorders
• Clinically:
• Enlarged lymph nodes
• Fever, night sweats, weight loss
• Splenomegaly
• High “curability” and low “mortality”
19
Hodgkin Lymphoma
• Histological diagnosis:
• Biopsy of lymph node / extra nodal tissue
• Presence of Reed-Sternberg cell (classical):
• Diagnostic large cell
• 2 or more nuclei with single nucleolus
• ~1% of tumour cells
• They are scarce and fragile!
• Haematological findings:
• Often normal FBC in untreated HD Kadin, M. ASH Image Bank 2002;2002:100484
• Normocytic normochromic anaemia Copyright ©2002 American Society of Hematology. Copyright
restrictions may apply.
• Elevated ESR
• Hypercelluar BM: Reed-Sternberg cells
20
Non-Hodgkin Lymphoma
• Tumours arising from lymphoid cells at different stages of maturation
• Many different types and classification systems:
• Rappaport classification (most clinically useful)
• Working formulation (morphology only)
• Kiel classification (morphology & cellular immunology)
• Unknown aetiology (?familial, ?viral, ?drug-related)
• Clinically:
• Localised or generalised lymphadenopathy
• Primary GIT involvement
21
• Diagnosis of lymphoma is dependent on:
• Lymphoid tissue architecture:
• Follicular (i.e., localised / nodular)
• Diffuse (i.e., spreading)
• Cell morphology (small vs large lymphocytes)
• Cell markers (B-cells vs T-cells)
• Cytogenetic analysis
• e.g., t(8,14) c-myc Burkitt’s lymphoma
• Gene rearrangement Kadin, M. ASH Image Bank 2003;2003:100692
• e.g., Ig heavy & light chain rearrangement in B cell NHL Copyright ©2003 American Society of Hematology.
22
• Haematological Findings:
• May be normal
• Circulating lymphoma cells (small, intermediate or large)
• Anaemia, neutropaenia, thrombocytopaenia
• BM aspirate: lymphoma cells may be present
• Treatment:
• Depends on classification, staging, size and symptoms
• Low dose chemotherapy for good prognosis
• High dose chemotherapy for poor prognosis
• Autologous bone marrow transplantation
Rodak, p636
23
Summary
lesions of the lymphoid system
• CLL is characterised by proliferation of lymphocytes in the bone
marrow and peripheral blood (exclude other causes of lymphocytosis)
• The vast majority of cases are B-cell CLL
• Plasma cell neoplasms are B cell lymphoproliferative disorders
characterised by clonal expansion of plasma cells and excess
production of Ig’s or their chain components
• Lymphomas manifest mainly in the lymph nodes, but variably
progress in extranodal sites, BM or peripheral blood
24
References
• Chapter 36, pp 619 – 640.
• Parsons B. Gundersen Health System Center for Cancer and Blood
Disorders.
25

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1BMS326 1-16 Combined &amp; Compressed

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Introduction to Anaemia

BMS326 Clinical Haematology I

1. Describe and categorise causes of anaemia

RBC usually decreased

Compare laboratory result of parameters to reference intervals

Nuclear Defects Cytoplasmic Defects

Affect all cell lines Abnormal Hb production

e.g. Megaloblastic Anaemia e.g. Iron Deficiency Anaemia

NB Causes of anaemia may be multi-factorial

Rodak 5th Ed. Page 287 16

1. Describe and categorise causes of normocytic anaemia

Biopsies from patients with Bone Marrow Aspirate

• Bone marrow biopsy demonstrates infiltrating cells

• Decreased haemoglobin synthesis may be due to:

• The 2 main variants of classical thalassaemia are:

Hb Portland Hb Gower I Hb Gower II HbF HbA2 HbA Hb Barts HbH

• Genotype permutations result in 4 catergories of clinical manifestion:

“Slow” Hbs “Fast” Hbs

Cathode (-) Anode (+)

Sickle Cell Trait (AS)

• Two forms of disease:

RBC with HbS (inflexible)

As more RBCs with HbS give up oxygen

Lazarchick, J. ASH Image Bank 2005;2005:101384 Rodak. p440

Schrier, S. ASH Image Bank 2001;2001:100208

• Ferric iron is reduced to ferrous iron, bound to HMW chelators e.g.

• Ferrous iron is more stable and easily absorbed

TIBC ↓TIBC ↑TIBC

Blood Vessel Fe3+

Fe2+ Fe2+ Fe3+ Fe2+

Mucosal Fe3+ Ferritin

• RBC life span 120 days

• 85% of iron is recycled to plasma

• Transferrin delivers iron back to

Serum iron decreased

Plasma Ferritin (µg/L)

Plasma Iron (µg/dL)

Transferrin Saturation (%)

Sideroblasts (%) 40 - 60 40 - 60 40 - 60 40 - 60 40-60 <10 <10

RBC Protoporphyrin (µg/dL RBC) 30 30 30 30 30 100 200

Erythrocytes Normal Normal Normal Normal Normal Normal Microcytic

• An arbitrary classification of patients with:

• Iron accumulates inside the cristae of mitochondria:

Rodak, page 305 8

HFE-Associated HFE2-Related HAMP-Related TFR2-Related Ferroportin-Related

Affected gene HFE HFE2 (HJV) HAMP TFR2 SLC40A1 FTH1

Rodak, page 307, Table 20.3 13

2. HFE protein interacts with

Transferrin HFE β2-microglobulin

1. Normal HFE protein binds β–microglobulin intracellularly

2. HFE protein doesn’t interact with

1. Abnormal HFE protein doesn’t bind

Serum Iron reduced reduced normal raised

TIBC raised reduced normal normal

Serum ferritin reduced normal normal raised

• 76 year old female Hb (g/L) 94 (115 – 155 g/L)

• History: Fatigue 2/12 WCC (x109L) 5.2 (4.0 – 11.0 x 109/L)

• Doctor requested: MCV (fL) 79 (80 – 98 fL)

RCC (x1012/L) 3.12 (3.50 – 5.50 x 1012/L)

Hb (g/L) 87 (115 – 155 g/L)

WCC (x109L) 8.8 (4.0 – 11.0 x 109/L)

MCV (fL) 71 (80 – 98 fL)

Platelets (x109/L) 358 (150 – 400 x 109/L)

1BMS326 1-16 Combined & Compressed

1BMS326 1-16 Combined & Compressed