Blood

Approach to hematological disorders

History Physical findings Blood findings
Weakness Pallor Anemia Approach to a
Fatigue Icterus patient with anemia
Exertional dyspnea

Recurrent Spleenomegaly Leukocytosis Disorders of

infections Enlarged lymph Leukopenia phagocytic and
Unexplained fever nodes Abnormal immune system
leukocytes

Bleeding Petechiae Thrombocytopenia Approach to

Bruising Ecchymosis Prolonged bleeding bleeding disorders
Hemarthrosis time
Mucosal bleeding Abnormal
prothrombin or
partial
thromboplastin
time
 Blood: Physical characteristics
Average amount: 8% body weight
(70 kg man‐ 5.6L)
Specific gravity: 1055‐1065 (viscosity is 5 times
that of water)
pH: 7.35‐7.45
Osmolarity: 300 mOsm
Salanity: 0.9%
Colour: Bright red to deep red
 Components of Whole Blood

Plasma
(55% of whole blood)

Buffy coat:
leukocyctes and
platelets
(<1% of whole blood) Formed
elements
Erythrocytes
1 Withdraw blood 2 Centrifuge (45% of whole blood)
and place in tube

• Hematocrit
• Males: 47% ± 5%
• Females: 42% ± 5%
 Components of blood

Composition

Plasma Cells
55% 45%

WBC
Water 4000‐ 11000/mm3
Solids & Platelets
90 % RBC
Gases 1.5‐4 lakh/mm3
4.8– 5.4
million/mm3
 Components of plasma
(90% water rest solids)
Solids

Plasma Proteins Regulatory &

protective Organic and inorganic substances
6‐8 gm/dL proteins

Albumin Waste material

Hormones
3‐4 gm/dL
Antibodies Nutritive material
Globulin
2.5‐4 gm/ dL Enzymes Ions

Fibrinogen
0.3 gm/ dl
 Functions of blood
Distributive
• Carries O2 (from lungs) and nutrients (from
GIT and body stores) to all cells
• Carries wastes from all cells to elimination
sites (lungs for CO2, liver for bilirubin and
kidneys for nitrogenous wastes)
• Carries hormones (chemical signals) from
endocrine organs to target tissues.
 Functions of blood…
Regulatory functions
• Body T° by absorbing and distributing heat
• pH by virtue of its many buffers
• Maintains adequate fluid volume in the body
Protective functions
• Prevents blood loss by initiating clotting
mechanisms in response to blood vessel damage
• Prevents infection via WBCs and plasma immune
proteins
 Separation of plasma proteins
• Solubility
Salting in
Salting out
• Molecular size
Dialysis
Gel filtration chromatography
Ultracentrifugation
Sodium dodecyl sulfate (SDS) polyacramide gel
electrophoresis
 Separation of plasma proteins
• Molecular charge
Ion exchange chromatography
High performance liquid chromatography
Electrophoresis

• Specific binding of the protein to a specific

substance
Affinity/ absorption chromatography
Precipitation by antibodies
 Separation procedures
• Protein solubility
 Salting out
Adding divalent salts eg. Ammonium
sulphate
 Salting in
Dialysis against a solution with low salt
concentration
 Separation procedures…
• Molecular size
Dialysis
Gel filtration chromatography
Ultracentrifugation
Sodium dodecyl sulfate (SDS) polyacramide gel
electrophoresis
Dialysis
Gel filtration chromatography
Gel filtration chromatography…
SDS‐PAGE electrophoresis
 Separation procedures…
• Molecular charge
 Ion exchange chromatography
 High performance liquid chromatography
 Electrophoresis
 Separation procedures…
• Specific affinity binding
Affinity/ absorption chromatography
Precipitation by antibodies
 Column chromatography
• Gel filtration
• Ion exchange
• Affinity
Chromatography: Matrices
 Separation procedures…
• Immunoelectrophoresis
• ELISA(Enzyme linked immunosorbant assay)
 Immune electrophoresis
• This is a double immunodiffusion technique
• Sample is placed in a well on a glass slide coated with
agarose or cellulose acetate, and electrophoresed to
separate the proteins according to their charge
• A trough is cut in the agarose parallel to the axis of the
electrophoresed proteins into which is placed monospecific
antibodies against IgG, IgM, IgA, or kappa or lambda light
chains
• The slide is incubated for 18 to 24 hours to allow the
antibodies to diffuse from the trough into the agarose,
forming precipitin arcs with their respective antigens
• The relative size of the precipitin arcs is proportional to the
quantity of immunoglobulin or light chains
ELISA
 ELISA
• Binding the antigen to the surface of the wells of
a ELISA plate
• Blocking any non‐specific antibody binding sites
with a generic protein (BSA.
ovalbumin, gamma‐globulin, skimmed milk)
• Adding primary antibody solution to each well to
allow specific antigen‐ antibody
recognition/binding to occur
• Removing unbound primary antibody.
 ELISA…
• Adding a secondary antibody (e.g. goat anti‐mouse anti
IgG that is covalently linked to a protein such as horse
radish peroxidase or alkaline phosphatase). This
antibody binds to the primary antigen that remained
bound to antigen after washing the wells
• Remove unbound secondary antibody
• Determine the amount of secondary antibody bound
to the wells by adding a chemical reagent that is
converted by the peroxidase or phosphatase to a
colored product whose absorbance is measured using a
ELISA plate reader
Separation of proteins by
electrophoresis
Plasma protein fractions
 Plasma protein fractions…
• α1 zone: α1 anti trypsin, TBG, HDL
• α2 zone: α2 macroglobulin,caeruloplasmin,
VLDL, haptoglobin
• β zone: Transferrin, LDL, fibrinogen, C3 & C4
complement
• γ zone: Immunoglobulins, Factor VIII,
C‐reactive proteins, α feto protein
 Origin of plasma proteins
In embryo
Mesenchymal cells
In adults
Liver: albumin, α and β globulin , fibrinogen
B lymphocytes in lymph nodes, bone marrow: γ
globulins(immunoglobulins)
Albumin
 Albumin
• 60% of total plasma proteins(3.4‐4.7 g/dL)
• 40% intravascular and 60 % extra vascular
• T1/2 = 19 days
• Molecular weight= 66000 Da
• Shape ellipsoid
• Catabolism: receptor mediated trans cytosis
and then pinocytosis by tissue cells
 Functions of albumin

Exerts colloidal osmotic pressure of 25 mmHg (80%)

 Functions of albumin…
• Regulates blood volume & body fluid balance
• Viscosity: One of the determinants of
resistance to blood flow
• Blood pressure maintenance
• Protein reserve
 Functions of albumin…
• Binding to various ligands helps in transport of
free fatty acids, bilirubin, calcium
• Secondary carrier for thyroxin, cortisol &
heme
• Drug binding eg. Sulfonamides, penicillin G,
dicumarol, aspirin
• Buffering action: Helps maintain pH of blood

OH‐ H+ NH2 CO O‐ H+
 Variations in plasma albumin
• Increase
• Secondary to burns , dehydration
• Decrease
• Infants and newborns
• Pregnancy
• Hepatitis
• Cirrhosis
• Nephrosis
• Protein losing enteropathies
 Alpha Globulins
• Alpha 1 antitrypsin
• Antithrombin III
• Antiplasmin
• Caeruloplasmin
• Haptoglobin
• Progesterone binding globulin
• Retinol binding proteins
• Transcortin
 Beta globulins
• Beta 2 microglobulin
• Hemopexin
• Plasminogen
• Sex hormone binding globulin
• Transferrin
Functions of alpha & beta globulins
• TBG: Carrier protein for thyroid hormone in blood
• α2 macroglobulin: Inhibitor of serum
endoprotease
• Caeruloplasmin: Transports copper
• Haptoglobin: Transports free hemoglobin
• Transferrin: Transports iron
• Fibrinogen: Precursor of fibrin
• C3 & C4 : Proteins of complement ssystem
• α feto protein: Osmotic regulation, carrier protein
 Transferrin
• Transport of iron: from catabolism of heme
and from food (gut) to the sites where iron is
required, i.e. to the bone marrow and other
organs
• 2 moles of Fe3+ per 1 mol of transferrin
Receptor mediated transferrin
endocytosis
 Ceruloplasmin
• Carries 90% of copper in plasma (copper –
cofactor for a variety of enzymes);
• 1 molecule binds 6 atoms of copper
• binds copper more tightly than albumin that
carries other 10%
of copper
• albumin may be more important in copper
transport (donates copper to tissues more
readily)
 Wilson’s disease
• accumulation of copper in liver, brain, kidneys
• Liver disease, neurologic symptoms
 Haptoglobin
• Binds free hemoglobin and delivers it to the
reticuloendothelial cells
• complex Hb‐Hp is too large to pass through
glomerulus prevention of loss of free Hb

• Free Hb passes through glomeruli, enters

tubules and precipitates leading to kidney
damage
 Act as antioxidants
• transferrin
• ferritin
• ceruloplasmin
• haptoglobin
• hemopexin (binds heme and transfers it to the
liver)
• remove Fe 2+ and thus prevent the Fenton
reaction: H2O2 + Fe 2+  Fe 3+ + OH∙ + OH‐
 1‐ ANTITRYPSIN
(1‐antiproteinase)
• Principal plasma inhibitor of serine protease
(inhibits trypsin, elastase)
• Deficiency has a role in emphysema – proteolytic
damage of the lung
• methionine involved in AT binding to proteases is
oxidized by smoking
• AT no longer inhibits proteases
• increased proteolytic damage of the lung,
particularly devastating in patients with AT‐
deficiency
Smoking

Acyl
Protease α 1 antitrypsin
transferase
α 1 antiproteinase

Proteoltyic damage of lungs

Emphysema
• Functions of gamma globulins
• Functions of fibrinogen
 Variations in gamma globulins
• Increase
TB
Leukemia
Cirrhosis and acute hepatitis
Nephritis
• Decrease
Immune deficiency
 Variations in fibrinogen
• Increase
 Pregnancy
 Menstruation
 Malaria
 Tissue injury
• Decrease
 Congenital
 Carcinoma prostate
 Intravascular coagulation
 Acute phase proteins
Tissue injury or infection

Local inflammation
Activated
macrophage

IL 1 and TNF α
Systemic acute phase response

Fever Hepatocyte

Acute phase reactants

Acute phase reactant response
 Acute phase proteins
• α1 antitrypsin
• Fibrinogen
• Complement
• Haptoglobin
• C‐reactive protein
Mammalian embryonic blood
formation

Human embryo at various stages

 Formation of cellular elements of
blood
In fetus: Extramedullary hematopoiesis(3 stages)
• Mesoblastic: (16‐19 days): Clusters of
mesenchymal cells in yolk sac ends by 12
weeks
• Hepatic (Second trimester) Liver, spleen and
lymph node
• Myeloid(Last month of gestation+ after birth):
Bone marrow
Bone marrow is the site of synthesis of
all formed elements of blood after
birth
Red bone marrow
 Formation of cellular elements of
blood
Upto 5 yrs: all bone marrow
Upto 20 yrs: bone marrow of membranous +
ends of long bones
After 20 yrs: bone marrow of membranous bone
Granulocyte vs erythroid development
• Development of granulocytic system lags
behind
• Number of mature neutrophils stored in the
marrow is less
Sensitivity of newborns to bacterial sepsis
Classical studies in developmental
biology have used amphibians to examine
embryogenesis and the general principles
of embryonic development are maintained in
higher organisms.
• 1924( Maximow) postulated that blood cells were
derived from a single class of progenitors
• 1938(Downey) added the concept that progenies
of pleuripotent cells were progressively more
committed to a single lineage
• 1961(Till & Mc Culloch) demonstrated that single
cells were capable of establishing nodules of
hemopoitic growth in spleens of irradiated mice
& that such colonies displayed multilineage
differentiation
Developmental models for
hematopoiesis
 Terminology of stem cells

Stem
Cell

Lymphoid Non-lymphoid
LIne line
(myeloid)

erythroid granulocyte megakaryocyte monocyte/macrophage

Hematopoiesis
Stages in
differentiation off
blood cells
Hematopoiesis

Figure 17.9
Erythropoiesis: Formation of RBCs

This development takes about 7 days

and involves three to four mitotic cell
divisions, so that each stem cell gives
rise to 8 or 16 cells.
 Erythropoiesis

Basophilic Polychromatophil Orthochromatic

erythroblast erythroblast erythroblast

Early Intermediate Late

Megaloblast normoblast normoblast normoblast

7‐10 µ m
15‐20µ m Nucleus: size 10‐14 µ m Nucleus: cart
Nucleus: Big Nucleoli: absent Nucleus: Size wheal‐ pyknotic
Hb: absent Hb: absent Hb: Starts appearing Hb: increases
 Q
• Why does the cytoplasm become more
eosinophilic as RBCs mature
M:E ratio = 3:1(2.3)
In acclerated erythropoiesis the ratio
• Increases
• Decreases
• Doesn’t change
• None of the above
Reticulocyte
 Reticulocyte
• Why the name?
• Size: 8 microns
• Shape: irregular & polylobulated
• More adhesive
• Contain ribosomes, mitochondria & golgi
complex
• Produce 30% of total hemoglobin
• Reticulocytes have transferrin receptors
 Percentage in circulation
In newborn: 30‐40%
In infants up to first week of life: 2‐6%
In children & adults: 0.2‐2.0 %(Ave. 1%)
Absolute count: 20000‐ 90000/ mm3
 Reticulocytes VS mature RBC
• Size: 8 μm • Size: 7 μm
• Shape:Polylobulated • Shape: Biconcave disc
• Adhesiveness more • Adhesiveness less
• RNA and ribosomes • RNA and ribosomes
present absent
• Transferrin receptors • Transferrin receptors
present absent
• Hemoglobin synthesis • No more hemoglobin
synthesis
Reticulocytosis
Physiological causes: Newborn, high altitude
Pathological causes:
During t/t of deficiency anemias
After hemorrhage

Reticulocytopenia
Aplastic anemia
Post spleenectomy
 Q
• How do reticulocytes differ from mature
erythrocytes?
Erythrocytes (RBCs)

Figure 17.3
PBS under low power
PBS under high power
 Mature Erythrocyte
Shape Biconcave disc ?
Mean diameter 7.5µ(7‐8 microns)

Thickness at periphery 2.5 µm

Thickness at center 1 µm
Number: Males 5.2million ± 3lakh
Females 4.7 million±3 lakh
Children 4.4 million
Hemoglobin(g/dl of whole blood) Males 14‐ 18,
Females 12‐15.5
At birth 23
At end of 3 m 10.5, Children upto 1 yr 12
MCV 90±9fl
MCH 32±2 pg
MCHC (g/100ml of packed cells) 32‐ 34
Principle of automated cell counting
 RBC indices
• MCV gives you the average volume of
erythrocytes.
• MCH gives you the average weight of
hemoglobin per eyrthrocyte.
• MCHC gives you the average hemoglobin
concentration per erythrocyte.
 RBC indices
MCV: Average volume of the RBC
MCV= PCV in % X 10 cubic microns
RBC count in
millions/mm3
MCH: Average Hb concentration of a RBC
MCH= Hb(gm/dl) X10 picogram
RBC count in million/mm3
MCHC: Ave. Hb concentration per RBC
MCHC= Hb(gm/dl) X100
PCV(%)
 Biconcave disc shape of RBC
• Increased ratio of surface area : volume(40%
more membrane)
Facilitates gas transport
• More deformable
 Maintenance of biconcave shape
• Elastic forces within the membrane
• Surface tension
• Electrical forces on the membrane surface due
to albumin adsorption
• Osmotic /hydrostatic forces
 Mature erythrocyte
Lacks: Ribosomes,mitochondria and nucleus
thus
• Unable to synthesize new protein
• Unable to carry out oxidative reactions a/w
mitochondria
• Unable to undergo mitosis
 RBC metabolism
Require energy to
• Maintain shape & flexibility of cell membrane
• Maintain iron in Fe++ form
• Preserve the milieu of RBC (high K+ , low Na+ & Ca++)
Thus must have constant access to glucose
Glucose enters the RBC via facilitated diffusion
Mature RBCs do not have a Citric aid cycle for glucose
utilization
Less efficient pathways
 Anaerobic glycolysis (EMP)(95%)
 Pentose phosphate pathway(HMP shunt/
phosphogluconate pathway)
Anaerobic glycolysis(EMP)
HMP shunt
ATP production in anaerobic glycolysis
NADPH synthesis
• Normally H2O2 is disposed off by catalase &
glutathione peroxidase. The latter leads to an
increase in production of GSSG(oxidized
glutathione)
• Reduced glutathione(GSH) is regenerated
from GSSG by action of glutathione reductase
which depends on the availability of NADPH
• NADPH synthesis protect sulfhydryl groups in
erythrocyte membranes and hemoglobin
 Heinz bodies
• Appear inside the RBC when it has been
subjected to oxidative stress as a result of
oxidation and subsequent precipitation of –SH
groups of hemoglobin
• They stain purple with cresyl violet
Meth hemoglobin reduction
2, 3 DPG generation

G
 Role of 2,3 DPG
α2 α1 O2
α2 α1O2
O2
α2O2 α1O2

D D D
β1 P
G
β2 β1 P
G
β2 β1 P
G
β2
T state
Taut /Tense

α2O2 α1O2 α2O2 α1O2

O2 O2

β1O2 β2 β1O2 β2O2

R state
Relaxed
Energy metabolism in RBC
Glucose

G‐6‐P Pentose
F‐6‐P CO2
phosphate Ribose
pathway
Ga‐3‐P phosphate

Anaerobic
Glycolysis

2ATP 2ADP Rapoport‐

lubering 2,3 diphospho glycerate
shunt

Lactate
 Products of metabolism in RBC
• NADH
• ATP
• 2, 3 DPG
• NADPH,major reducing agent in the RBC
• Conversion of hexoses to pentoses
 Erythrocyte membrane and fragility
Broken by certain physical stimuli
• Mechanical fragility:RBCs shaken with glass bead X 1hr, 2‐5%
lysis
• Autohemolysis: blood kept at 370C for 24 hrs
< 0.5% hemolysis
• Osmotic fragility: RBCs in physiological saline remain intact for
hours
 RBC membrane
1. Peripheral proteins ‐‐ spectrin, ankyrin, (band
4.1), actin. Comprise peripheral cytoskeleton,
which supports membrane. All cells are
thought to have a similar structure under the
plasma membrane.
 RBC membrane…
2. Intrinsic proteins
Examples
(1). Multipass (band 3/anion exchanger) ‐‐ Catalyzes reversible
exchange of the anions HCO3‐ (bicarb) and Cl‐ between RBC and
plasma. Exchange allows max. transport of CO2 in blood (as bicarb
in solution
(a) Basic point: Bicarb is much more soluble in plasma than CO2, so
lots of bicarb (but not much CO2) can be carried in the blood.
Therefore need to covert CO2 to bicarb when want to carry CO2 in
blood; need to do reverse to eliminate the CO2 (in lungs).
(b) Conversion of CO2 to bicarb (& vice versa) can only occur inside
RBC, where the enzyme carbonic anhydrase is. Carbonic anhydrase
catalyzes:
CO2 + H2O ↔ HCO3‐ + H+
 RBC membrane…
(c). Gases can pass through membranes by diffusion ‐‐ CO2 can exit or
enter RBC as needed. However bicarb cannot pass through
membranes. You need the anion exchanger to get bicarb in and out
of RBC.
(d). Where CO2 is high, as in tissues, CO2 diffuses into RBC and is
converted to bicarb inside the RBC. (Reaction above goes to right.)
Then bicarb leaves RBC in exchange for chloride using the anion
exchanger.
(e). In lungs, the process is reversed ‐‐ bicarb reenters the RBC in
exchange for chloride using the anion exchanger. The bicarb is
converted back to CO2 inside the RBC (reaction above goes to left).
Then the CO2 diffuses out of the cells and is exhaled.
(2). Single pass (glycophorin) ‐‐ function of protein
not known.
(a). Large amount of (‐) charged modified
carbohydrate ‐‐ sialic acid ‐‐ may cause RBC to
repel each other and prevent clumping of RBC.
(b). Loss of terminal sugars may occur with age and
trigger destruction of "old" RBC.
(c). Glycophorins make up a gene family; variations
in glycophorin A are responsible for MN blood
type differences. Variations in glycophorin C are
correlated with resistance to malaria.
RBC membrane structure
 RBC membrane structure

Protein 4.1 binds spectrin to glycophorin C

Protein 2.1(Ankyrin) binds spectrin to anion exchanger band 3
 Q
• Your patient is a 44 year ‐old woman
complaining of "exhaustion".
• hematocrit is 0.15
• RBC count 1.0 million
• reticulocyte count 2%.
What do these findings indicate to you
Patient is recovering from anemia
Patient is going into marrow failure
• When expressed as a % of total RBCs the reticulocyte
count may overstate the actual number of
reticulocytes. Therefore:
• In this case:
• Thus the retic count is not increased (normal being 0.5‐
1.5%), but is in fact relatively low in an anemic patient
indicating no marrow response and suggesting marrow
failure.
• The use of the absolute rectic count avoids this
problem. In this case:
• (0.02 x 1,100,000) = 22,000 retics, which is abnormally
low for someone with such a low Hct
 Rouleaux formation

Increased amount of fibrinogen in the

blood can cause rouleaux formation
 Anisocytosis
Variations in erythrocyte size
• Microcytic : MCV = <80 fL & size = <6 μM
Eg.iron‐deficiency anemias.
• Normocytic : MCV = 80 ‐ 100 fL & size = 6 ‐ 9
μM
• Macrocytic: MCV = >100 fL & size = >9 μM
Eg. hepatic diseases & vitamin B12 and folic
acid deficiency anemias
 Poikilocytosis
Variation in the shape of erythrocytes
Due to chemical or physical alteration in the
red blood cell membrane or the actual
contents of the cell
 Spherocyte

Observed in immune induced hemolysis,

post blood transfusions, and congenital
anemia
 Target cell

Observed in hemoglobinopathy, hepatic

diseases, iron deficiency anemia, hemolytic
anemia, and splenectomy.
 Schistiocytes

Uremia, microangiopathic hemolytic anemias,

hemolytic anemias cause by physical agents,
and disseminated intravascular coagulation
(DIC)
 Ovalocytes

Hereditary defect present in the RBC

cytoskeletal proteins (the spectrin chain),
iron deficiency anemia, leukemia
associated anemias, thalassemia, and
dyserythropoiesis
 Poikilocytosis

Indicator of abnormal erythropoiesis due to

bone marrow effects and/or abnormal RBC
destruction
 Howell‐Jolly bodies

Observed in hemolytic anemias, pernicious

anemia, post‐operative conditions,
splenectomy, or splenic atrophy
 Echinocyte

Observed in uremia, acute blood loss,

stomach cancer, and pyruvate kinase
deficiency
 Burr cell

Observed in uremia, acute blood loss,

stomach cancer, and pyruvate kinase
deficiency
 Basophilic stippling

Observed in lead poisoning, alcoholism

megaloblastic anemias
 Erythrocytes hemoglobinization
• Normochromic RBC normal amount of
hemoglobin which stains uniformly
MCH = 27 to 32 pg & MCHC = 31 to 37%.
• Hypochromasia / hypochromia
MCH = <27 pg & MCHC = <31
Eg.iron‐deficiency anemia and thalassemia,
any hemoglobinopathy
 Hematocrit/ Packed cell volume
Percentage of the total volume of blood that is
occupied by packed red blood cell
Normal values are as follows:
• Adult male = 42% to 53%
• Adult female = 36% to 46%
• Newborn = 50% to 62%
• One year = 31% to 39%
 Hematocrit
• Increased
Polycythemia
Shock associated with surgery, burns, or traumas
Dehydration
• Decreased
Anemias
Pregnancy
receiving IV fluids
Cardiac decompensation (a failure to maintain a good blood
circulation)
 Factors required for normal
erythropoiesis
• Dietary factors
Protein
Iron
Copper
Manganese Normo
Vitamin C blast
Folic acid
Vitamin B12
• Intrinsic factors
• Hormones: Thyroid & corticoid
IL‐1, IL3 ,IL6,
GM‐CSF, G‐CSF, SCF

GM‐CSF GM‐CSF
EPO TPO

GM‐CSF
IL‐5 GM‐CSF

M‐CSF
IL‐3
IL‐4
G‐CSF
Dietary factors affecting erythropoiesis
Protein: all 10 are important
Histidine, Valine, Leucine, Isoleucine, Lysine,
Arginine, Methionine, Tryptophan,
Phenylalanine, Threonine, Glycine
In PEM anemia results
Normocytic, normochromic, reticulocyte count
normal, slightly hypocellular bone marrow
 Iron

Pinocytosis
 Iron metabolism
Why is iron required?
Hemoglobin: 65%
Myoglobin: 4%
Cytochromes
Cytochrome oxidases
Peroxidase 1%
Catalase
Transferrin: 0.1%
Ferritin(Liver &RES): 15‐30%
 Iron metabolism
Total body iron 4‐5gm
Daily losses
Males: 1mg/day
Females: 2 mg/day
Absorption of iron: 3‐6 % of ingested amount
Site of absorption: Duodenum
Inhibitors of absorption:
Phytic acid, phosphates, oxalates & carbonates
Promoters of absorption: Citric acid
 Iron absorption from the gut
2 pathways for iron absorption
• Heme iron
• Non heme iron
2 factors determine absorptive rate
1. Amount of storage iron
2. Rate of erythropoiesis
Mucosal block theory
Modified mucosal block theory
Unaccepted Fe

Fe in gut lumen Body Fe

Normal

Fe defficient

Fe overload
Copper: Promotes absorption, mobilization &
utilization of iron
Vitamin C
Folic acid
Vitamin B12
Regulation of RBC production
 Erythropoietin Mechanism

Start
Normal blood oxygen levels Stimulus: Hypoxia due to
decreased RBC count,
decreased availability of O2
to blood, or increased
Increases tissue demands for O2
O2-carrying
ability of blood

Reduces O2
levels in blood

Erythropoietin
Kidney (and liver to a
Enhanced stimulates red
smaller extent) releases
erythropoiesis bone marrow
erythropoietin
increases RBC
count

Figure 17.6
Mechanism of action of erythropoietin
Erythroid cell most sensitive to EPO is
proerythroblast
EPO+ receptor

Ca++
Intracellular cAMP, cGMP
Tyrosine specific protein kinase
Phosphatidylinositol
Protein kinase C
 Erythropoietin
Site of synthesis
90% kidney, interstitial cells in the peritubular capillaries
10% liver, Perivenous hepatocytes
Factors influencing erythropoietin production
• Hypoxia:Hypobaric, bleeding, cardio respiratory
disturbance, carboxyhemoglobin
• Vasoconstrictors: 5‐HT, PG E1(By inducing renal
hypoxia)
• Nucleotides: cAMP, NAD, NADPH
• Products of RBC destruction
• Hormones: Androgens, ACTH, TSH, GH, Prl, PTH
 Formation of hemoglobin

Mitochondria

Cytoplasm
141 a.a. 146 a.a.

Characteristics of oxygen combination with hemoglobin

•Oxygen is carried in molecular form
•Forms coordinate bond with iron atom
Structure of hemoglobin
Structure of Hemoglobin

Figure 17.4
Life Cycle of
Red Blood
Cells
Sites of erythropoiesis
Genes regulating hemoglobin
synthesis
 Normal hemoglobin types
In the embryo
• Gower 1 (ζ2ε2)
• Gower 2 (α2ε2)
• Hemoglobin Portland (ζ2γ2)
In the fetus
• Hemoglobin F (α2γ2)
In adults:
• Hemoglobin A (α2β2) The most common with a normal amount over 95%
• Hemoglobin A2 (α2δ2) ‐ δ chain synthesis begins late in the third trimester
and in adults, it has a normal range of 1.5‐3.5%
• Hemoglobin F (α2γ2) ‐ In adults Hemoglobin F is restricted to a limited
population of red cells called F‐cells
Elevated in persons with sickle‐cell disease.
Time course of appearance of different
hemoglobins
• Gower 1 (ζ2ε2): First 3 months of embryo
• Gower 2 (α2ε2): Most important embryonic
hemoglobin, first 3 months
• Hemoglobin Portland (ζ2γ2)
• Hemoglobin F (α2γ2): Appears in 5th week of IUL
peaks at 7th month(95%), at birth (80%), by 6
months totally replaced
• Hemoglobin A1 (α2β2): Appears in 5th month of
IUL
• Hemoglobin A2 (α2δ2): makes up 3 % of adult hb
Developmental profile of hemoglobins
 Variant forms of hemoglobin which
cause disease
• Hemoglobin H (β4) ‐ tetramer of β chains, which may be
present in variants of α thalassemia
• Hemoglobin S (α2βS2) ‐ β‐chain gene, causing a change in
the properties of hemoglobin which results in sickling of
red blood cells.
• Hemoglobin C(α2βC2) ‐ Variation in the β‐chain gene. This
variant causes a mild chronic hemolytic anemia.
• Hemoglobin AS ‐ A heterozygous form causing Sickle cell
trait with one adult gene and one sickle cell disease gene
• Hemoglobin SC disease ‐ Another heterozygous form with
one sickle gene and another encoding Hemoglobin C.
 Hemoglobin types

• HbA IC: Glycated hemoglobin(Glucose

attached to terminal valine in each beta chain
• Meth Hb: Fe++ changed to Fe+++
• Carboxy hemoglobin
Differences between adult and fetal
hemoglobin
 Age related changes in RBC
[1] Increased membrane bound IgG
[2] Increased cell density
[3] Increased intracellular sodium
[4] Decrease enzyme activity
[5] Decrease hb affinity for oxygen
[6] Decreased cell cholesterol
[7] Changes in MCHC and MCV
[8] Cell becomes more spherical
[9] Increased intracellular viscosity
10] Increased methemoglobin
[11]Decrease intracellular potassium
[13]Decrease in sialic acid
Mechanism of red cell destruction
Averege life span 120 days
4 major mechanisms of destruction
1. Osmotic lysis
2. Erythrophagocytosis
3. Complement induced cytolysis
4. Fragmentation
Heme oxygenase system responsible for
hemoglobin degradation is located in the
phagocytic cells of Liver, Spleen& Bone marrow
 Sites of erythrocyte destruction
• Extravascular hemolysis(80‐90 %)
Spleen, Liver, Macrophages, Lymph node and
Bone marrow
• Intravascular hemolysis
Hemoglobin is discharged directly into the
circulation & is removed by several
mechanisms
 Hemoglobin catabolism
Heme Globin

Heme oxygenase Amino acids

Biliverdin+iron+CO
Reductase
Excreted via lungs
Bilirubin
(Plasma levels: <1mg/dl)
Conjugated/Unconjugated
 Hepatic handling of bilirubin
3 steps
1. Uptake
2. Conjugation
Regurgitation
3. Excretion
Blood
1 2 3
GST
B‐Alb B BG BG Bilirubin BG BG
glucuronide Large
Alb GST intestine

Stercobilinogen

Feces 80% EH
circulation
20%
Hemoglobin catabolism: Intravascular
Hemoglobin
Haptoglobin Liver

Free hemoglobin

Methemoglobin
Kidney
Globin

Heme
Hemopexin
Hemopexin‐heme

Methemalbumin
 Jaundice
Definition : Yellowish discolouration of skin and
eyes due to an elevation in the concentration of
bilirubin in blood
Clinically detected only when bilirubin > 2.5 mg/dL
First site where it is detected : sclera
Types:
• Hemolytic
• Hepatic
• Obstructive
 Types of jaundice
Jaundice
Hemolytic Hepatic Obstructive
Fecal Increased Decreased Absent
stercobilinogen
Urinary Increased Decreased Absent
urobilinogen
Urinary bilirubin Absent Present Present
Liver function test Normal Impaired May be impaired
 Van den Berg test: Principle
Conjugated bilirubin + diazo reagent

Reddish violet coloured compound

Appears Immediately Doesn’t appear immediately

+
Alcohol
Direct positive Appearance of reddish violet colour
Indirect positive
Obstructive jaundice Hemolytic jaundice
 Jaundice
Hemolytic Hepatic Obstructive
Fecal Increased Decreased Absent
stercobilinogen
Urinary Increased Decreased Absent
urobilinogen
Urinary bilirubin Absent Present Present
Liver function test Normal Impaired May be impaired
Van den Bergh test Indirect Biphasic Direct
 Physiological jaundice
Neonatal jaundice
More common in premature & LBW babies
Appears on 2‐3rd day of life
Disappears within a week
Rarely exceeds 12 mg/dL
Cause: immaturity of Liver function
In utero the bilirubin formed is excreted mainly
by the placenta
 Oxygen transport by blood
• Dissolved oxygen is consumed first by cells in organs and tissues.
• Heme‐bound oxygen, which begins a sequential unloading of its
four oxygen molecules.
• During oxygen unloading, the hemoglobin tetramer undergoes
intramolecular conformational changes called cooperativity.
• Once the first oxygen has been unloaded, the unloading of the
second oxygen is facilitated. The second oxygen can dissociate after
a much smaller change in oxygen pressure than was needed to
unload the first. Another conformational change facilitates
dissociation of the third oxygen.
• Cooperativity is an important phenomenon that permits the loading
and unloading of large amounts of oxygen at physiologically
relevant oxygen pressures.
 Physiological significance of the
shape of the oxygen dissociation
curve
Flat upper part
The flat upper part acts as a buffer in the sense that the pO2 can drop
to about 80 mmHg and yet the haemoglobin will still remain highly
saturated (96%) with oxygen. This keeps the arterial oxygen
concentration high despite impairment in saturation in the lung.
Steep lower part
If the tissues require more oxygen, substantial amounts of oxygen
can be removed from haemoglobin without much further drop in pO2.
The pressure gradient for diffusion of oxygen from capillary to cell
tends to be relatively well maintained despite the much increased
oxygen extraction
 Physiological significance of the
shape of the oxygen dissociation
curve
Summary, the shape of the ODC provides this
double buffering effect because:
• The flat upper part tends to ‘buffer’ haemoglobin
saturation against a substantial drop in pO2. This
is useful in the lungs to maintain the arterial
haemoglobin saturation.
• The steep lower part has 2 advantages: Large O2
unloading & a maintained O2 diffusion gradient
(ie the pO2 gradient from capillary to cell).
• Haemoglobin binds with CO , 240 times more
readily than with oxygen.
• The presence of carbon monoxide on one of the
4 haem sites causes the oxygen on the other
haem sites to bind with greater affinity.
• This makes it difficult for the haemoglobin to
release oxygen to the tissues and has the effect of
shifting the curve to the left
• With an increased level of carbon monoxide, a
person can suffer from severe hypoxaemia while
maintaining a normal pO2.
Oxyhemoglobin dissociation curve
• When fully saturated with oxygen 1 gm of hb
carries 1.34 ml of O2
• 100 ml of arterial blood contains 20 ml O2
(hb = 15 gm%)
• 100 ml of venous blood contains 15 ml O2
• 5 ml or 25% O2 extracted by tissues
• If hb is only 7.5 gm % the O2 it contains is 10
ml
 Anemia
Definition
Deficiency of hemoglobin in blood as a result of
• too few RBCs(< 4million/cumm)
• too little hemoglobin
• WHO, 1992
• Hb < 7.0 g % severe anemia,
• 7.0 – 9.9 g % moderate anemia
• 10.0 – 10.9 g% mild anemia in pregnant women
and 10.0 –11.9g% for non‐pregnant women
 Manifestations of anemia
• Reduction in oxygen carrying capacity of blood
• Degree of change in the total blood volume
• Rate of development of the above two factors
• Associated manifestations of the underlying
disorder
• Capacity of the CVS and respiratory system to
compensate
 Mechanisms for compensation of the
loss of oxygen carrying capacity
• Increase in 2,3 DPG
• Redistribution of blood flow
• Increased cardiac output
• Insidious onset
Physiological adjustments in CVS
Changes in oxygen hemoglobin dissociation
curve
• Acute onset
Symptoms related to acute hemorrhage
Physical signs of chronic anemia
 Cardiac signs
In severe anaemia
Hyperdynamic circulation: a fast heart rate
(tachycardia), flow murmurs, and cardiac
enlargement. There may be signs of heart
failure
Atrial fibrillation
 Skin signs
• Pallor in the mucous membrane of the mouth,
• conjunctiva, lips & nail bed
• Skin may be pale in the absence of anemia or it may
fail to appear pallid in the presence of anemia
• Loss of normal skin elasticity & tone
• Thinning, loss of lusture & early greying
• Nails lose lusture, become brittle, cholioneychia
• Chronic leg ulcers
• Glossitis
• Fissures at the angles of mouth
• Jaundice in haemolytic anaemia
• Bone deformities (found in thalassaemia major) or
 Neuromuscular signs
Severe anemia
Headache, vertigo, tinnitus, fainting,
scotomas, lack of mental concentration,
drowsiness, restlessness, muscular weakness
Paresthesia
 GIT signs
Glosssitis & atrophy of the papilla of the tongue
Painful ulcerative & necrotic lesions
Dysphagia
 Genitourinary signs
Slight proteinuria
Microscopic hematuria
 Classification of anemia:
Based on underlying mechanism
• Blood loss
Acute: Trauma
Chronic: Lesions of GIT,hook worm infestation,
gynaecological
• Hemolytic anemia
Intracorpuscular defect: Membrane defect, Enzyme
defect, Hb defect
Extracorpuscular defect: Antibody mediated,
mechanical trauma, infections
• Impaired production
Disturbance of proliferation & differentiation
Disturbance of proliferation & maturation
 Signs
• Pallor (pale skin, mucosal linings and nail
beds)
• Koilonychia(in iron deficiency),
• Leg ulcers (seen in sickle cell disease).
Morphological classification of anemia
Normochromic Hypochromic
Normocytic Recent blood loss After chronic hemorrhage
All hemolytic anemias except
thalassemia
Aplastic anemia
Endocrine abnormalities

Macrocytic Megaloblastic anemia Secondary to Liver disease

MCV > 100
Microcytic Chronic infections Iron defficiency anemia
MCV < 80 Thalassemia
 Signs & symptoms of blood loss
anemia
Acute blood loss
• >30% loss sudenly: Postural hypotension, increase heart rate
• > 40% blood loss: Hypovolumic shock, confusion, air hunger,
diaphoresis, decrease hemoglobin, increase heart rate, CNS hypoxia
leading to headache, dimness of vision & faintness
Chronic blood loss
• Forceful apical pulse
• Strong peripheral pulse
• Wide pulse pressure
• Mid/holosystolic murmur
• Pallor: Skin & mucous membranes
 Hemolytic anemia
Intracorpuscular defect
 Membrane defect: Spherocytosis
 Enzyme defect: Pyruvate kinase def., hexokinase
def.
 Hb defect: Thalassemia, sickle cell anemia
Extracorpuscular defect
 Antibody mediated: transfusion mediated,
erythroblastosis
 Mechanical trauma
 Infections : Malaria
 Hemolytic anemia
Clinical features
• Hemoglobinemia
• Hemoglobinuria
• Methhemoglobinemia
• Jaundice: Unconjugated hyperbilirubinemia
• Hemosiderinuria
 Specific types of hemolytic anemias
Spherocytosis
Thalassemia
Sickle cell anemia
Extracorpuscular defect
Erythroblastosis
Mechanical trauma
Malaria
 RBC membrane structure

Protein 4.1 binds spectrin to glycophorin C

Protein 2.2(Ankyrin) binds spectrin to anion exchanger band 3
 Spherocytosis
• Deficiency of spectrin due to a primary defect
in
• Ankyrin gene
• Protein 3
Effect: reduced membrane stability/ plasticity
Premature lysis of the cells in spleen
C/F: anemia, spleenomegaly & jaundice
Spherocytosis
Spleenic architecture
Normal

Increased
fragility
 Increased osmotic fragility in
hereditary spherocytosis
100
80
% Hemolys

60
40
20
0
0.3 0.4 0.5 0.6
NaCl (%of normal saline)

Normal HS
 Lab investigations
• Anemia of increased destruction
– Normocytic, normochromic anemia
– Shortened RBC survival
– Reticulocytosis ‐ Response to increased RBC
destruction
– Absent haptoglobin
 Thallasemia
• Beta
• Alpha
 Thalassemia
• Genetic blood disorder resulting in a mutation or
deletion of the genes that control globin production.
• Normal hemoglobin is composed of 2 alpha and 2 beta
globins
• Mutations in a given globin gene can cause a decrease in
production of that globin, resulting in deficiency
• aggregates become oxidized  damage the cell
membrane, leading either to hemolysis, ineffective
erythropoiesis, or both.
• 2 types of thalassemia: alpha and beta.
 Demographics
• The thalassemia gene may be maintained in
the human population, in part because of the
greater immunity of heterozygous individuals
against malaria and is found in parts of the
world where malaria is common
• These include Southeast Asia, China, India,
Africa, and parts of the Mediterranean.
Inheritance of thalassemia
 Alpha Thalassemia
• mutation of 1 or more of the 4 alpha globin
genes on chromosome 16
• severity of disease depends on number of
genes affected
• results in an excess of beta globins
 Silent Carriers (heterozygotes ++/+‐)

• 3 functional alpha globin genes

• No symptoms, but thalassemia could
potentially appear in offspring
 Alpha Thalassemia Trait (++/‐‐)
• 2 functional globin genes
• results in smaller blood cells that are lighter in
colour
• no serious symptoms, except slight anemia
 Hemoglobin H Disease (+‐/‐‐
• 1 functional globin gene
• results in very lightly coloured red blood cells
and possible severe anemia
• hemoglobin H is susceptible to oxidation,
therefore oxidant drugs and foods are avoided
• treated with folate to aid blood cell
production
 Alpha Thalassemia Major (‐‐/‐‐
• no functional globin genes
• death before birth (embryonic lethality):
Hydrops fetalis, Hb Bart
Alpha thallasemia
 Beta Thalassemia
• mutations on chromosome 11
• results in excess of alpha globins
 Beta Thalassemia Trait 0
(β / β)
• slight lack of beta globin
• smaller red blood cells that are lighter in colour
due to lack of hemoglobin
• no major symptoms except slight anemia
• Beta thalassemia trait is seen most commonly in
Mediterranean (including North African, and
particularly Italian and Greek), Middle Eastern,
Indian, African, Chinese, and Southeast Asian
(including Vietnamese, Laotian, Thai,
Singaporean, Filipino, Cambodian, Malaysian,
Burmese, and Indonesian
 Beta Thalassemia Intermedia (β+/ β+)
• lack of beta globin is more significant
• bony deformities due to bone marrow trying to make
more blood cells to replace defective ones
• causes late development, exercise intolerance, and high
levels of iron in blood due to reabsorption in the GI tract
• if unable to maintain hemoglobin levels between 6 gm/dl
– 7 gm/dl, transfusion or splenectomy is recommended
 Beta Thalassemia Major 0
β/ β0

• complete absence of beta globin

• enlarged spleen, lightly coloured blood cells
• severe anemia
• chronic transfusions required, in conjunction
with chelation therapy to reduce iron
(desferoxamine)
 Clinical features
• Severe hypochromic anemia with spleenomegaly and
markedly elevated levels of HbF.
• Family studies show both parents as carriers of the
beta‐thalassemic trait, which is marked by mild,
microcytic, hypochromic anemia and high levels of
HbA2.
• Early signs and symptoms are associated with the
anemia, which is characterized by hypochromic,
microcytic red cells with variable numbers of nucleated
erythrocytes and reticulocytes.
• Pallor, icterus, and cardiac enlargement occur
frequently.
 Clinical features
• Marrow hypertrophy and extramedullary
hematopoiesis may result in
hepatosplenomegaly
• Skull deformities
• Facial deformity: Prominence of malar
eminences and mal alignment of teeth, which
gives rise to the characteristic "rodent facies."
 Clinical features
• Iron overload
• Infections such as hepatitis
• Bone deformities
• Enlarged spleen
• Slowed growth rate
• Heart problems
Widening of the calvarium
New bone formation producing a "hair‐on‐end" appearance
 More Permanent Options
 Bone Marrow Transplants
 Replacing patient’s marrow with donor marrow
 First performed on thalassemia patient in 1981
 Difficult, because donor must be exact match for
recipient
 Even a sibling would only have a 1 in 4 chance of
being a donor
 Cord Blood Transplants
 Rich in stem cells
 Also needs to be an exact match
Sickle cell anemia or HbSS
or SS disease
 Heterozygous population
• Hb AS : Sickle cell trait
• Hb SC: Sickle Hb C disaease
• HbS/ β+ : Sickle beta plus thalassemia
• HbS/ β0 : Sickle beta zero thalassemia
 Signs & symptoms
• Anemia
• Crisis
Vaso‐occlusive
Sequestration
Aplastic
Hemolytic
• Complications
 Vaso‐occlusive crisis
• Due to sickled RBCs which obstruct blood flow
and lead to ischemia in several organs
• Bone, lung, spleen, brain, spinal cord, digits
• Painful dactylitis is the first manifestation
 Spleenic sequestration crisis
• Acute painful enlargement of the spleen
• May occur in the liver also
• An emergency
 Aplastic crisis
• Transient arrest of erythropoiesis resulting in
reticulocytopenia
• Typically preceeded by fever & upper
respiratory or GIT infection
• Often due to Parvo virus B19 infection
 Hemolytic crisis
• Increased rate of hemolysis with fall of
hemoglobin but increase in reticulocyte count
• Usually accompany a painful crisis
Complications of sickle cell disease
Chronic hemolysis Vaso‐occlusive
• Anemia • Pain syndrome
• Pigment gall stones • Acute chest syndrome
• Aplastic episodes • Priaprism
• Jaundice • Stroke
• Retinopathy
• Delayed growth
• Avascular hip necrosis
• Spleenic sequestration
• Leg ulcers
• Chronic nephropathy
 Early symptoms & complications
• Typically in 1st year of life
• Dactylitis
• Fever
• Pain in chest, abdomen, limb & joints
• Enlargement of, heart, Liver, spleen
• Frequent URI
• Chronic anemia as child grows older
Clinical features of sickle cell disease
 Sickle cell anemia
• Symptoms experienced throughout childhood
and adulthood can include: fatigue,
breathlessness, jaundice, paleness,
susceptibility to infections, hand and foot
swelling, painful joints, hands, arms, legs, and
back, chest syndrome (pain in the chest wall),
priapism (prolonged, painful erections),
anemia and “pain crises
Prevalence of disease
Sickling of RBC in circulation
Polymers of Hemoglobin S
Sickle cell
 Prevention
• Daily folic acid supplement
• Daily Pencillin till 6 yrs
• Plenty of water
• Avoid over exertion & stress
• Plenty of rest
• Regular check ups
 Treatment
• Analgesia for painful episodes
• IV antibiotics
• Vaccination
• Correction of NO deficit
• Induce HBF synthesis
• Bone marrow transplantation
• Gene therapy
Malarial parasite within RBC
 Malaria and sickle cell anemia
• It is likely that P. falciparum, the parasite
responsible for malaria, decreases oxygen in red
blood cells it infects.
• As a result of low oxygen concentrations,
hemoglobin S within cells polymerizes, forming a
sickled cell.
• These cells are then marked for cell death since
they are unhealthy, and the parasite‐infected cells
are destroyed before they can cause harm
 Anemia due to impaired production
Disturbance of proliferation & differentiation
Aplastic anemia, anemia of renal failure,
anemia of endocrine disorders
Disturbance of proliferation & maturation
Defective DNA synthesis: Megaloblastic
anemia
Defective heme synthesis: Iron defficiency
Defective globin synthesis: Thalassemia
Classification of megaloblastic anemia
• Cobalamine deficiency
Inadequate intake : rare
Malabsorption: Pernicious anemia
Decreased intrinsic factor Gastrectomy
Congenital absence
Disorders of terminal ileum: tropical sprue,
regional enteritis
Competition for cobalamine: fish tape worm,
blind loop syndrome
 Vitamin B12 absorption
• Daily requirement 2‐3 µg
• Source: Animal products
• Absorption: Protein bound Vit. B12
Pepsin
Stomach
Vit. B12
R binders
R‐B12 complex
Duodenum Pancreatic protease
Vit. B12
Intrinsic factor
Vit. B12‐IF complex
Ileum
Absorbed in ileum
 Folic acid
• Folic acid defficiency (can develop quickly)
Inadequate intake
Increased requirement: pregnancy, infancy,
malignancy, hemodylasis
Malabsorption: Tropical sprue, phenytoin,
barbiturates
Impaired metabolism: Methotrexate
 Folic acid/ pteroylglutamic acid
• Animals can not synthesise folates
• Source: Plants (Polyglutamate conjugates)

Diet Intestine Intestinal cell

Carboxy Folate
peptidase reductase
Polyglutamates Monoglutamates Tetrahydro folate
 Role of folic acid in DNA synthesis:
DNA cycle

DNA

Methyl group
dUMP dTMP

Glycine Serine
Methyl

THF transfers methyl group from serine to uridine

 DNA cycle
dUMP dTMP

DHF
Folate reductase

Methylene THF THF

Glycine Serine

Serine transfers methylene group to THF

Methylene THF transfers the methyl group to uridine
Regeneration of THF from DHF occurs in the presence of folate reductase
Unlike the methylation cycle, the DNA
cycle does not depend on vitamin B12.
Folic acid can thus maintain the supply of
intracellular folate required for DNA synthesis. DNA
synthesis, and hence cell replication, can therefore
take place in people with vitamin B12 deficiency,
provided that folic acid is available as a source of
folate. This is why, in people with vitamin B12
deficiency, folic acid supplementation will treat the
megaloblastic anaemia (due to deficient cell
replication), but will not affect the neurological
complications which occur as a result of the
disruption of the methylation cycle.
 Methyl trap
dUMP dTMP

DHF
Folate reductase

Methylene THF THF

Methyl
THF Glycine Serine
 Action of B12
Methyl
THF
THF

B12
Methyl
B12

Methionine Homocystein

Excess methionine
S‐adenosyl
methionine

Recovery of THF from the methyl trap

S‐adenosyl methionine production
 The methylation cycle
Depends on both folate and vitamin B12 to
produce methionine
An example of a methylation reaction is the
methylation of the protein in myelin (the
insulation cover on nerves). When this process
is interrupted, as it is during vitamin B12
deficiency, one of the clinical consequences is
the demyelination of nerves, resulting in a
neuropathy, which leads to ataxia, paralysis
and, if untreated, ultimately to death.
Another methylation reaction involves the
degradation of methionine.
Any excess methionine is degraded to
homocysteine.
Homocysteine can be either degraded to form
pyruvate which can then be used as a source of
energy, or it can be remethylated to form
methionine
Vitamin B6 is essential in the former reaction, and
vitamin B12 and folate in the latter.
 Features common to all forms of
megaloblastic anemia
• Anisocytosis
• Normochromic
• Macrocytes(MCV > 100) and oval shaped
• Lower reticulocyte count
• Hypersegmented neutrophil
• Dissociation between nuclear and cytoplasmic
maturation
 Features common to all forms of
megaloblastic anemia….
• Accumulation of megaloblasts in bone marrow
• Inefective erythropoiesis
• Increased hemopoietic destruction

• Leucopenia
• Thrombocytopenia
 Pernicious anemia
Immunologically mediated ,autoimmune
destruction of gastric mucosa
Morphological changes in
• Alimentary system
Tongue: shiny, glazed &“beefy”(atrophic
glossitis)
Stomach: Atrophy of fundic glands
• Bone marrow: Hemosiderosis
 Pernicious anemia...
• Classical neurological features
• Poly neuropathy progressively involving the
peripheral nerves and the posterior and
eventually the lateral columns of the spinal cord
(subacute combined degeneration)
• Symmetrical paraesthesiae in the fingers and toes
• early loss of vibration sense and proprioception
• progressive weakness and ataxia
• Paraplegia
• Dementia and optic atrophy
 Clinical course of megaloblastic
anemia
Insidious in onset
• Moderate to severe megaloblastic anemia
• Leukopenia with hypersegmented neutrophil
• Thrombocytopenia
• Neurological changes associated with involvement of
posterolateral spinal cord
• Achlorhydria
• Inability to absorb an oral dose of cobalamine (Schillings
test)
• Low Vitamin B12 levels
• Methyl malonic acid excretion in urine
• Dramatic response on parenteral administration of Vit. B12
 Investigations
• Haematological findings show the features of a
megaloblastic anaemia
• Bone marrow shows the typical features of megaloblastic
erythropoiesis
• Serum bilirubin may be raised as a result of ineffective
erythropoiesis.
• Serum vitamin B12 is usually well below 160 ng/L, which is
the lower end of the normal range. Serum vitamin B12 can
be assayed using radioisotope dilution or immunological
assays.
• Serum folate level is normal or high, and the red cell folate
is normal or reduced owing to inhibition of normal folate
synthesis
 Investigations
• FIGLU test
Histidine FIGLU Glutamate

THF

Oral challenge of Histidine

Increased urinary excretion of FIGLU in folate defficiency
as well as B12 defficiency
 Investigations
• Schilling’s test
Radiolabelled B12 orally
Measuring radioactivity in urine
 Folic acid/pteroylmonoglutamic acid
Neurological changes not seen
Prime function: To act as intermediates in
transfer of 1 C moieties i.e. methyl & formyl
groups to various organic compounds
1 C moieties are used as building blocks in the
synthesis of biological macromolecules
• Daily requirement of folic acid 50‐200 micro g
• Source: green vegetables i.e. lectuce, spinach,
asparagus & broccoli
Fruits: lemons, banana, melons
Polyglutamate form in diet
Absorbed as 5‐methyltetrahydrofolate
 Iron deficiency anemia
• Most common form of nutritional deficiency
in developed & developing countries
• 1.0 ml of blood may be considered to contain
0.5 mg iron
 Iron balance
Unique:
Balance achieved by a control of absorption
Absorption
In proximal jejunum
Only 5% of ingested iron is absorbed
 Absorption of iron
• Non heme iron
Mainly in Fe 3+ form
Must be converted to Fe 2+ before absorption
• Dietary factors enhancing non heme iron absorption
 Acsorbate
 Meats & fish
 Human breast milk
 Acidic gastric juice

• Dietary factors inhibiting non heme iron absorption

 Phytates in grains and vegetable food
 Polyohenols in legumes, tea, coffee & wine
 Phosphates
 Calcium
 Egg white & bovine milk proteins
 Absorption of iron…
• Heme iron
10 – 15% of iron in non vegetarian diets
Unaffected by composition of the diet Ferritin

Apoferritin
Heme +apoprotein Acid & protease Hemin Heme oxygenase Free Fe2+ pool
(enterocyte)

Hemopexin bound Fe
(Plasma)
 Regulation of mucosal absorption
• Intestinal mucosal cell is programmed to
absorb iron in proportion to the body’s iron
requirement esp. rate of erythropoiesis
Modified mucosal block theory
Unaccepted Fe

Fe in gut lumen Body Fe

Normal

Fe deficient

Fe overload
 Iron

Pinocytosis
• Total Iron Binding Capacity (TIBC)
Clinically the amount of transferrin is expressed
in terms of amount of iron it will bind
• Storage iron proteins
Ferritin
Hemosiderin
 Etiology
Negative iron balance
Decreased Fe intake Increased Fe loss
Inadequate diet GIT bleeding
Impaired absorption Excessive menstrual flow
Blood donation
Disorders of hemostasis

Idiopathic hypochromic anemia

Increased requirement
Infancy
Pregnancy
Lactation
 Etiology…..
• GIT infection with hookworm: Necator
americanus or Ancylostoma duodenale(0.2
ml/worm/day)
• Other worms: Schistosoma mansoni & S.
hematobium , Trichuris trichura
• Excessive menstruation: use of > 12 pads /d,
Passage of clote > 2 cm diameter after the first
day, Duration> 7 days
 Etiology….
• Blood donation Each unit of blood donated
contains approx. 250 mg of iron
• Pergnancy and lactation : Most of the Fe loss
occurs during the third trimester (3‐7.5
mg/day)
• Lactation : Daily blood loss 0.5‐1.0 mg
 Stages in the development of iron
deficiency
• Depletion of the iron stores in the hepatocytes
and macrophages of spleen, liver and bone
marrow
• Decrease in plasma iron content leading to
inadequate supply of iron to bone marrow for
regeneration of hemoglobin
• Increase in free erythrocyte protoporphyrin
and decrease in blood hemoglobin levels
 Clinical manifestations
• Growth: Impaired growth in infancy
• Neuromuscular system: Impaired muscular performance as
measured by standardized exercise tests
• Epithelial tissue
• Nails: Brittle, longitudinally ridged, thinning, flattening,
kolionychia(Spoon shaped nails)
• Tongue & mouth: Atrophy of the lingual papillae, angular
stomatitis
• Dysphagia: Difficulty in swallowing solid foods but little
problem in swallowing liquids
• Stomach: Presence of gastritis & reduction in gastric
secretion
 Clinical manifestations…
• Immunity & infection: Defective cell mediated
immunity & impaired bacterial killing by
phagocytes
• Pica
• Spleen: Enlarged in 10% patients
• Genitourinary system: Frequent disturbances
in micturition
• Skeletal system: Changes similar to those
found In Thalassemia
 Laboratory investigations
• Microcytic hypochromic anemia
• MCV <80 Fl, MCH<25 gm/dl
• Serum iron < 30 micro grams/dL(Normal:50‐150)
• Total iron binding capacity: raised
• % saturation of transferrin:<10%(30‐50)
• S. ferrritin: <15 microgram/L
• Cigar / pencil RBCs
• With iron treatment, reticulocyte counts increase
after 3‐4 days, peak at 10 days
 Aplastic anemia
• Acquired
Chemical &physical agents: Benzene,
radiations, antifolic compounds
Other causes: Viral infections(Hepatitis, EBV,
HIV)
• Familial
Fanconis anemia, pancreatic deficiency in
children
 Symptoms & signs
• Anemia , bleeding, fever , infections
• Weakness, fatigue
• Bleeding from the, nose, mouth, GIT
• Ulcerations in the mouth & pharynx
 Polycythemia/Erythrocytosis
Increase RBC & Hb levels
Classification
• Relative: Reduced plasma volume
• Absolute
Primary: Abnormal proliferation of myeloid
stem cells(polycythemia vera)
Secondary: Lung disease, high altitude,
erythropoietin secreting tumour
 Polycythemia vera
• Increase RBC
• Increase blood volume
• Increase viscosity
• Increase hematocrit
Effect of polycythemia on function of the
cardiovascular system
Cardiac output
Arterial pressure
Colour of skin