Dunedin Basic Medical Sciences Course 
 CELLULAR HAEMATOLOGY

The Mature Red Cell
Dunedin Basic Medical
Sciences Course CELLULAR HAEMATOLOGY
► Flexible
biconcave disk ~7-8 um diam
► Cytoplasm
▪ Viscous haemoglobin solution 320-350 g/L
▪ Hb packaged in red cells to minimise viscosity of blood
▪ Compare concentration with plasma ~70 g/L
Presented by Prof Ian Morison
FRCPA(Haem)
Slides by Dr Jim Faed
▪ Energy handling
▪ Glycolytic (Embden-Meyerhoff) pathway
▪ Hexose monophosphate shunt
▪ No mitochondria, endoplasmic reticulum or ribosomes in
mature red cells
2
1
Blood – the transport medium
► Normal
Haematopoiesis
blood volume
▪ Various formulae – simplest: mL/kg
95% ranges + 2sd
Red cell volume
►
Plasma volume
► Total blood volume
►
►
men
25-35
40-50
65-85
women
20-30
40-50
60-80
Blood volume (mL/kg) with varying body build
Gender
Obese
Thin
Normal
Muscular
Male
60
65
70
75
Female
55
60
65
70
Neonate
100
3
Normal bone marrow
4
Haemopoietic cell lineages
Mixed myeloid
progenitor stem cell
Pluripotent stem cell
(Haemopoietic stem/progenitor cell)
Lymphoid
stem cell
Erythroid
progenitor
(red cells)
5
Megakaryocyte
progenitor
Granulocyte
(platelets)
progenitor
Monocyte
progenitor
Dendritic
cell
progenitors
(antigenpresenting
cells)
B cell
T cell NK cell
progenitors
6
Hemopoiesis - regulation
Haemopoiesis - regulation
► Control
mechanisms -
▪ Cytokine signals
▪ induce growth and differentiation
▪ produced by peripheral tissues and cells of marrow
▪ Marrow stromal cells
▪ cell surface signals
S Haemopoiesis is an integrated process
▪ Results in self renewal of pluripotent stem cells
▪ Differentiation of some pluripotent stem cells into
specific cell lineages - irreversible
7
8
Regulatory signals in haemopoiesis
Haemopoiesis
Pluripotent stem cell
(Haemopoietic stem cell)
Mixed myeloid
progenitor stem cell
Erythroid
Progenitor
Regulation of cell
pools by
cytokines
Lymphoid&
stem
cell
marrow
stromal
cells
Megakaryocyte
Progenitor
Granulocyte
Progenitor
Blood Red cells
Blood Platelets
Monocyte
progenitor
Blood Neutrophils Monocytes
Basophils
Eosinophils
Dendritic
cell
progenitors
Lymphoid
tissues,
skin, etc
1
2
3
B cell T cell NK cell
Proliferation
progenitors
and
differentiation
of
Thymus
Bone committed cells
Act on stromal cells
▪
IL-1, TNF, others
Act on pluripotent stem
cells
▪
▪
Stem cell factor (SCF)
FLT ligand
▪
IL-3, GM-CSF, G-CSF,
IL-6, TPO
Act on multipotential
progenitor cells
• Act on committed
progenitor cells
▪ G-CSF - neutrophils
▪ M-CSF - monocytes
▪ IL-5
- eosinophils
▪ EPO
- red cells
▪ TPO
- platelets
▪ (plus IL-3 & GM-CSF)
marrow
Lymphoid tissues & marrow
9
Erythroblasts
10
Erythropoiesis - committed cells
► Proerythroblast
1-2d
erythroblast
1-2 days
▪ rapid division, iron uptake
► Polychromatic erythroblast
3-4 days
▪ divides, Hb synthesised, then starts
clearance of RNA, mitochondria, etc,
and buds off nucleus to become a ...
► Reticulocyte
3-4 days
▪ immature red cell: completes clearance of RNA
mitochondria, golgi, etc
▪ migrates into blood after 1-3d
Marrow stages - 8-9 days
► Erythrocyte
~120 days
► Basophilic
Polychromatic erythroblasts & neutrophil
Key issues:
Polychromatic erythroblasts
1. Proliferation
surrounding a macrophage
2. Iron uptake
3. Haemoglobin production
4. and finally, removal of mitochondria, ER, ribosomes, golgi
and loss of nucleus.
11
12
Expansion of cell numbers
proerythroblasts
1-2 days
!
!
3-4 days
!
!
basophilic
erythroblasts
~4 days
reticulocytes
!
polychromatic
erythroblasts
{
mature erythrocytes
RNA and remnants of other cell
organelles in young red cells are
precipitated by new methylene blue
dye, resulting in a blue reticulated
pattern.
Erythropoiesis
haemopoietic stem
cells
1-2 days
Reticulocytes
Manual (historical) retic count
Normal: 1-2% ~ 20-90x109/L
Current (large) haematology analysers
are able to provide accurate reticulocyte
counts and identify the proportion of
young reticulocytes (increased in
haemolysis)
nucleus lost
migrate
into blood
Mature erythrocytes - circulate ~120 days
Red cells produced by an average adult: ~2.3 x 1011/day
13
Obtaining diagnostic bone marrow
specimens
(This film is from a case of haemolytic
anaemia and has a raised reticulocyte
count.)
Automated retic count by
fluorescence
14
Bone marrow biopsy
▪ Aspirates – for cytology
► Morphology
of haemopoietic cells
▪ Recognise abnormal maturation
► Detection
of malignancy (if >5% blast cells)
▪ Cytochemistry – films
▪ Immunological detection of cell surface markers to characterise
acute leukaemias
▪ Trephine biopsy for histology
► Assess
marrow stroma
lymphoid and other malignancies in marrow
► Identify metastatic tumour deposits
► Detect
Patient lying in recovery position
15
Red cell indices: NZ reference ranges
Newborn –
22 d–3 m
cord blood
7-10 y
Female
adult
Male adult
RBC
3.5-6.7
2.8-4.3
3.5-5.5
3.9-5.6
4.5-6.5
Hb (g/L)
137-201
95-130
101-139
115-165
130-180
MCV (fL)
99-117
72-90
76-96
MCH (pg)
30-37
26.33
80-98
27-32
17
Red Cell indices: international data
Adult males
NZ
UK
USA
RBC (x1012/L)
4.5-6.5
4.5-6.5
4.3-5.9
Hb (g/L)
130-180
135-175
135-172
Ht
0.40-0.54
0.40-0.52
0.39-0.49
MCV (fL)
78-98
80-95
76-100
MCH (pg)
27-32
27-34
27-33
18
How to approach the red cell indices
▪ Carbohydrate (10%) - on external surface
▪ Lipid bilayer (40%) - fluid at 37oC
▪ Protein cytoskeleton (50%)
Examples
Evaluate the Red
Cell Indices in this
sequence -
A
B
C
D
4
RBC
4.4
5.0
4.8
3.2
1
Hb
140
140
110
110
5
Ht
0.41
0.43
0.36
0.33
2
MCV
93
85
75
102
3
MCH
31.8
28
22.9
34.4
Red cell membrane
from
Essential Haematology,
AV Hoffbrand, et al. 4e
19
Red Cell Aging
20
Red Cells - transport function
▪ Oxygen uptake in lungs
▪ Changes with age ~450 km circulation
▪ saturation >95% occurs by ~1/3 distance travelled in alveolar
capillaries
▪ Loss of membrane lipid
▪ Loss of Decay acceleration factor (CD55) & CD59
► (Complement inactivation)
▪ Loss of CD47 – inhibits phagocytic clearance
▪ Some enzymes decrease
▪ Oxidative damage to lipids & proteins
▪ Cross-linking of structural components
▪ CO2 transport from tissues
▪ CO2 enters red cells
► rapidly converted to H2CO3 – carbonic anhydrase
► HCO3-, diffuses out, H+ bound by Hb - minimises pH shift
!
▪ Nitric oxide – transport & reserve for BP control
▪ Structural anatomy critical -
▪ End result:
► Increased
rigidity and surface changes
▪ Reduced ability to repair oxidative damage
▪ Leading to phagocytosis by macrophages
► splenic cords, marrow & liver sinusoids
21
Normal haemoglobins
▪ large alveolar area
▪ thin layer for diffusion ► epithelial cell - ground substance - endothelial cell
▪ red cells squeeze through capillaries (<8 um diam.)
22
O2 uptake & release by Haemoglobin
Bohr effect
▪ Tetrapeptide - Mr 68,000
❖ Sigmoid curves – affinity
changes as Hb binds O2
▪ Adult Hb: Hb A: α2β2
▪ 4 haem groups
▪ 4 oxygen (O2) molecules carried
❖ pH lower in tissues – shifts
dissoc curve to right.
▪ HbA2
▪ HbF
!
!
❖ CO2 enters RBC: HCO3released, globin binds H+
▪ Minor haemoglobins in adult:
α2δ2
1.5-3.2%
α 2γ 2
0.5-0.8%
Relaxed
high affinity
Tense
low affinity
23
Mean
oxygen
tension
Mean
oxygen
saturation
Arterial
95 mm Hg
95%
Venous
40 mm Hg
70%
24
Synthesis of Hb: critical nutrients
Iron Metabolism
▪ Iron - an essential nutrient, but two key issues:
► Iron
►For
► Folate
& Vit. B12
►Required
!
►
▪ A toxic metal that must be kept chelated / sequestered in ferritin
▪ ability to absorb iron is restricted because excess iron cannot
be excreted
haem production
► Hard
for thymidine synthesis
▪ Iron deficiency is the commonest cause of
anaemia
Vit B6 - pyridoxine
►
►
to absorb
excrete
► Cannot
Required for haem synthesis
But deficiency is very rare (alcoholics, and isoniazid therapy)
▪ Causes differ in different cultures and socio-economic
environments
25
26
Iron distribution in the body
Dietary iron
▪ Iron in food -
DISTRIBUTION OF IRON IN THE BODY COMPARTMENTS - AVERAGE ADULT (mg)
Male
Female
2400 mg
1700 mg
65%
Stores: ferritin, haemosiderin
1000
300
30% variable
Myoglobin
150
120
3.5%
Haem enzymes
20
15
0.5%
Transferrin
4
3
0.1%
Circulating red cells
▪ ferric – hydroxides – limited absorption
▪ ferric - protein complexes – moderately absorbed
▪ haem-protein complexes - most readily absorbed
% Total iron
▪ Western non-vegetarian diet
▪ ~10-15 mg iron
▪ ~5-10% absorbed
▪ increased to 20-30% if iron deficient or pregnant
▪ Vegetarian diet
▪ Lower iron availability, 2-5% absorbed
27
28
The mucosal (enterocyte) iron block
Iron absorption
Iron absorption is regulated at two levels
▪ Mucosal block exists:
► Fe
*** DMT-1 Divalent
metal transporter (Fe+
+, Ca++, Zn++)
DMT expression is
regulated by iron
status
Fe++
Fe+++
Stored in ferritin
HCP-1 transporter Haem absorption
in duodenum and proximal jejunum
iron – controlled: DMT-1
► enterocytes take up Fe++
► Haem uptake – HCP-1
► Fe+++ absorption (minor)
▪ Fe stored in apoferritin (ferritin) - Fe+++
▪ Fe released to plasma transferrin – controlled: ferroportin
► Hepcidin regulates ferroportin concentration (inversely)
*** Ferroportin
Expression is increased
by low levels of
Hepcidin
▪ Enterocytes shed into gut lumen at end of lifespan - ferritin iron lost
▪ Fe not absorbed in distal small bowel: insoluble at alkaline pH
► Enteric
Transferrin
Iron uptake is restricted by ionic state and release to transferrin is strictly controlled.
A specific pathway exists for haem absorption – facilitated uptake of iron.
transport rate determined when enteric stem cells divide
► Absorption
29
cell mechanisms: iron regulatory proteins bind to iron
response elements of mRNA
► high Fe level increases translation of ferritin mRNA
► low Fe status increases translation of mRNA for Tf & ferroportin
30
Basics of iron handling
►
Iron absorption
►
Iron status of individual
►
Iron transport in plasma
The Iron Cycle
Daily loss ~1(-1.5) mg/d
Duodenum - food
▪ Some foods promote and others reduce iron absorption
urine, faeces, nails, hair, skin
Iron not absorbed
from food: 70-95%
▪ Absorption increased if iron stores low
~1(-1.5) mg/d
absorbed
▪ Transferrin – up to 2 Fe++ atoms per molecule
▪ Saturation: 15-50% (about 15-50% of the available bi
►
Iron storage
►
Assess iron status of patients before all major
elective surgery
Tissue iron
liver, kidney, muscle, etc, ~0.15g)
Transferrin
(~1/3 saturated with Fe)
!
!
Plasma
~4mg
▪ Ferritin (soluble)
▪ Haemosiderin (particulate)
Bone marrow
(erythroblasts ~20mg iron/d)
`
Macrophages (~0.5-1.5g)
Erythrocytes
circulating haemoglobin
Haemorrhage
31
(menstrual loss ~0.5-1mg/d)
-
Another way of viewing Iron status
modified from Essential
Haematology, Hoffbrand, Pettit
&
32
Moss, 4th ed
Laboratory Tests for Iron Status –
Modified from Essential Haematology, Hoffbrand, Pettit & Moss, 4th ed
Daily loss ~1mg/d
Duodenum
~1(-1.5) mg/d
urine, faeces, nails,
hair, skin
Transport iron
!
Transferrin
Iron pools
storage iron
Transferrin
red cell &
tissue iron
normal
iron
overload
-
33
Laboratory Tests for Iron Status –
urine, faeces, nails,
hair, skin
!
Transferrin
(~1/3 saturated)
!
► Blood
Tissue iron
(menstrual loss ~0.5-1 mg/d)
(menstrual loss ~0.5-1mg/d)
34
screen – helpful screening test: MCV, MCH, Hb
► Serum
iron, transferrin and % saturation of transferrin
▪ Not quite as reliable as ferritin for iron deficiency
▪ Helpful for assessing iron overload
!
!
Macrophages (~0.5-1.5 g)
(Ferritin)
Erythrocytes !
!
circulating haemoglobin
Haemorrhage
Haemorrhage
Serum ferritin
leakage from
stores
▪ Best overall test but elevated if inflammation or hepatocellular
injury present
liver, kidney, muscle, etc, ~0.15 g)
Plasma iron
~4mg
Transferrin
receptors
- free in plasma
Erythrocytes !
!
circulating haemoglobin
► Ferritin
!
!!
Bone marrow
(erythroblasts ~20 mg iron/d)
Macrophages (~0.5-1.5g)
(Ferritin)
Laboratory tests for iron status
Daily loss ~1 mg/d
Transport iron
Plasma iron
~4mg
Transferrin
receptors
- free in plasma
Modified from Essential Haematology, Hoffbrand, Pettit & Moss, 4th ed
Duodenum
~1(-1.5) mg/d
!
!!
Zinc protoporphyrin
Cheap, not as specific as
ferritin (in the absence of
inflammation)
Bone marrow
(erythroblasts ~20mg iron/d)
low iron
iron
iron
stores:
deficiency deficiency
incipient
anaemia
iron
deficiency
progressive iron depletion
(~1/3 saturated)
!
Tissue iron
liver, kidney, muscle, etc, ~0.15g)
Serum ferritin
leakage from
stores
►
35
Complex cases where inflammation / necrosis is present
▪ Zinc protoporphyrin
► in iron deficiency – Zn replaces Fe in protoporphyrin
▪ Soluble Transferrin Receptors – helpful in complex cases where there is a need to
confirm a diagnosis of iron deficiency
► Derived mainly from erythroblasts
36
Iron status in health
Iron storage
- Daily iron losses –
▪ Ferritin
Vary with age, sex, pregnancy and frequency of blood donation
▪ Outer protein shell - apoferritin
soluble
by all cells to store
excess iron
► Largest amounts in macrophages and liver cells
Urine, sweat Menstr. Pregnancy Growth
& faeces
► Water
► Produced
Adult male
▪ Iron (Fe+++)-phosphate-hydroxide core
▪ Varying saturation with iron
► Up
to 5000 iron atoms per molecule of ferritin
0.5-1
Post menopause female
0.5-1
Menstruating female
0.5-1
Pregnant female
0.5-1
Children (average)
▪ Trace amounts of ferritin leak out of cells: plasma ferritin
Female 12-15 yrs
▪ Haemosiderin
Adult blood donor:
▪ aggregated ferritin, insoluble, visible in light microscope
– 200 - 250 mg iron in 500 mL blood
tract
► Uterus - menstruation
► Trauma
► insufficient
to cope with losses and growth
1.1
0.6
1.6-2.6
0.6 mg/day
2
1.2
3
1.8
4
2.4
platelet count may be increased
- bleeding / inflammation
In this film –
a small number of red cells are
well-filled with haemoglobin
suggesting that this patient is
now receiving iron replacement
treatment.
►Malabsorption
► Coeliac
disease
are uncommon
39
▪ Oral iron for up to 3-6 months
film - dimorphic population of red cells
iron to replace Hb and stores
▪ Anaemia of chronic disease
► sufficient
▪ Parenteral: Fe polymaltose
► Rapid,
▪ chronic infection, inflammation or malignancy
► mild
or moderate anaemia
microcytosis
► Ferritin is normal or increased
► Serum iron and TIBC are decreased
► Serum iron saturation is usually decreased
► Mediated by Hepcidin: binds to Ferroportin, causing removal &
internalisation of Ferroportin
quantified, useful where heavy iron loss is ongoing
▪ Prophylaxis: treatment to prevent Fe deficiency
► Pregnancy
► Premature
40
Other anaemias: normocytic
(normochromic) but with a tendency to
hypochromic microcytic features
Treatment of iron deficiency
► blood
38
some pencil cells
►Diet
► Others
additional
0.6
anisocytosis and some bizarre
cells
►Growth
neonate, childhood
0.5-1
1.5-3
microcytic hypochromic rbc
► Gastrointestinal
► pregnancy,
1-2
1-2
Iron deficiency – blood screen
Causes of iron deficiency
►Bleeding
0.5-1
1 donation / year -
37
0.5-1
0.5-1
0.5
0.5-1
Avge
Total
► Often
– where testing shows a need
infants
▪ Causes of failure of treatment: Lab monitoring of
treatment is essential
► bleeding
► not taking iron
► wrong diagnosis
► malabsorption
▪ Oral iron has no benefit - not iron deficient
► Problem
► Hepatic
41
- reduced iron release from macrophages
hepcidin increased – iron absorption reduced
42
Other microcytic anaemias
Examples: Inflammation & infection
▪ Thalassaemias
▪ Genetic defect in globin production (b or a)
► Variable
severity
anaemia
► Serum iron normal or raised
► Serum ferritin and iron stores usually increased
► Common in parts of Mediterranean, Africa &
Asia
▪ Surgery
► Microcytic
▪ acute fall in serum iron
level and serum
transferrin
▪ a transient effect in
acute inflammation
▪ but persistent in
chronic inflammation
The film shows a mild form of beta thalassaemia in
which there is microcytosis with pencil cells and mild
anisochromia. Compare the red cell size with the
nucleus of the small lymphocyte.
Hb 121, MCV 60, MCH 19.0
▪ Iron is unavailable to
developing red cells
43
Other hypochromic anaemias
Metabolism in the Red Cell - 1
▪ Myelodysplasia (can have microcytic cells but
macrocytic overall)
▪ No mitochondria in mature red cells
▪ Energy is derived from -
▪ Premalignant disorder
► Abnormal
clone haemopoietic stem cell clone
► Refractory anaemia often with ringed sideroblasts
► Defect in haem synthesis – iron persists as a ring of
granules around erythroblast nucleus
► Mixture of hypochromic and hyperchromic cells – usually
macrocytic
► Raised ferritin, raised serum iron, and low TIBC
► Leucocyte and platelet dysfunction may be present
►Glycolysis
▪ For each molecule of glucose catabolised:
► Used:
► Produced:
►Hexose
▪ Lead poisoning
► mild
cells show basophilic stippling
anaemia
Fe+++ Methaem
reduced to Fe
++ Haem
!
ADP
!
ATP
!
ADP
!
NAD
!
NADH
!
46
Metabolism - 2
Glucose
!
G-6-P
Fructose-6-P
!
Fructose-1,6-DP
!
Glyceraldehyde-3-P
!
1,3-BPG
3-BPG
Hexose monophosphate
shunt
monophosphate shunt
45
Major enzyme pathways in red cells
ATP
2 molecules ATP
4 molecules ATP
2 molecules NADH
(2 molecules lactate)
▪ 2 molecules NADPH produced / molecule glucose
▪ Defect in haem and globin synthesis
► Red
44
NADP
NADPH
Redox
reactions
H2O2
cleared
Dihydroxyacetone-P
▪ ATP required for
►ion
pumps
▪ High osmotic pressure
▪ Cell volume, shape, flexibility
▪ Cell membrane ATPases (Na-K) & Ca:
1 molecule of ATP used to pump
3 Na+ ions out & 2 K+ ions in
2,3-DPG
! !!
! Pyruvate
2x ATP
!
2x ATP
Lactate
47
48
Megaloblastic anaemia
Metabolism - 3
▪ NADH
►In
the normal red cell, haemoglobin Hb(Fe++) is
slowly oxidised to Methaemoglobin - Hb(Fe+++)
►MetHb + NADH -> Hb to permit
(Methaemoglobin reductase)
oxygen transport
▪ NADPH
►Needed
to reverse oxidative damage to the red cell
membrane and proteins
▪ Maintains a pool of reduced Glutathione
49
Haemolytic anaemia
► Increased
50
Haemolytic anaemia: laboratory features
rate of rbc destruction
► Increased
(normocytic or macrocytic – reticulocytes are big)
serum unconjugated bilirubin
► ↓ serum haptoglobin
► ↑ LDH but non-specific
lifespan <120 days
►Extravascular haemolysis (spleen usually)
►Intravascular haemolysis uncommon as a dominant
feature
► Clinical
rbc breakdown
► anaemia
►RBC
►↑
!
► Increased
rbc production
► Polychromasia
in blood film
– immature reticulocyte fraction increased
► marrow erythroid hyperplasia
features
► reticulocytosis
►Anaemia,
jaundice, splenomegaly
gallstones - if chronic
►“Aplastic” crises: parvovirus, folate deficiency
►Pigment
►
►
!
Damaged rbc – red cell fragments (in some forms of microvascular or mechanical
haemolysis)
!
(Folate deficiency)
51
Haemolytic anaemia: classification
Intravascular haemolysis
► mismatched
► G6PD
► hereditary
▪ membrane e.g. hereditary spherocytosis & ovalocytosis
▪ metabolic e.g. G6PD deficiency
▪ haemoglobinopathy e.g. thalassaemia, sickle cell anaemia
► acquired
▪
▪
▪
▪
▪
52
immune (transfusion, fetal; autoimmune)
rbc fragmentation
infection
chemical, drug
secondary (liver, renal – usually milder disease)
blood transfusion
deficiency (splenic destruction)
▪ acute episodes of oxidative stress
► rbc
fragmentation syndromes (microangiopathic)
haemolytic anaemia (splenic destruction)
► immune
▪ some autoimmune
▪ some drug-induced
► infection
(C. perfringens, et al)
Hb (splenic destruction)
► March haemoglobinuria (rare)
► Paroxysmal Nocturnal Haemoglobinuria – cells
susceptible to complement-mediated lysis
► unstable
53
54
Microangiopathic haemolysis
Thrombotic microangiopathy
Intravascular haemolysis
Hbaemia
Fragmented red cells: normally < 1% of red
cells
methaemalbumin
haemoglobinuria
urinary haemosiderin 55
56
Hereditary spherocytosis -
Hereditary spherocytosis
inherited haemolytic membrane disorder
►
Polychromatic
red cell
Commonest inherited haemolytic
anaemia in Europeans
▪ Autosomal dominant
▪ Cytoskeletal protein defect – can’t
hold onto membrane
► spectrin
Thrombotic microangiopathy –
fibrin production following
damage to endothelium
Spherocyte
defect most common
▪ Loss of cell membrane
► Reduced
volume results in RBC
becoming spherocytes
▪ Loss of cell flexibility
▪ Trapped and destroyed in splenic
cords
57
58
Enzyme abnormality – oxidative haemolysis
Membrane structure
Glucose 6 phosphate dehydrogenase deficiency
► G6PD:
required to protect rbc from oxidative
damage
Spectrin lattice intersecting with
actin and band 4.1 proteins to
form a loose hexagonal lattice.
!
Oxidative radicals neutralised
The lattice structure is defective
in hereditary spherocytosis and
elliptocytosis.
glucose
reduced
glutathione
!
Caused by several different
membrane protein
abnormalities, resulting in loss
of lipid from the membrane and
increased Na+ leakage.
NADP
G6P
oxidised
glutathione
membrane damage!
Heinz bodies
NADPH
G6PD
6PG
NADP
NADPH
59
lactate
pentose 5-P
60
G6PD deficiency - blood film during a
severe haemolytic crisis
G6PD deficiency
►
►
►
X-linked disorder
Mediterranean, Middle East, SE Asia, West Africa
Intravascular haemolysis in the face of oxidant stress
1. Severe anaemia
▪ Fava beans: precipitate haemolysis – some cases
▪ Acute infection: oxidative stress - neutrophils
▪ Oxidative drugs: antimalarials, sulphonamides, etc
•Sphered and retracted cells
•Pale ‘blister’ zone on one side of some cells
(see Heinz body below)
•Reticulocytes increased (marrow response)
and occasional erythroblasts may be seen
1
2. Special (reticulocyte) stain:
Heinz bodies: blue stained mass of oxidised,
denatured and aggregated haemoglobin attached to inside of red cell membrane.
2
Precipitated haemoglobin (Heinz bodies) getting stuck in the
spleen
Reticulocyte
61
Sickle cell anaemia – haemolysis
62
Sickle cell anaemia
α2βs2
► Single amino acid substitution in β globin chain
(6Glu→Val)
► Homozygotes have severe HA with crises
► HbS crystallises under low oxygen tension
► HbS
▪ infection, surgery
► Importance
of dehydration
(sickle cell trait) only symptomatic under
severe anoxia (anaesthesia, pregnancy)
► Heterozygotes
►Vascular obstruction
►Infection
63
64
Thalassaemia
Sickle cell anaemia
Beta globin gene
Beta thal
X
X
65
Alpha globin gene
Alpha thal
66
Thalassaemia
Normal Hb production
► genetic
defect in production of either α or β
globin chains leading to:
▪ Microcytic anaemia
►plus shortened rbc survival and ineffective
erythropoiesis
►β-thalassaemia - α-globin/heme aggregates
►α-thalassaemia - β4 tetramers precipitates
α
HbA
β
HbF
γ
% total
globin
synthesis
Early death of developing red cells = ineffective
erythropoiesis
HbA α2β2
HbF α2γ2
HbA2 α2δ2
ε, ζ
67
β Thalassaemia
6
δ Hb A2
birth
weeks
30
68
β thalassaemia homozygote
► Usually
point mutation
► Heterozygous state: trait (asymptomatic)
► Homozygous or compound heterozygous:
thalassaemia major
▪ Symptomatic after six months of age
▪ Transfusion dependent
▪ Skeletal abnormalities
▪ Iron overload despite chelation therapy
69
70
β thalassaemia homozygote
Haematopoiesis
Massive expansion of bone marrow to compensate – bony
deformities
71
72
β thalassaemia homozygote
α Thalassaemia
► Four
α globin genes: 2 from each parent
► Deletion of one or more α globin genes:
▪ 1 gene deletion: silent carrier
▪ 2 gene deletion trait: – mild
►but
beware of risk to offspring if partner also has 2 gene
deletion
▪ 3 gene deletion: HbH disease (β chain tetramers)
►severe
anaemia ± transfusion dependency
▪ 4 gene deletion: hydrops fetalis syndrome
►lethal
in utero
73
74
Haemoglobinopathies
Alpha thalassaemia
75
76
Examples of acquired haemolytic
anaemias
malaria
burns
autoantibody
Immune haemolysis
warm type (IgG)
►
►
►
Red cells parasitised haemolysis occurs after
parasite grows to
maturity
Thermal injury to red cells microspherocytes produced:
cleared rapidly
Opsonisation of red cells
by macrophages in
spleen. Spherocytes,
reticulocytosis and
erythroblasts in blood
77
idiopathic
secondary
▪ SLE etc
▪ CLL, lymphomas
drugs
IgG (+IgM, IgA, C3b/d)
cold type (IgM)
►
►
►
idiopathic
secondary
▪ Mycoplasma
▪ infectious mononucleosis
▪ lymphoma
IgM + peripheral agglutination &
slow Complement activation
Direct Coomb’s test = Direct Antiglobulin Test (DAT)!
detects IgG or Complement on rbc’s: anti-IgG + anti-C3b/d
78
To answer the question about direct and indirect antiglobulin test from yesterday
Immune haemolysis: drug induced autoantibody
Anaemia
Spherocytes
Polychromasia
Reticulocytes raised
Bilirubin increased
79
Acute lymphoblastic
leukaemia – immature
lymphoids/
lymphobasts
Acute myeloid
leukaemia – immature
myelobasts
Lymphomas and
mature lymphocyte
cancers (CLL, myeloma
etc)
}
}
Myeloproliferative
neoplasms – too many
mature forms
80
Acute leukaemia
Lymphoid series
(lymphocytes)
► accumulation
of malignant blast (primitive)
cells, that fail to differentiate.
► replace normal marrow and cause bone
marrow failure
► clonal proliferation
normal cells
Myeloid series
Also myelodysplastic syndrome
– odd and ineffective myeloid
cells
Acute leukaemia
Marrow
population
(log scale)
symptom
threshold
somatic mutation
∗
tumour cells
82
time
Acute lymphoblastic leukaemia
83
84
Acute myeloid leukaemia (AML)
► all
age groups
► commonest leukaemia in adults
► Usually de novo but
► may follow –
WHO: ALL
▪ myelodysplasia,
▪ myeloproliferative disease:
o
Polycythaemia, Chr. Myeloid Leuk., Myelofibrosis
► blast
WHO: Burkitt leukaemia
cells of some types have Auer rods
85
86
Acute leukaemia: investigations
► Peripheral
!
► Bone
Treatment Concepts
blood screen
marrow aspirate
!
► Immunological
classification by flow cytometry
▪ Immunophenotyping – cell surface marker molecules
► Cytogenetics
/ Molecular genetics
▪ Disease type characterised – WHO classification
87
AML classification -
AML – other variant types
AML, not otherwise categorised
► AML
WHO
88
FAB
minimally differentiated
M0
► AML without maturation
M1
► AML with maturation
M2
► Acute promyelocytic leukaemia
M3
► Acute myelomonocytic leukaemia
M4
► Acute monoblastic/monocytic leukaemia M5
► Acute erythroid leukaemia
M6
► Acute megakaryoblastic leukaemia
M7
► ….
► AML
- with recurrent cytogenetic
abnormalities, eg
► AML
with t(8;21)
with t(9;22)
► AML with t(16;16) or inv(16)
► AML with 11q23 abnormalities
► AML with t(15;17)
► AML
▪ Acute Promyelocytic Leukaemia
► AML
- with multilineage dysplasia
► AML
- treatment related
► abnormal
89
► Alkylating
maturation in at least 2 cell lines
agents, Topoisomerase inhibitors
90
Auer rods in AML (M2)
Acute myeloid leukaemia
► AML
M3 acute promyelocytic leukaemia
► AML
M4 (myelomonocytic) and M5 (monocytic)
▪ associated with chronic DIC
▪ t(15;17) RAR-PML gene sites
▪ tissue infiltration
► Remission
can be achieved in a majority of
patients, but it is generally short-lived
► Long term survival 20%
► Bone marrow transplantation (either autologous or
allogeneic) improves the prognosis
91
92
CML: t(9;22)
Chronic myelogenous leukaemia (CML)
► Philadelphia
chromosome the translocation
results in a fusion between BCR (chr 22) and
abl (Abelson oncogene) (chr 9)
► middle
aged
► massive expansion of granulocyte mass
▪ Most of the leukaemic cells are differentiated
▪ hypermetabolism
► sweats,
!
!
!
weight loss, anorexia
▪ splenomegaly
▪ marrow failure
►New
fusion gene that codes for a novel tyrosine kinase
I
XI
to Glivec (imatinib): blocks hybrid
▪ Responsive BCR-abl
tyrosine kinase
► undergo
a terminal transformation to AML
► mean survival 3-4 years – with conventional
cytotoxic treatment
►Cells
differentiate and die
further mutations later result in drug resistance
(not very often)
►But,
93
LYMPHOID CANCERS
Myelodysplasia
► neoplastic / pre-neoplastic marrow
► abnormalities of all three cell lines
94
& blood condition
Chronic lymphocytic leukaemia
▪ stem cell disorder
▪ most arise de novo, some occur in patients with previous
chemotherapy
► ineffective
cell maturation
despite increased marrow cellularity
► cytopenias,
▪ anaemia with macrocytosis usually
▪ may have thrombocytopenia + impaired platelet function
▪ may have neutropenia + impaired neutrophil function
► tendency
to progress to AML (~ 30%)
95
96
Multiple (plasma cell) myeloma
Chronic lymphocytic leukaemia
97
Multiple (plasma cell) myeloma
Hodgkin lymphoma
▪ Anaemia
▪ Bone erosion & fractures (lytic lesions)
►stimulate
osteoclasts (IL-6)
immunoglobulin in most but ~20% only λ or κ
light chains (light chain myeloma)
► presence
►Monoclonal
▪ Reduced levels of polyclonal Ig – bacterial infection risk
▪ hypercalcaemia,
▪ renal failure – tubule obstruction
►Ca
+ light chains + urinary mucoprotein: precipitates
▪ Therapy
►Cytotoxic
98
therapy / autologous BMT in younger age group
of Reed Sternberg cells
▪ bi-or multi-nucleate
▪ owl’s eye appearance
▪ malignant cell is a B cell
► often
present in single node area
► disseminates to others
► splenomegaly 50%
► prognosis and treatment depends on stage
▪ > 90% survival stage I-II
99
100
Staging for Hodgkin’s disease
Hodgkin lymphoma
► Classic
mass
presentation = young adults with mediastinal
► Usually
nodular sclerosing HD
► Constitutional
symptoms
► Fever,
night sweats, weight loss, pruritus
► Reduced T cell immunity: viral & fungal inf.
► antibody responses preserved until late
► Early
Stage I
Stage II
Stage III
stage disease: excellent prognosis
► Advanced disease > 50% 5 year survival
!
► Risk of chemotherapy-induced myelodysplasia & AML
Stage IV
101
102
Non-Hodgkin lymphoma
►
►
►
►
►
►
►
Hodgkin
lymphoma
Majority arise from germinal follicle centre cells
Architecture may be either follicular or diffuse
90% are B cell derived
More malignant forms have larger cells with high rates of
proliferation and diffuse infiltration of node
Many many types: Diffuse lymphoma most common, then
follicular lymphoma.
Burkitt lymphoma
Nodes at
presentation
Spread
Mesenteric
nodes, Waldeyer
ring
▪ African children, jaw mass, EBV,
Adult T cell leukaemia / lymphoma
▪ HTLV-1, CD4+ T cells
▪ Caribbean and Southern Japan
Extranodal
involvement
103
single group,
axial
contiguous node
areas
Non-Hodgkin
lymphoma
multiple areas
non-contiguous
rare
more common
uncommon
common
104
`