ANEMIA

(Definition , Classification , Clinical

features)

Dr. PRIYANKA SACHDEV , MD

 Definition
• Anemia is defined as a reduction of the total circulating
red cell mass below normal limits

• In practice, the measurement of red cell mass is not easy,

and anemia is usually diagnosed based on a reduction in
hemoglobin concentration

• Anaemia is defined as reduced haemoglobin

concentration in blood below the lower limit of the
normal range for the age and sex of the individual.
• The lower extreme of the normal haemoglobin is taken
as →

➢13.6 g/dl for males

➢12.0 g/dl for females
➢15 g/dl for newborns
Morphology of RBC
• Normal RBC is biconcave with diameter of 7 to 8 um.
• Biconcave shape is due to spectrin protein.

• The hemoglobin of red cells is located peripherally, leaving an area of

central pallor equal to approximately 30-35% of diameter of the cells
( Central 1/3rd pallor)

• The lifespan of red cells is 120 + 30 days.

 VARIATIONS IN SIZE

• RBC of normal size → Normocytic

• When red cell diameter is greater than 9 μm they are

referred as macrocytes

• When red cell diameter is less than 6 μm they are

referred as microcytes
 REMEMBER

• On smear size of RBC is compared with small

lymphocyte
 VARIATIONS IN COLOUR

• RBC with normal hemoglobin content (color) ( Central

1/3rd pallor) → Normochromic

• When area of central pollar is greater than 50% of

diameter, it is referred as hypochromic
• Variation is size of RBCs is know as anisocytosis

• Variation in shape of RBCs is know as poikilocytosis

 • Thalassemia, Hemoglobinopathies
• Post splenectomy
Target cells
• Liver disease
• Artefact
• G6PD deficiency
Bite cells • Unstable haemoglobin disorders
• Oxidative drugs
• Sickle cell anemia
Sickle cell
• S-beta thalassemia
• Myelofibrosis
Teardrop cell
• Underlying marrow infiltrate
• Abetalipoproteinemia
• Liver disease
Spurr cell (Acanthocyte)
• Post splenectomy
• McLeod blood group phenotype
 • Usdaia
Burr cell (Echinocyte) • Artefact
• Liver disease
• Haemoglobin C disease
Burr cell (Echinocyte)
• Haemoglobin SC disease

• Chronic liver disease

• Malignant lymphoma, Multiple
Rouleaux formation
myeloma
• Chronic inflammatory disease

• Cold autoimmune haemolytic

anemia
RBC agglutination • Paroxysmal cold hemoglobinuria
• IgM associated lymphoma
• Multiple myeloma
 CLASSIFICATION OF ANAEMIAS

1. MORHOLOGICAL CLASSIFICATION

2. PATHOPHYSIOLOGICAL CLASSIFICATION
 MORPHOLOGIC CLASSIFICATION

i. Microcytic, hypochromic
ii. Normocytic, normochromic
iii. Macrocytic, normochromic
 MORPHOLOGIC CLASSIFICATION
1.Microcytic, hypochromic
• MCV, MCH, MCHC are all reduced

2.Normocytic, normochromic
• MCV, MCH, MCHC are all normal

3. Macrocytic, normochromic
• MCV is raised
PATHOPHYSIOLOGICAL/ ETIOLOGICAL
CLASSIFICATION
 Anemia

Blood Loss ↓Production ↑Destruction

Acute Chronic

Cytoplasmic Nuclear Stem cell

defect defect proliferation defect

Heam deficit Globin deficit Megaloblastic Aplastic anemia

Anemia
Iron deficiency anemia Thalassemia
Sideroblastic anemia
Anemia of chronic disease
 HEMOLYTIC ANEMIAS

Hereditary Acquired

Ab in RBC membrane Ab in RBC Interior AIHA

- Spherocytosis MANA
PNG
Direct toxin
Defect in enzyme Defect in Hb Splenomegaly
G6PD

Qualitative Quantitative
SCC Thalassemia
 Classification of anemias according to MCV

Microcytic Normocytic Macrocytic

S - Sideroblastic
I - Iron deficiency S – Sickle cell anemia
T - Thalassemia H – Hereditary spherocytosis
A - Anemia of A – Autoimmune hemolytic
chronic disease anemia
P – Paroxysmal noctumal
hemoglobinuria
E – Enzyme deficiency
(most important is
G-6PD deficiency
L - Liver disease
H - Hypothroidism
M - Megalobastic anemia
C - Cytotoxic drugs like
Methotrexate and other
drugs like phenytoin
 HEMOLYTIC ANEMIA
(Definition , Classification , lab
diagnosis)

Dr. PRIYANKA SACHDEV , MD

 HAEMOLYTIC ANAEMIAS

• Haemolytic anaemias are defined as anaemias resulting

from an increase in the rate of red cell destruction
 Characteristic features of haemolytic anaemia

• 1. A shortened red cell life span, i.e. premature destruction

of red cells.

• 2. Elevated erythropoietin level and inceased

erythropoiesis in the marrow and other sites, to
compensate the loss of red cells → characterized by
increased reticulocyte count.

• 3. Accumulation of products of hemoglobin catabolism

 RBC destruction

Destruction inside blood Destruction inside mononuclear

Vessels or in the circulation phagocytic system

Intravascular hemolysis Extravascular hemolysis

 Intravascular hemolysis
RBC destruction in the blood vessels

↑ Free Hb in serum ↑ Unconjugated bilirubin

Jaundice
↑ Risk of
bilirubin
Hemoglobinemia ↑ Complex with ↑ Oxidation of or pigment
haptoglobin Hb into gallstones
↑ Hb excretion which is cleared methemoglobin
in urine in the RES
Methemoglobinuria
↓ Haptoglobin
Hemoglobinuria Methemoglobinemia
↓ Hemopexin
 Extravascular hemolysis

RBC destruction in reticuloendothelial system

• Anemia ↑ Destruction ↑ Destruction

• Jaundice in spleen in liver No free Hb released in
circulation
• No hemoglobinuria
• No hemoglobinemia
Splenomegaly Hepatomegaly • No methemoglobinuria
 HEMOLYTIC ANEMIAS

Hereditary Acquired

Ab in RBC membrane Ab in RBC Interior AIHA

- Spherocytosis MANA
PNG
Direct toxin
Defect in enzyme Defect in Hb Splenomegaly
G6PD

Qualitative Quantitative
SCC Thalassemia
MCQs
1. All are features of Intravasular hemolysis
except
a. Thrombocytopenia
b. Hemosiderinuria
c. Decreased haptoglobin
d. Raised indirect bilirubin
1. All are features of Intravasular hemolysis
except
a. Thrombocytopenia
b. Hemosiderinuria
c. Decreased haptoglobin
d. Raised indirect bilirubin
2. Extravascular hemolysis causes
a. Hemoglobinemia
b. Hemosiderinuria
c. Jaundice
d. Hemoglobinemia
2. Extravascular hemolysis causes
a. Hemoglobinemia
b. Hemosiderinuria
c. Jaundice
d. Hemoglobinemia
3. All are features of hemolytic anemia, except
a. Hemoglobinuria
b. Jaundice
c. Increased haptoglobulin
d. Hemosiderinuria
3. All are features of hemolytic anemia, except
a. Hemoglobinuria
b. Jaundice
c. Increased haptoglobulin
d. Hemosiderinuria
Hereditary Spherocytosis

Dr. PRIYANKA SACHDEV , MD

 Introduction

• Hereditary spherocytosis is a common type of hereditary

haemolytic anaemia
• It is autosomal dominant inheritance

• In this the red cell membrane is abnormal

 PATHOGENESIS

• Membrane cytoskeleton that lies closely opposed to the

internal surface of the plasma membrane, is responsible
for elasticity and maintenance of RBC shape.
 Membrane skeleton consists

• 1. Spectrin →The chief protein component responsible

for biconcave shape
• 2. Ankyrin and band 4.2 →Binds spectrin to band 3
• 3. Band 3 →A transmembrane ion transport protein.
• 4. Band 4.1 →Binds spectrin to glycophorin A, a
transmembrane protein.
Spectrin, which has two subunits a and β
• This spectrin is attached to cell membrane at two sites→

1. Spectrin binds to ion transporter, band 3 of membrane

with the help of ankyrin and band 4.2
2. Spectrin binds to glycophorin A of the membrane by
protein 4.1 and actin
• Mutation most commonly involves the gene coding for →
Ankyrin > Band-3 > Spectrin >Band 4.2 (also called paladin)
 REMEMBER

• Most common defect in hereditary Spherocytosis is in

Ankyrin.

• Most common defect in hereditary elliptocytosis is in

spectrin.
 Mutation

Loss of membrane cytoskeleton proteins (ankyrin, spectrin, Band 3, 4.2)

Reduced membrane stability

Loss of membrane relative to cytoplasm during exposure to shear stresses

Cells become microspherocytes (smallest possible diameter for a given volume )

Trapped in to spleen (Due to reduced deformability)

RBC are phagocytosed by RE cells

Extravascular hemolysis
 LABORATORY FINDINGS
Extravascular hemolysis

• 1. Mild to moderate chronic anaemia

• 2. Reticulocytosis.
• 3. Unconjugated (indirect) hyperbilirubinaemia.
• 4. Spleenomegaly
• 5. Increase in MCHC
 Morphology of RBC

• Blood film shows the characteristic abnormality of

erythrocytes in the form of microspherocytes
• Spherocytosis —> Peripheral smear shows
microspherocytes which are small RBCs without central
pallor (Normally central 1/3 pallor is present in red cells).
 Special test
• 1. Osmotic fragility test / Pink test is helpful in testing the
spheroidal nature of red cells

• RBCs lyses more readily in solutions of low salt

concentration i.e. osmotic fragility is increased
• 2. Autohaemolysis test is similar to osmotic fragility
test after incubation and shows increased
spontaneous autohaemolysis (10-15% red cells) as
compared to normal red cells (less than 4%).
• 3. Direct Coombs’ (antiglobulin) test is negative so as
to distinguish this condition from acquired
spherocytosis of AIHA in which case it is positive.
 Conditions with spherocytosis

• Autoimmune hemolytic anemias

• Infections (malaria)
• Burns
• ABO hemolytic disease (NOT with Rh hemolytic
disease)
• G6PD deficiency
• Hereditary spherocytosis
4. A characteristic feature of HS is increase in MCHC due
to dehydration caused by loss of K+ and water.

• Hereditary spherocytosis is the only important anemia in

which MCHC is increased.
2. Shape of RBC is biconcave due to?
a. Ankyrin
b. Spectrin
c. Band protein
d. Glycophorin-C
2. Shape of RBC is biconcave due to?
a. Ankyrin
b. Spectrin
c. Band protein
d. Glycophorin-C
3. Most common cause of hereditary
spherocytosis?
a. Spectrin
b. Glycophorin
c. Ankyrin
d. B and 4
3. Most common cause of hereditary
spherocytosis?
a. Spectrin
b. Glycophorin
c. Ankyrin
d. B and 4
7. In hereditary spherocytosis an inherited
abnormality is seen in which of the following
red blood cell component:
a. a-globin chain
b. b-globin chain
c. Phosphatidyl inositol glycan A
d. Spectrin
7. In hereditary spherocytosis an inherited
abnormality is seen in which of the following
red blood cell component:
a. a-globin chain
b. b-globin chain
c. Phosphatidyl inositol glycan A
d. Spectrin
8. Hereditary spherocytosis is due to:
a. Acquired membrane defect
b. Ankvrin deficiency
c. Defective hemoglobin synthesis
d. Mechanical trauma to red cells
8. Hereditary spherocytosis is due to:
a. Acquired membrane defect
b. Ankvrin deficiency
c. Defective hemoglobin synthesis
d. Mechanical trauma to red cells
PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA
(PNH)

• PNH is the only hemolytic anemia caused by an

acquired intrinsic defect in the cell membrane.

• PNH results from acquired mutation that inhibits the

synthesis of Glycosylphospatidylinositol (GPl)
2 anchoring proteins called complement regulating
proteins →
1. Decay accelerating factor (DAF, CD55)
2. A membrane inhibitor of reactive lysis (MIRL, CD59)

• These protective proteins prevent activation of

complement
• So they prevent complement mediated lysis of blood cells.

• These proteins are anchored to cell membrane of blood

cells by GPI
 Myeloid progenitor cell lineage (RBCs, WBCs, platelets)

Mutation of PIG-A (phosphatidyl inositol glycan) gene

Synthesis of glycosyl phosphatidyl inositol (GPI) does not occur

Deficiency of GPI results in absence of complement regulating proteins

(CD55 , CD59) on blood cells

Blood cells become unusually sensitive to complement mediated lysis

Intravascular hamolysis
 Clinical features

1. The hemolysis is→

a) Paroxysmal (intermittent attacks)

b) Nocturnal (occurs in the night) → Because during
sleep the pH of blood gets slightly reduced and
acidic medium leads to activation of the
complement.
2. Pancytopenia
• Because the causative somatic mutations occur in
pluripotent stem cells, all its clonal progeny i.e., red cells,
white cells, and platelets are deficient in GPI linked
protein.

• So, all these cells are sensitive to complement mediated

lysis that results in pancytopenia → Anemia, Leucopenia,
Thrombocytopenia
3. Thrombosis

• Due to absence of CD-59 on platelets, this results in

externilization of phosphotidylserine →thrombosis
• Intrabdominal veins are the most common sites of
thrombosis that may result in Budd chiary syndrome
due to hepatic vein thrombosis.
 Triad of PNH

1. Hemolysis
2. Pancytopenia
3. Thrombosis
 LABORATORY FINDINGS
Intravascular hemolysis

• 1. Mild to moderate chronic anaemia

• 2. Reticulocytosis.
• 3. Unconjugated (indirect) hyperbilirubinaemia.
• 4. Intravascular haemolysis → haemoglobinaemia,
methaemoglobinaemia, haemoglobinuria, and
haemosiderinuria
 Screening test

1. Ham test
2. Sucrose lysis test
 Ham test
• Acidic pH will activate complement pathway for RBC lysis.
 Sucrose lysis test

• Sucrose will reduce pH and this will activate complement pathway for hemolysis.
 Confirmatory test

Flow cytometry

• PNH is definitively diagnosed by flow cytometry

• It provides a sensitive means for detecting red cells that

are deficient in GPI-linked protein such as CD59 and CD55

• Bimodal distribution of the red cells

 REMEMBER
• PNH can lead to AML or MDS/aplastic anemia

• Bone marrow transplantation can be curative

MCQs
1. Triad of PNH includes all except
a. Pancytopenia
b. Renal failure
c. Hemolysis
d. Venous thrombosis
1. Triad of PNH includes all except
a. Pancytopenia
b. Renal failure
c. Hemolysis
d. Venous thrombosis
2. Ham test is done for
a. PNH
b. Megaloblastic anemia
c. Sickle cell anemia
d. Thalassemia
2. Ham test is done for
a. PNH
b. Megaloblastic anemia
c. Sickle cell anemia
d. Thalassemia
3. PNH associated with somatic mutation
affecting:
a. Decay accelerating factor
b. Membrane inhibitor of reactive lysis (MIRL)
c. Glycosylphosphatidylinositol (GPI)
d. C8 binding protein
3. PNH associated with somatic mutation
affecting:
a. Decay accelerating factor
b. Membrane inhibitor of reactive lysis (MIRL)
c. Glycosylphosphatidylinositol (GPI)
d. C8 binding protein
5. PNH due to defect in:
a. CD 59
b. CD 15
c. CD 100
d. CD 20
5. PNH due to defect in:
a. CD 59
b. CD 15
c. CD 100
d. CD 20
 Autoimmune Haemolytic Anaemia (AIHA)

• Autoimmune hemolytic anemia is caused by

autoantibodies directed against a red cell antigen.
 Types

1. ‘WARM’ ANTIBODY AIHA

2. ‘COLD’ ANTIBODY AIHA
 ‘WARM’ ANTIBODY AIHA
• This is the most common form of autoimmune hemolytic
anemia.

• Antibody react with RBC at 37°C.

• Most causative antibodies are of the IgG class, sometimes

IgA antibodies are culprit.

• Extravascular hemolysis may occur.

 IgG antibodies attach to red blood cells at 37 degree C

leaving there Fc portion sticking out

Antibody coated RBCs pass through spleen

The Fc portion of antibody RBC complex is recognized by Fc

receptor on phagocytes

Phagocytosis of RBCs in the spleen Membrane lost

Extravascular hemolysis Acquired spherocytosis

 Causes of warm AIHA

a) Autoimmune disorders → SLE, RA, Ulcerative colitis,

Scleroderma, Antiphospholipid antibodies.
b) Chronic lymphocytic leukemia (CLL)
c) Hodgkin disease
d) Non-Hodgkin lymphoma
e) Multiple myeloma
f) Thymoma
g) Drugs → Methyldopa, penicillin, Cephalosporine, quinidine.
h) PAN
 ‘COLD’ ANTIBODY AIHA

• 2 conditions:

• 1. Cold agglutinin disease

• 2. Paroxysmal cold haemoglobinuria (PCH)
 1. Cold agglutinin disease

• Antibodies react with RBC at 0° to 4°C.

• Caused by cold agglutinin IgM antibodies.

• Extravascular hemolysis occur

 IgM antibody directed against the I antigen of RBC

bind to I antigen of RBC at 4 degree C

Fix complement on RBC (transient reaction)

C3b acts as an opsonin

It enhances the phagocytosis of RBCs in phagocytic system of

liver and spleen

Extravascular hemolysis
 2. Paroxysmal cold haemoglobinuria (PCH)

• Antibodies react with RBC at 0° to 4°C.

• The autoantibodies are of IgG class, also known as

Donath-Landsteiner antibody

• Intravascular hemolysis occur

IgG (Donath-Landsteiner) antibody directed against the P
antigen of RBC

bind to P antigen of RBC at 4 degree C

Antigen antibody complex on the red blood cell activates

complement

Large amount of membrane attack complex (MAC)forms

Destroys red cells directly in blood vessel

Intravascular hemolysis
 Causes

• Syphilis
• Mycoplasma
• Mumps
• Measles
• Flu syndrome
 Cold AIHA Cold AIHA
Warm AIHA
CAD PCH

Temperature 370C 40C 40C

IgG
(Donath-
Ab. IgG IgM
Landsteiner)

Hemolysis Extrinsic Extrinsic Intrinsic

Warm antibody type Cold antibody type
Mostly IgG; rarely IgA Mostly IgM, rarely IgG
Causes Causes

• Primary (Idiopathic)
• Primary (Idiopathic)
• Mycoplasma infection,
• SLE, rheumatoid arthritis
• Infectious Mononucleosis Lymphoid
• B cell lymphoid neoplasms
neoplasms
• Drugs (a-methyldopa, penicillin)
• Paroxysmal cold hemoglobinuria (IgG)

Mechanism of hemolysis Mechanism of hemolysis

Extravascular hemolysis in cold agglutinin
Extravascular hemolysis (in spleen)
Intravascular hemolysis in cold hemolysins
Antibodies reacts at 4-6°C,
The antibody is active at 37°C
dissociate at 30°C or above
 LABORATORY FINDINGS
• Intravascular haemolysis as well as Extravascular
haemolysis

• 1. Mild to moderate chronic anaemia.

• 2. Reticulocytosis.
• 3. Unconjugated (indirect) hyperbilirubinaemia.
• 4. Intravascular haemolysis → haemoglobinaemia,
methaemoglobinaemia, haemoglobinuria, and
haemosiderinuria
 Morphology

• Prominent spherocytosis in the peripheral blood film.

 Special test

• 1. Positive direct Coombs’ (antiglobulin) test for

presence of warm antibodies on the red cell, best
detected at 37°C.

• 2. A positive indirect Coombs’ (antiglobulin) test at

37°C may indicate presence of large quantities of warm
antibodies in the serum.
• Direct Coombs test detects antibodies on RBC
surface.

• Indirect Coombs test detects antibodies in serum.

1. Cold hemagglutinin is associated with:
a. Anti IgM
b. Anti IgG
c. Anti IgA
d. Donath-Landsteiner antibody
3. Donath-Landsteiner antibodies are seen in:
a. Warm agglutination
b. Cold agglutination
c. Paroxysmal nocturnal hemoglobinuria
d. ITP
4. 'Warm' autoantibodies are seen in:
a. SLE
b. Mycoplasma
c. Syphilis
d. Varicella
4. 'Warm' autoantibodies are seen in:
a. SLE
b. Mycoplasma
c. Syphilis
d. Varicella
Warm antibody type Cold antibody type
Mostly IgG; rarely IgA Mostly IgM, rarely IgG
Causes Causes

• Primary (Idiopathic)
• Primary (Idiopathic)
• Mycoplasma infection,
• SLE, rheumatoid arthritis
• Infectious Mononucleosis Lymphoid
• B cell lymphoid neoplasms
neoplasms
• Drugs (a-methyldopa, penicillin)
• Paroxysmal cold hemoglobinuria (IgG)

Mechanism of hemolysis Mechanism of hemolysis

Extravascular hemolysis in cold agglutinin
Extravascular hemolysis (in spleen)
Intravascular hemolysis in cold hemolysins
Antibodies reacts at 4-6°C,
The antibody is active at 37°C
dissociate at 30°C or above
 Sickle Cell Anaemia

• Sickle cell anaemia (SS) is a homozygous state of HbS in

the red cells

• In this an abnormal gene is inherited from each parent.

 Haemoglobin

β-globin chain gene mutation (chromosome11)

Single point mutation at sixth position of β-globin chain (mis-

sense mutation)

Substution of a valine residue for a glutamic acid residue

Sickle hemoglobin (HbS) instead of HbA

 Consequences of Sickling

1. Vaso occlusion of microcirculation

2. Haemolytic anaemia
3. Increased MCHC
 Deoxygenation of HbS-containing RBC

Polymerisation of deoxygenated HbS

Elongated rod-like polymers fibres align

Distort the RBC into classic sickle shape (Sickling)

Express higher levels of adhesion molecules (abnormally sticky)

Vaso occlusion of microcirculation

 With repeated episodes of deoxygenation and sickling

Membrane damages permanently

RBC become irreversibly sickled

Difficulty in passing through splenic sinusoids → Spleenic Sequestration

Rapid phagocytosis

Extravascular Haemolysis → Haemolytic anaemia

 With membrane damage

Water comes out of the cell

Intracellular dehydration

Increased MCHC
 Factors determining rate of sickling

• 1) Amount of HbS and interaction with other Hb

• 2. Hemoglobin concentration of RBC
• 3. Intracellular dehydration
• 4. Decrease in pH
• 5. The length of time red cell are exposed to low oxygen
tension
 1) Amount of HbS and interaction with other Hb
• Most important factor for sickling is the amount of HbS

➢In homozygous individuals, all Hb. in RBC is HbS and undergo

sickling.

➢In heterozygous, only 40% of Hb. is HbS → Sickling does not occur
because HbA (remaining 60%) has an inhibitory effect on
polymerization of HbS

➢Fetal hemoglobin HbF also has inhibitory effect on sickling of HbS

→ Newborns do not manifest the disease until 6 months of age,
when the amount of HbF in the cells falls close to the adult level.
 2. Hemoglobin concentration of RBC

• Hb concentration of the cell, i.e. MCHC affects

polymerization to a great extent.

• Greater the MCHC→ greater is the sickling

 3. Intracellular dehydration

• Intracellular dehydration →increases the MCHC →

faciliatates sickling

• Conditions that decrease MCHC →Reduces sickling

 4. Decrease in pH
Decrease in pH(Acidosis)

Reduces the oxygen affinity of Hb.

Increasing the fraction of deoxygenated HbS

faciliatates sickling
 5. The length of time red cell are exposed
to low oxygen tension

• Organs having slow or sluggish circulation (bone and

spleen) have an increased chance of sickling
 CLINICAL FEATURES

1. Anaemia
2. Vaso-occlusive phenomena
 1. Anaemia
• Irreversible sickle cells have difficulty in passing the
splenic sinusoids, sequestration, and rapid
phagocytosis.

• Anemia is associated with Jaundice reticulocytosis.

 2. Vaso-occlusive phenomena

• Reversible sickle cells express higher levels of

adhesion molecules and are abnormally sticky

• So responsible for occlusion of microcirculation

• Vasoocclusive symptoms are the most common

manifestations of sickle cell anemia.
A. Bone
a) It presents as dactylitis or inflammation of the bones of hands and
feet, so called Hand foot syndrome
b) Avascular necrosis of femoral head
c) Prominent cheek bones
d) Crew cut appearance of skull → due to marrow expansion
causing bone resorption and secondary new bone formation
e) Fish mouth appearance of vertebra → due to vaso occlusive crisis
of vertebral arteries.
B. Lungs → Acute chest syndrome characterized by cough, fever and
chest pain

C.Brain →seizures or stroke

D. Skin →leg ulcers

E. Penis → stagnation in corpora cavernosa leads to priapism

F. Aplastic crises
• Occurs from the infection of red cell progenitors by parvovirus
B19→ sudden worsening of the anemia
G. Spleen
• In the initial stages, there is splenomegaly due to
congestion and trapping of red cells in the vascular sinusoids

• Prolonged hypoxia and infarction can lead to

autosplenectomy which increases susceptibility to infection
with capsulated organisms like Hemophilus influenzae,
Pneumococcus, etc.
 Protection against falciparum malaria
Patients with HbS are relatively protected against falciparum malaria

Malarial parasites consume O2

Decrease intracellular pH

Promote sickling

Distorted RBCs are cleared rapidly by phagocytes in the spleen

Keep parasite loads down

 Screening

Sickling Test
Method
1. Sample – Venous (from arm)/ Capillary blood
(Fingertips, Ear lobes in adults/ Heel in Infants)
 Venous (Arm)
Blood Sample Capillary (Fingertips, Ear lobes in adults/
Heel in Infants)
Mix with

Sodium Metabisulphite (Reducing agent)

Wait for 20 minutes

Microscope

Normal RBC Sickled RBC

Negative test HbA Positive test HbS

Diagnosis
 1. Peripheal smear

The blood film shows→

1. Sickle cells
2. Target cells
3. Features of splenic atrophy such as presence of
Howell-Jolly bodies
 2. ESR

• ESR is usually increased in all anemia except in sickle cell anemia where
ESR is decreased because there is no rouleaux formation.
 3. Haemoglobin electrophoresis

• HbS moves Slowly towards Anode.

• HbA moves faster towards Anode

Interpretation→
• Formation of 1 band is suggestive of sickle cell anemia
• Formation of 2 bands are suggestive of sickle cell carrier or
trait.
MCQs
1. In sickle cell anaemia defect is in which chain

a. a-chain
b. b-chain
c. Both the chains
d. None of these
1. In sickle cell anaemia defect is in which chain

a. a-chain
b. b-chain
c. Both the chains
d. None of these
2. Sickle cell mutation is
a. Point mutation
b. Select mutation
c. Frame shift mutation
d. Nonsense mutation
2. Sickle cell mutation is
a. Point mutation
b. Select mutation
c. Frame shift mutation
d. Nonsense mutation
3. Person having heterozygous sickle cell trait
is protected from infection of -
a. Plasmodium falciparum
b. P. vivax
c. Pneumococcus
d. Salmonella
3. Person having heterozygous sickle cell trait
is protected from infection of -
a. Plasmodium falciparum
b. P. vivax
c. Pneumococcus
d. Salmonella
4. Autosplenectomy is seen in?
a. Hereditary spherocytosis
b. G6 PD deficiency
c. Sickle cell anemia
d. Thalassemia major
4. Autosplenectomy is seen in?
a. Hereditary spherocytosis
b. G6 PD deficiency
c. Sickle cell anemia
d. Thalassemia major
5. In sickle cell trait, number of bands found in
Hb
a. 2
b. 1
c. 4
d. 5
5. In sickle cell trait, number of bands found in
Hb
a. 2
b. 1
c. 4
d. 5
6. In sickle cell anaemia true is
a. Autosplenectomy due to thrombosis &infarction
b. Microcytosis
c. Microcardia
d. Splenomegaly
6. In sickle cell anaemia true is
a. Autosplenectomy due to thrombosis &infarction
b. Microcytosis
c. Microcardia
d. Splenomegaly
 THALASSAEMIAS

• Thalassaemias are quantitative abnormalities of

polypeptide globin chain synthesis

• The thalassaemias are a diverse group of hereditary

disorders in which there is reduced synthesis of one
or more of the globin polypeptide chains.
 Classification

• α Thalassaemia→ Absent or reduced synthesis of α globin

chain (chromosome 16) with normal β -chain synthesis.

• β Thalassaemia→ Absent or reduced synthesis of β globin

chain (chromosome 11) with normal α -chain synthesis
 Classification of a-thalassaemias
Clinical
TYPE HB Electrophoresis Genotype
Syndrome
Fatal in utero or
Deletion of
1. Hydrops foetalis 3-10 gm/dl Hb Barts in early infancy
four a-genes

Deletion of Haemolytic
2. HbH disease 2-12 gm/dl HbF , HbH
three a-genes anaemia
Microcytic
3. a-Thalassaemia hypochromic
Deletion of
trait 10-14 gm/dl Almost normal blood
two a-genes
picture but no
anaemia
4. a-Thalassaemia Deletion of
Carrier Normal Normal One a-genes Asymptomatic
 Classification of b-thalassaemias
TYPE HB Electrophoresis Genotype Clinical Syndrome

Severe anaemia,
HbA(0-50%),
1. b-Thal major <5 gm/dl
HbF(50-98%) bthal/bthal requires
transfusions

Severe anaemia,
Multiple but regular
2. b-Thal intermedia 5-10 gm/dl Variable
mechanisms transfusions not
required

10-12 HbA2(4-9%), Usually

3. b-Thala minor
gm/dl HbF (1-5%) bA/bthal asymptomatic
 β thalassemia
• Absent or reduced production of β globin chain

• Reduced formation of HbA in the red cells.

 Molecular pathogenesis
Point Mutations of β-globin gene resulting from single base
changes

3 types of mutations →
i) Splicing mutations (most common)
• Mutations leading to aberrant splicing are the cause of β -
thalassemia.

ii) Chain terminator mutations

iii) Promoter region mutations

 TYPES

• Depending upon the extent of reduction in β -chain

synthesis, there are 3 types of β -thalassaemia:

1. β -Thalassaemia major
2. β -Thalassaemia intermedia
3. β -thalassaemia minor (trait)
 Classification of b-thalassaemias
TYPE HB Electrophoresis Genotype Clinical Syndrome

Severe anaemia,
HbA(0-50%),
1. b-Thal major <5 gm/dl
HbF(50-98%) bthal/bthal requires
transfusions

Severe anaemia,
Multiple but regular
2. b-Thal intermedia 5-10 gm/dl Variable
mechanisms transfusions not
required

10-12 HbA2(4-9%), Usually

3. b-Thala minor
gm/dl HbF (1-5%) bA/bthal asymptomatic
 β -Thalassaemia major
• It is the most severe form of congenital haemolytic anaemia.
• Also termed Mediterranean or Cooley’s anaemia
• It is the most common form of congenital haemolytic
anaemia.

• 2 Types→

1. β ° thalassemia - Characterized by total absence of β chains

in the homozygous state.

2. β + thalassemia - Characterized by reduced synthesis of β

chains in the homozygous state.
 β -Thalassaemia intermedia

• Intermediate degree of severity

• Does not require regular blood transfusions.

 β -thalassaemia minor (trait)

• Mild asymptomatic
Clinical features of β -Thalassaemia major
 b chains not produced

a chain accumulate

Combine with y chain Destruction of normoblasts in the bone marrow

to form a2 g2
Ineffective erythropoiesis

Anemia
↑HbF
Hypoxia in tissues
Repeated ↑EPO secretion by renal cells
blood
transfusion Erythroid hyperplasia in bone marrow

↑Iron absorption Bone changes because of

medullary cavity expansion
Iron overload
Death
• 1. Anaemia starts appearing within the first 4-6 months of life

• 2. Marked hepatosplenomegaly → due to extramedullary

haematopoiesis and iron overload.

• 3. Expansion of bones → due to marked erythroid hyperplasia

leading to thalassaemic facies and malocclusion of the jaw.

• 4. Iron overload due to repeated blood transfusions causes

damage to the endocrine organs (slow rate of growth, delayed
puberty, diabetes mellitus) and damage to the liver and heart.
Thalassaemic facies
Lab Diagnosis of β -Thalassaemia major
 1. Perpheral smear

1. Severe microcytic hypochromic RBC

2. Marked anisopoikilocytosis
3. Basophilic stippling
4. Target cells
5. Tear drop cells
6. Nucleated RBCs
 2. Bone marrow examination

a) Hypercellular with erythroid hyperplasia

b) Reversal of normal M:E ratio (1:3)
c) Pink inclusions are seen in the normoblasts
(caused by a chain accumulation).
 3. Osmotic fragility test

Increased resistance to saline haemolysis i.e. decreased

osmotic fragility
 4. NESTROF Test
Naked Eye Single Tube Red cell Osmotic Fragility (NESTROF) test

• In this test 2 blood samples (1 of a normal person serving as

control and 1 of patient) are added to 2 tubes with 0.35% saline.
• After 30 min a white paper with a black line is placed behind
both the tubes.
• The RBCs in control sample undergo hemolysis so the black line is
visible
• The RBCs in thalassemia trait are resistant so black line is not
clearly visible.
 5. Haemoglobin electrophoresis

a) Since β chains are not produced but y chains are

synthesized normally → increased amounts of HbF
(90%).
b) Increased amount of HbA2
c) Almost complete absence of HbA
 6. Skull X-ray
Widening of the diploe gives rise to →
✓Crew cut appearance on skull X-ray
✓Hair on end appearance
 α thalassemia
• Absent or reduced production of α globin chain

• Reduced formation of HbA in the red cells.

 Molecular pathogenesis

• Deletion of one or more of the α -chain genes located on

short arm of chromosome 16.
 TYPES

• 1. Four a-gene deletion: Hb Bart’s/hydrops foetalis.

• 2. Three a-gene deletion: HbH disease.
• 3. Two a-gene deletion: α-thalassaemia trait
• 4. One a-gene deletion: α –thalassaemia carrier
 Classification of a-thalassaemias
Clinical
TYPE HB Electrophoresis Genotype
Syndrome
Fatal in utero or
Deletion of
1. Hydrops foetalis 3-10 gm/dl Hb Barts in early infancy
four a-genes

Deletion of Haemolytic
2. HbH disease 2-12 gm/dl HbF , HbH
three a-genes anaemia
Microcytic
3. a-Thalassaemia hypochromic
Deletion of
trait 10-14 gm/dl Almost normal blood
two a-genes
picture but no
anaemia
4. a-Thalassaemia Deletion of
Carrier Normal Normal One a-genes Asymptomatic
 a thalassemia

Normal Silent a Thalassemia 3-a 4-a

aa | aa carrier trait chain deletion chain deletion
-a | aa - - / -a --/--
cis Trans-
100% a chain - - | aa -a | -a ↑b chain ↑g chain
• 70% a chain Tetramer of
• Asymptomatic Tetramer of g chain
b chain
Asians • Africans g4
• American Barts hemoglobin
b4
• Hydrops fetalis
HbH • Lethal in utero
Clinical features of α-thalassaemia
 α –thalassaemia carrier

• Clinically asymptomatic
 α-thalassaemia trait

• Minimal or no anemia
• No abnormal physical signs
 a-chains not produced

Formation of b and g chains is normal

b-chains forms tetramers g- chains forms tetramers

(b4 or HbH) (g4 or Barts Hb)

Ineffective erythropoiesis Not able to transport

oxygen properly
HbH inclusion in red cells not
able to release oxygen to tissue
Fetus develops
Trapping of these cells in spleen intrauterine hypoxia

Extravascular hemolytic anemia Fetal death or Still birth

 HbH

• HbH has extremely high affinity for oxygen and

therefore is not useful for oxygen exchange, leading to
tissue hypoxia
• This produces a moderately severe anemia
• Require occasional blood transfusion.
 Hydrops fetalis

• Hydrops fetalis is the most dangerous form of α-thalassemia

• In the fetus, excess y-globin chains form tetramers, known as

hemoglobin barts.
• Hemolgobin bart has such a high affinity for oxygen that it
delivers almost no oxygen to tissues
• Severe tissue anoxia leads to Intrauterine fetal death.
• The fetus shows severe pallor, generalized edema, and massive
hepatosplenomegaly.
MCQs
1. Deficiency in globin synthesis
a. Thalassemia
b. Sickle cell disease
c. Hereditary spherocytosis
d. PN11
1. Deficiency in globin synthesis
a. Thalassemia
b. Sickle cell disease
c. Hereditary spherocytosis
d. PN11
2. In Beta thalassemia, there is
a. Increase in beta chain, decrease in alpha chain
b. Decrease in beta chain, increase in alpha chain
c. Decrease in beta chain, decrease in alpha chain
d. Increase in beta chain, increase in alpha chain
2. In Beta thalassemia, there is
a. Increase in beta chain, decrease in alpha chain
b. Decrease in beta chain, increase in alpha chain
c. Decrease in beta chain, decrease in alpha chain
d. Increase in beta chain, increase in alpha chain
3. NESTROFT test is used in screening of
a. Thalassemia
b. Autoimmune hemolytic anemia
c. Spherocytosis
d. G6PD deficiency
3. NESTROFT test is used in screening of
a. Thalassemia
b. Autoimmune hemolytic anemia
c. Spherocytosis
d. G6PD deficiency
4. In a-thalasscmia
a. Excess a-chain
b. No a-chain
c. Excess b-chain
d. No b-chain
4. In a-thalasscmia
a. Excess a-chain
b. No a-chain
c. Excess b-chain
d. No b-chain
5. HbH is associated with
a. Deletion of 3 alpha genes
b. Deletion of 4 alpha genes
c. Deletion of 3 beta genes
d. Deletion of 4 beta genes
5. HbH is associated with
a. Deletion of 3 alpha genes
b. Deletion of 4 alpha genes
c. Deletion of 3 beta genes
d. Deletion of 4 beta genes
Iron deficiency anaemia

Dr. PRIYANKA SACHDEV , MD

 Introduction

• It is the most common cause of anemia worldwide and

in India

• Its prevalence is higher in the developing countries.

 Normal iron metabolism
• Iron is absorbed from upper small intestine mainly
duodenum

• In diet iron occurs in two forms

1. Haeme iron (animal products)
2. Inorganic (non-haeme) iron (vegetables)
 Iron in diet is in Fe3+ (ferric) state

Reduced to Fe2+ (ferrous) iron in gastric acid

Transported across apical membrane by divalent metal transporter 1 (DMT1)

Transported from the cytoplasm across the basolateral membrane by Ferroportin

This is coupled to oxidation of Fe2+ iron to Fe3+ iron

Fe3+ iron binds to the plasma protein transferrin

Transferrin delivers iron to red cell progenitors marrow

Extra iron is stored in liver in the form of feritin

 Regulation of iron absorption
• Iron absorption is regulated by hepcidin
 Increases in intrahepatic iron levels

Hepcidin is synthesized and released from the liver

Hepcidin binds to ferroportin

Degrade it

Inhibits iron transfer from the enterocyte to plasma

Iron becomes trapped within duodenal cells

Iron is lost as these cells are sloughed

When body is replete When body iron stores is
with iron low

High hepcidin levels Hepcidin synthesis falls

Inhibit iron absorption Facilitates iron absorption

into the blood into the blood
 Factors of Iron absorption
Factors increasing absorption
• Ferrous form (Fe2+)
• Acid (HCI) in the stomach
• Ascorbic acid
• Amino acid and sugars in the food
• Iron deficiency
• Physiological conditions (pregnancy
and hypoxia)
Factors decreasing absorption
• Ferric form (Fe3+)
• Achlorhydria (absence of HCI secretion)
• Alkaline food (pancreatic secretions)
• Phytates, tannates and phosphates in
diet
• Iron overload
• Tetracyclines and EDTA
• Inflammatory disorders
 Don’t Forget

• Iron is transported is blood in combination with a

glycoprotein transferrin.

• Iron is stored as ferritin or haemosiderin.

 ETIOLOGY

1. Increased blood loss

2. Increased requirement
3. Inadequate dietary intake
4. Decreased absorption
Dietary lack Impaired Increased Chronic blood loss
absorption requirement
• Infants • Steatorrhea • Growing infants • GIT (peptic ulcer,
• Children • Sprue and children gastric cancer,
• Low • Chronic diarrhea • Pregnant females hemorrhoids,
socioeconomic • Gastrectomy • Premenopausal hookworm
status women disease)
• Elderly • Urinary tract
(renal, pelvic or
bladder cancers)
• Genital tract
(uterine cancer,
menorrhagia)
 LABORATORY FINDINGS

1. Blood picture and red cell indices

2. Bone marrow findings

3. Biochemical findings
 Lab diagnosis

Blood Bone Marrow Biochemistry

a) Hb a) Cellularity a) Serum iron

b) RBC b) Erythropoiesis b) Serum Ferritin
c) Retic c) Marrow Iron c) TIBC
d) Indices d) Transferrin saturation
e) WBC
f) Platelets
 1. Blood picture
a) Haemoglobin concentration → Fall

b) RBCs
• Hypochromic
• Microcytic
• Anisocytosis
• Poikilocytosis
• Target cells
• Ring / pessary cells (central pallor occupies whole cell and only
peripheral rim of hemoglobin is seen).
c) Reticulocyte count → normal but may be slightly low

d) Indices
• Diminished MCV (below 50 fl)
• Diminished MCH (below 15 pg)
• Diminished MCHC (below 20 g/dl)

e) Leucocytes → TLC and DLC normal

f) Platelets → normal
 2. Bone marrow findings
a) Marrow cellularity
• Increased due to erythroid hyperplasia
• Myeloid-erythroid ratio decreased

b) Erythropoiesis
• Small normoblasts. (micronormoblast)
• Cytoplasmic maturation lags behind nuclear maturation
(compared from megaloblastic anaemia in which the nuclear
maturation lags behind).

c) Marrow iron (Prussian blue reaction) → Deficient

 3. Biochemical findings

a) Serum iron level → Low (50 μg/dl)

b) Serum ferritin level → Low (Indicating poor tissue

iron stores)

c) Total iron binding capacity (TIBC) → High

d) Transferrin saturation → Low (below 15%)

 Lab diagnosis

Blood Bone Marrow Biochemistry

a) Hb a) Cellularity a) Serum iron

b) RBC b) Erythropoiesis b) Serum Ferritin
c) Retic c) Marrow Iron c) TIBC
d) Indices d) Transferrin saturation
e) WBC
f) Platelets
 Differential diagnosis

1. Anemia of chronic disease

2. Thalassemia
3. Sideroblastic anemia
 IRON CHRONIC THALASSAEMIA SIDEROBLASTIC
TEST
DEFICIENCY DISORDERS MINOR ANAEMIA
Very low (except
1. MCV, MCH, Low normal-to-
Reduced Very low MCV raised in
MCHC reduced
acquired type)
2. Serum iron Reduced Reduced Normal Raised
3. TIBC Raised Low-to-normal Normal Normal
Raised (complete
4. Serum ferritin Reduced Raised Normal
saturation)
5. Marrow-iron
Absent Present High High
stores
6. Iron in
Absent Absent Present Ring sideroblasts
normoblasts
7. Hb
Normal Normal Abnormal Normal
electrophoresis
MCQs
1. Iron absorption is decreased by A/E-
a. Calcium
b. Tetracycline
c. Phytate
d. Ascorbic acid
1. Iron absorption is decreased by A/E-
a. Calcium
b. Tetracycline
c. Phytate
d. Ascorbic acid
2. Which of the following glycoproteins is
transported in plasma in iron metabolism:
a. Spectrin
b. Transferrin
c. Ferritin
d. Hemosiderin
2. Which of the following glycoproteins is
transported in plasma in iron metabolism:
a. Spectrin
b. Transferrin
c. Ferritin
d. Hemosiderin
3. Which of the following is the indicator for
body iron Stores?
a. Perretin
b. Transferrin
c. TIBC
d. Serum iron levels
3. Which of the following is the indicator for
body iron Stores?
a. Perretin
b. Transferrin
c. TIBC
d. Serum iron levels
4. The earliest sign of iron deficiency anaemia -
a. Increase in iron binding capacity
b. Decrease in serum ferritin level
c. Decrease in serum iron level
d. All the above
4. The earliest sign of iron deficiency anaemia -
a. Increase in iron binding capacity
b. Decrease in serum ferritin level
c. Decrease in serum iron level
d. All the above
5. Which is not seen in iron deficiency anaemia
a. Hyper-segmented neutrophils
b. Microcytosis preceeds hypochromia
c. MCHC < 50 %
d. Commonest cause of anaemia in India
5. Which is not seen in iron deficiency anaemia
a. Hyper-segmented neutrophils
b. Microcytosis preceeds hypochromia
c. MCHC < 50 %
d. Commonest cause of anaemia in India
6. Iron deficiency anemia is characterized by
a. Increased porphyrin
b. Increased MCHC
c. Increased ferritin level
d. Increased TIBC
6. Iron deficiency anemia is characterized by
a. Increased porphyrin
b. Increased MCHC
c. Increased ferritin level
d. Increased TIBC
7. Best test for assessment of iron status is -
a. Transferrin
b. Ferritin
c. Serum iron
d. Hemoglobin
7. Best test for assessment of iron status is -
a. Transferrin
b. Ferritin
c. Serum iron
d. Hemoglobin
8. The commonest cause of microcystic
hypochromic anaemia is
a. Thallassemia
b. Megaloblastic anaemia.
c. Iron deficiency anaemia
d. Sideroblastic anaemia
8. The commonest cause of microcystic
hypochromic anaemia is
a. Thallassemia
b. Megaloblastic anaemia.
c. Iron deficiency anaemia
d. Sideroblastic anaemia
9. Response to iron in iron deficiency anemia is
denoted By
a. Restoration of enzymes
b. Reticulocytosis
c. Increase in iron binding capacity
d. Increase in hemoglobin
9. Response to iron in iron deficiency anemia is
denoted By
a. Restoration of enzymes
b. Reticulocytosis
c. Increase in iron binding capacity
d. Increase in hemoglobin
Megaloblastic anaemias
(Vitamin b12 and folate deficiency)

Dr. PRIYANKA SACHDEV , MD

 Introduction
• The megaloblastic anaemias are disorders caused by
impaired DNA synthesis

• Vit B12 and folic acid are required for DNA synthesis.

• So megaloblastic anaemia is due to deficiency of vit. B12

and folic acid
 Pathogenesis
• Vit B12 and folic acid are required for DNA synthesis.
 THYMIDYLATE SYNTHETASE REACTION
dUMP dTMP DNA

Methylene THF DHF PGA

DHF reductase
THF
Methionine
Methyl HOMOCYSTEINEMETHIONINE
vitamin B12 REACTION
Homocysteine
Methyl THF = Block due to folate deficiency
(Plasma folate) = Block due to drug with normal tissue folate level
 Inadequate DNA synthesis

Defective nuclear maturation

Maturation of the nucleus lag behind to that of the cytoplasm

Nuclear/Cytoplasmic asynchrony

Formation of megaloblasts and macrocytes

ERYTHROID SERIES
• Vit. B12 is also involved in the conversion of
methylmalonyl CoA to succinyl CoA which is required
for the formation of normal neuronal lipids.

• So, deficiency of vitamin B12 (but not of folic acid)

results in neurological features
 CLINICAL FEATURES
• 1. Anaemia (In both vit.B12 and folate def.)

• 2. Neurologic manifestations (only in vit.B12 def. Not in

folate def.) →
➢Subacute combined degeneration of the spinal cord
➢Peripheral neuropathy
➢Numbness, paraesthesia, weakness, ataxia, poor finger
coordination and diminished reflexes.
 Laboratory findings

1. Blood picture and red cell indices

2. Bone marrow findings

3. Biochemical findings

4. Special Tests for Cause of Specific Deficiency (vit B12 or

Folic acid)
 Lab diagnosis

Blood Bone Marrow Biochemistry Special Tests

a) Hb a) Cellularity a) Serum iron a) Schilling

b) RBC b) Erythropoiesis b) Serum Ferritin test
c) Retic c) Marrow Iron b) FIGLU
d) Indices
e) WBC
f) Platelets
 1. Blood picture
a) Haemoglobin concentration → Fall

b) RBCs
• Macrocytosis
• Macroovalocytes
• Anisocytosis
• Poikilocytosis
• Tear drop cells
• Basophilic stippling
• Cabott Ring
• Howell-jolly bodies (Evidence of defective erythropoiesis)
c) Reticulocyte count → Low to normal

d)Absolute values
• Elevated MCV (above 120 fl)
• Elevated MCH (above 50 pg)
• Normal or reduced MCHC (because hemoglobin content in the cell
is increased proportiante to increase in the size of RBC)

e) Leucocytes
• Hypersegmented neutrophils (having more than 5 nuclear lobes) →
First manifestation of megaloblastic anemia

f) Platelets → Bizarre forms of platelets may be seen.

 2. Bone marrow findings

a) Marrow cellularity
• Hypercellular → erythroid hyperplasia
• Decreased myeloid-erythroid ratio

b) Erythropoiesis
• Megaloblastic erythropoiesis
• Nuclear maturation lags behind that of cytoplasm

c) Marrow iron →Increase

 3. Biochemical findings

• a) Serum iron → Normal or elevated

• b) Serum ferritin → Normal or elevated
 Special Tests for for vit. B12 deficiency

Schilling test

a) To detect vitamin B12 deficiency

b) To distinguish and detect lack of IF and malabsorption
syndrome.

• Radioisotope used for labeling B12 is either 58Co or 57Co.

3 stages
 Stage I
1. 1 mg of unlabelled vitamin B12 (‘cold’ B12) is given by
intramuscular route (to saturate binding sites)

2. 1 mg of radioactively labelled vitamin B12 (‘hot’ B12) is

given by oral dose

3. The patient is kept fasting for a further period of 2 hours

4. Urinary excretion of B12 is estimated

 Interpretation

➢In normal individuals → 24-hour urinary excretion is

>10% of the oral dose of ‘hot’ B12

➢Patients with B12 deficiency → 24-hour urinary

excretion is < 10% of ‘hot’ B12
 Stage II: With IF
If the 24-hour urinary excretion of ‘hot’ B12 is low→
• Test is repeated using the same procedure as in stage I but in
addition high oral dose of IF

Interpretation→
If the 24-hour urinary excretion of ‘hot’ B12 is now normal
(>10% of the oral dose of ‘hot’ B12 ) → IF deficiency
(Pernicious anaemia)
 Stage III: with antibiotics

If the 24-hour urinary excretion of ‘hot’ B12 is still

low→
• Test is repeated after a course of treatment with
antibiotics or anti-inflammatory drugs.

Interpretation→
If the 24-hour urinary excretion of ‘hot’ B12 is now
normal (>10% of the oral dose of ‘hot’ B12 ) →
Malabsorption
 Schilling Test
58Co-Cbl W/intrinsic After 5 Days W/Pancreatic
Factor of Antibiotics Enzymes

Pernicious
Reduced Normal Reduced Reduced
Anemia

Bacterial
Reduced Reduced Normal Reduced
overgrowth

Chronic
Reduced Reduced Reduced Normal
pancreatitis
 Special Tests for for folate deficiency

Urinary excretion of FIGLU

• Folic acid is required for conversion of

formiminoglutamic acid (FIGLU) to glutamic acid in the
catabolism of histidine.

• Thus, on oral administration of histidine, urinary excretion

of FIGLU is increased if folate deficiency is present
 Histidine

Formiminoglutamic acid (FIGLU)

Folic acid

Glutamic acid
 Lab diagnosis

Blood Bone Marrow Biochemistry Special Tests

a) Hb a) Cellularity a) Serum iron a) Schilling

b) RBC b) Erythropoiesis b) Serum Ferritin test
c) Retic c) Marrow Iron b) FIGLU
d) Indices
e) WBC
f) Platelets
MCQs
1. Megaloblastic anemia is due to
a. Defect in DNA synthesis
b. Defect in RNA synthesis
c. Defect in protein synthesis
1. Megaloblastic anemia is due to
a. Defect in DNA synthesis
b. Defect in RNA synthesis
c. Defect in protein synthesis
2. Characteristic blood picture in megaloblastic
anemia –
a. Macrocytosis and increased reticulocyte count
b. Macrocytosis and decreased reticulocyte count
c. Microcytosis and increased reticulocyte count
d. Microcytosis and decreased reticulocyte count
2. Characteristic blood picture in megaloblastic
anemia –
a. Macrocytosis and increased reticulocyte count
b. Macrocytosis and decreased reticulocyte count
c. Microcytosis and increased reticulocyte count
d. Microcytosis and decreased reticulocyte count
3. Which of these does not indicate
megaloblastic anemia?
a. Increased reticulocyte count
b. Raised Bilirubin
c. Mild splenomegaly
d. Nucleated RBC
3. Which of these does not indicate
megaloblastic anemia?
a. Increased reticulocyte count
b. Raised Bilirubin
c. Mild splenomegaly
d. Nucleated RBC
4. Macrocytosis incomplete blood count can be
diagnosed by:
a. ↑ MCV
b. ↑ TMCHC
c. ↑ Hematocrit
d. ↑ Red cell distribution width
4. Macrocytosis incomplete blood count can be
diagnosed by:
a. ↑ MCV
b. ↑ TMCHC
c. ↑ Hematocrit
d. ↑ Red cell distribution width

• MCV > 100fL indicates macrocytosis

5. Abnormality in Schilling test can be seen in
all of the following except:
a. B12 deficiency
b. Folic acid deficiency
c. Ileal disease
d. Bacterial overgrowth
5. Abnormality in Schilling test can be seen in
all of the following except:
a. B12 deficiency
b. Folic acid deficiency
c. Ileal disease
d. Bacterial overgrowth
6. FIGLU test is done for:
a. Cyanocobalamin deficiency
b. Folic acid deficiency
c. Thiamine deficiency
d. Riboflavin deficiency
6. FIGLU test is done for:
a. Cyanocobalamin deficiency
b. Folic acid deficiency
c. Thiamine deficiency
d. Riboflavin deficiency
7. Hypersegmented neutrophils are present in
which of the following anemia?
a. Hemolytic
b. Iron deficiency
c. Megaloblastic
d. Aplastic
7. Hypersegmented neutrophils are present in
which of the following anemia?
a. Hemolytic
b. Iron deficiency
c. Megaloblastic
d. Aplastic
8. Schilling test is used for identification of
which of the following?
a. Fat absorption.
b. Vit K absorption
c. Vitamin B12 absorption
d. Vitamin D absorption
8. Schilling test is used for identification of
which of the following?
a. Fat absorption.
b. Vit K absorption
c. Vitamin B12 absorption
d. Vitamin D absorption
Leukemias and Lymphomas
(Definition and Classification)

Dr. PRIYANKA SACHDEV , MD

 WBC

Granulocytes Agranulocytes

Neutrophils Eosinophils Basophils Lymphocytes Monocyte

 HSC

Lymphoid stem cells Myeloid (trilineage) stem cells

Prolymphocyte

Granulocyte-monocyte p. Erythroid p Megakaryocytes

Lymphocytes Neutrophils RBC Platelet

Eosinophils
Basophils
Monocytes
• Leukemia is used for lymphoid neoplasms presenting with
widespread involvement of the bone marrow usually
accompanied by the presence of large number of tumour
cells in the peripheral blood.

• Lymphoma is used to describe proliferations arising as

discrete tissue masses (lymph nodes, spleen, or
extranodal tissues).

• Lymphoma may have leukemia and vice versa.

 Classification of Leukemias
1) Lymphoid:
• (i) Acute Lymphoid leukemia (ALL)
• (ii) Chronic Lymphoid leukemia (CLL)

2) Myeloid:
• (i) Acute Myeloid leukemia (AML)
• (ii) Chronic myeloid leukemia (CML)
 Leukemias

Lymphoid Myeloid

Acute Chronic Acute Chronic

ALL CLL AML CML

 Myeloblast Lymphoblast

Size Larger Smaller

Cytoplasm Moderate Scanty
Auer rod May be present Absent
Nuclear chromatin Fine Coarse
Nucleoli Prominent, 1-4 Indistinct
• Acute leukemias have a high rate of proliferation
without differentiation and their clinical course is
rapid.

• Chronic leukemias have a low rate of proliferation of

tumor cells with good differentiation and their clinical
course is slow.
 Classification of Lymphomas

WHO categorise various lymphoid neoplasms into five

categories based on clinical features and morphology
 WHO classification of lymphoid neoplasm
Precursor Precursor Peripheral Peripheral T-cell Hodgkin’s
B-cell neoplasm T-cell neoplasm B-cell neoplasm and NK-cell lymphoma
neoplasm
• B-cell acute • T-cell acute • Chronic lymphocytic • Mycosis • Classical
lymphoblastic lymphoblastic leukemia/small fungoides/Sezary ▪ Nodular
leukemia/lymphoma leukemia/lymphoma lymphocytic syndrome sclerosis
(B-ALL) lymphoma • Large granular ▪ Mixed
• Mantle cell lymphocytic cellularity
lymphoma lymphoma ▪ Lymphocyte-
• Follicular lymphoma • Anaplastic large cell rich
• Marginal zone B-cell lymphoma ▪ Lymphocyte
lymphoma • Enteropathy depleted
• Burkitt's lymphoma associated T-cell • Lymphocyte
• Hairy cell leukemia lymphoma predominant
• Diffuse large B-cell • Hepatosplenic xdT-
lymphoma cell lymphoma
• Splenic and nodal • NK-cell leukemia
marginal zone B-cell • Angioimmuno blastic
lymphoma T-cell lymphoma
• Plasmacytoma/plas • Extranodal NK/T-cell
ma cell myeloma lymphoma
• Lymphoplasmacytic
 Lymphomas

Hodgkin's Non-Hodgkin's
Lymphoma Lymphoma
FEATURE HODGKIN'S NON-HODGKIN'S
1. Cell derivation B-cell 90% B
10% T
2. Nodal involvement Localised, may Disseminated nodal
spread to Spread
contiguous nodes
3. Extranodal spread Uncommon Common
4. Bone marrow Uncommon Common
involvement
5. Constitutional symptoms Common Uncommon
6. Chromosomal defects Aneuploidy Translocations, deletions
7. Spill-over Never May spread to blood
8. Prognosis Better Bad
(75-85% cure) (30-40% cure)
Chronic Myeloid Leukemia (CML)

Dr. PRIYANKA SACHDEV , MD

 CML AML ALL CLL
Introduction
Age
Pathogenesis
Classification
Clinical features

Lab diagnosis
Treatment

Prognosis
 Chronic Myeloid Leukaemia (CML)

• CML is an acquired disease of haemopoietic stem cell that is

characterized by→

1. Leucocytosis with granulocytic immaturities

2. Basophilia
3. Splenomegaly
4. Distinct chromosomal abnormality - Philadelphia (Ph’)
chromosome.
 Age

• CML presents in old age (> 50 years )

 Pathogenesis

• Translocations t(9;22) / ABL-BCR Translocation →

Philadelphia chromosome
ABL gene is a protooncogene having tyrosine kinase
activity.

• ABL gene → normal location on chromosome 9

• BCR gene→normal location on chromosome 22
 In Translocation
ABL gene (normal location on chromosome 9)

Translocated to chromosome 22

It fuses with BCR (breakpoint cluster region) gene

ABL-BCR hybrid gene → Philadelphia chromosome

210 KD fusion protein

Abnormal signal transduction even without growth factors

Uncontrolled mitosis

CML

I. Chronic phase
1. Bone marrow or peripheral
blood < 10% of blast cells.
2. BCR-ABL [t(9;22)] fusion genes 2. Basophilia ≥ 20%
present
Phases of CML
CML (Triphasic leukemia)
II. Accelerated phase III. Blast crisis
(one or more of the following)
1. Blast cells 10-19% 1. Blast count > 20%
2. Extramedullary blast cells
(-chloromas)
3. Thrombocytopenia or 3. Large clusters of blast cell on
thrombocytosis, nonresponsive bone marrow biopsy
to treatment
4. Leukocytosis or splenomegaly-
non- responsive to treatment
5. Cytogenetic changes - Trisomy
8, isochromosome some 17q;
Philadelphia chromosome
 Clinical Features
• I. Due To Bone Marrow Failure→
a) Anaemia producing pallor, lethargy, dyspnoea.
b) Bleeding manifestations causing spontaneous bruises,
petechiae, bleeding from gums and other bleeding
tendencies.
c) Infections

• II. Symptoms due to hypermetabolism such as weight loss,

lassitude, anorexia, night sweats.

• III. Splenomegaly is massive.

 Lab diagnosis

Blood Bone Marrow Cytogenetics Cytochemistry Others

a) Hb a) Cellularity t(9;22) NAP scores S. uric acid

b) Platelets b) Myeloid cells
c) WBC c) Erythropoiesis
d) Megakaryopoiesis
 I. Blood Picture
a) Anaemia→ Normocytic normochromic

b) Thrombocytopenia

c) White blood cells →

• Marked leucocytosis (approx 200,000/μl or more)
• Immature Neutrophils ((band forms, metamyelocytes,
myelocytes, promyelocytes)
• Basophilia
• Eosinophilia
 II. Bone Marrow Examination
a) Cellularity→ Hypercellularity (proliferating myeloid
cells)

b) Myeloid cells The myeloid cells predominate in the bone

marrow with increased myeloid-erythroid ratio

c) Erythropoiesis → Reduced

d) Megakaryocytes → Megakaryocytes are normal but are

usually smaller in size
 III. Cytogenetics

• Cytogenetic studies on blood and bone marrow cells

show the characteristic chromosomal abnormality called
Philadelphia (Ph) chromosome seen in 90-95% cases of
CML.

• Ph chromosome is formed by reciprocal balanced

translocation between part of long arm of chromosome
22 and part of long arm of chromosome 9 → t(9;22) →
BCR/ ABL fusion gene
 IV. Cytochemistry

• Reduced scores of neutrophil alkaline phosphatase

(NAP) which helps to distinguish CML from myeloid
leukaemoid reaction (in which case NAP scores are
elevated )
 V. Other Investigations

• Elevated serum uric acid (hyperuricaemia)

 Lab diagnosis

Blood Bone Marrow Cytogenetics Cytochemistry Others

a) Hb a) Cellularity t(9;22) NAP scores S. uric acid

b) Platelets b) Myeloid cells
c) WBC c) Erythropoiesis
d) Megakaryopoiesis
 Treatment
1. Treatment Of Anaemia And Haemorrhage
a) Anaemia and haemorrhage → fresh blood transfusions and
platelet concentrates.

b) Patients with severe thrombocytopenia → regular platelet

transfusions

2. Imatinib oral therapy

a) Competitively inhibit ATP binding site of the ABL kinase→ inhibits
signal transduction BCR/ABL fusion protein
b) Induces apoptosis in BCR/ ABL-positive cells and eliminates them

3. Allogenic bone marrow (stem cell) transplantation

 Index to assess prognosis of CML

Sokal index Hasford system

a) Platelet count a) Age
b) Age b) Basophils and eosinophils (%)
c) Spleen size c) Circulating blasts (%)
d) Circulating blasts d) Platelet count
c) Cytogenetic changes e) Spleen size
MCQs
1. BCR-ABL hybrid gene is present in
a. Biukitt's lymphoma
b. Retinoblastoma
c. Breast carcinoma
d. CML
1. BCR-ABL hybrid gene is present in
a. Biukitt's lymphoma
b. Retinoblastoma
c. Breast carcinoma
d. CML
2. A 60-year-old man presented with fatigue, weight
loss and heaviness in left hypochondrium for 6
months. The hemogram showed Hb, 10gm/dL, TLC
5 lakhs/mm , platelet count 4 lakhs/mm3 , DLC,
neutrophil 55%, lymphocytes 4%, monocytes 2%,
basophils 6%, metamyelocytes 10%, myelocytes
18%, promyelocytes 2% and blasts 3%. The most
likely cytogenetic abnormality in this case is:

a. a. t (1:21) b. t (9:22)
b. c. t (15, 17) d. Trisomy 21
2. A 60-year-old man presented with fatigue, weight
loss and heaviness in left hypochondrium for 6
months. The hemogram showed Hb, 10gm/dL, TLC
5 lakhs/mm , platelet count 4 lakhs/mm3 , DLC,
neutrophil 55%, lymphocytes 4%, monocytes 2%,
basophils 6%, metamyelocytes 10%, myelocytes
18%, promyelocytes 2% and blasts 3%. The most
likely cytogenetic abnormality in this case is:

a. a. t (1:21) b. t (9:22)
b. c. t (15, 17) d. Trisomy 21
Acute Myeloid Leukaemia(AML)

Dr. PRIYANKA SACHDEV , MD

 CML AML ALL CLL
Introduction
Age
Pathogenesis
Classification
Clinical features

Lab diagnosis
Treatment

Prognosis
 Acute Myeloid Leukaemia

• Acute myeloid leukaemia (AML) is a heterogeneous

disease characterised by infiltration of malignant
myeloid cells into the blood, bone marrow and other
tissues.
 Age

• Peak age group is 15 to 40 years.

 Pathophysiology
• AML develops due to inhibition of maturation of
myeloid stem cells due to mutations

a) t(8;21)
b) t(15;17)
c) inv(16)
 FAB Classification
FAB CLASS OLD NAME
M0: Minimally differentiated AML
M1: AML without maturation
M2: AML with maturation
M3: Acute promyelocyte leukaemia
M4: Acute myelomonocytic leukaemia (Naegeli type)
M5: Acute monocytic leukaemia (Schilling type)
M6: Acute erythroleukaemia (DiGuglielmo's syndrome)
M7: Acute megakaryocyte leukaemia
M0:
• Myeloid lineage blasts
• MPO Negetive

M1:
• Myeloblasts without maturation
• > 3% blasts MPO or SBB positive

M2:
• AML is the Commonest type of AML
• t (8;21) is present
• maximum incidence of chloroma
M3:
• t (15; 17) seen
• Auer rods are seen
• AML is associated with disseminated intravascular coagulation
(DIC)

M4:
• Inversion 16 present
• Presence of both myeloblasts and monoblasts

M5:
• t (9;11)
• Highest incidence of tissue infiltration, organomegaly, and lymphadenopathy
M6:
• Abnormal erythroid precursors are seen

M7:
• Least common type of AML
• Commonest type of AML in Down syndrome
• Megakaryocytes are seen
• Release of platelet derived growth factor (PDGF) causes myelofibrosis
 Clinical Features
I. Due To Bone Marrow Failure→

a) Anaemia producing pallor, lethargy, dyspnoea.

b) Bleeding manifestations causing spontaneous bruises,
petechiae, bleeding from gums and other bleeding
tendencies.
c) Infections
II. Due to organ infiltration by leukaemic cells →

a) Pain and tenderness of bones (e.g. sternal tenderness)

b) Lymphadenopathy and enlargement of the tonsils may occur.
c) Splenomegaly
d) Hepatomegaly
e) Leukaemic infiltration of the kidney
f) Gum hypertrophy due to leukaemic infiltration of the gingivae is a
frequent finding in AML M4 and AML M5
g) Chloroma or granulocytic sarcoma is a localised tumour forming mass
occurring in the skin of orbit (Proptosis) , frequent finding AML M2
h) Meningeal involvement
 Lab diagnosis

Blood Bone Marrow Cytogenetics Cytochemistry Others

a) Hb a) Cellularity a) t(8;21) a) MPO S.uric

b) Platelets b) Leukaemic cells b) t(15;17) b) Sudan black acid
c) WBC c) Erythropoiesis c) inv(16) c) NSE
d) Megakaryopoiesis d) PAS
e) Acid phosphatase
 I. Blood Picture
a) Anaemia→ Normocytic normochromic

b) Thrombocytopenia (Acute promyelocytic leukaemia (M3)

associated with DIC).

c) White blood cells →

• Exceed 100,000/μl
• Myeloblasts with Ayur rods present (Faggot cells→ Maximum in
M3)
 II. Bone Marrow Examination
a) Cellularity → Hypercellular

b) Leukaemic cells
• The diagnosis of AML is based on myeloblasts more than 20% of
cells in the marrow
• Auer rods (Represent abnormal azurophilic granule) present in
myeloblasts → definite evidence of myeloid differentiation.

c) Erythropoiesis → Reduced

d) Megakaryocytes → Reduced or absent

 III. Cytogenetics

a) M2 → t(8;21)
b) M3 → t(15;17)
c) M4 → inv(16)
 IV. Cytochemistry

a) Myeloperoxidase (MPO) → Positive (Negative in M0

myeloblasts)

b) Sudan Black → Positive

c) Non-specific esterase (NSE) → Positive in monocytic series (M3

,M4 and M5).

d) Periodic acid-Schiff (PAS) → Negative (Positive in M6).

e) Acid phosphatase → Negative (Diffuse reaction in M4 and

M5).
 V. Others

• Serum uric acid → Raised

 Lab diagnosis

Blood Bone Marrow Cytogenetics Cytochemistry Others

a) Hb a) Cellularity a) t(8;21) a) MPO S.uric

b) Platelets b) Leukaemic cells b) t(15;17) b) Sudan black acid
c) WBC c) Erythropoiesis c) inv(16) c) NSE
d) Megakaryopoiesis d) PAS
e) Acid phosphatase
 Treatment
1. Treatment Of Anaemia And Haemorrhage
a) Anaemia and haemorrhage → fresh blood transfusions and platelet
concentrates.
b) Severe thrombocytopenia → regular platelet transfusions

2. Cytotoxic Drug Therapy

a) Cytosine arabinoside
b) Anthracyclines (daunorubicin, adriamycin)
c) 6-thioguanine

• Promyelocytic leukaemia (M3) is treated with tretinoin (retinoic acid)

3. Allogenic bone marrow (stem cell) transplantation

 Prognostic factors of AML
Good prognosis
• Age <40 years
• M2,M3,M4 forms of AML
• Blast cell with Auer rods
• TLC < 25 X 109/L
• t(15;17),t(8;21), inv 16
• Rapid response to therapy
• Leukemia without preceding MDS
Bad prognosis
• Age <2 years or >55 years
• M0,M6,M7 forms of AML
• Complex karyotypes
• TLC > 100X109/L
• Deletions 5q, 7q
• Delayed response to therapy
• AML with preceding MDS or
anticancer drug exposure
MCQs
1. All the following are poor prognostic
indicators in AML except:
a. Inv 16
b. Complex karyotype
c. AMLM7
d. Deletion 7q
1. All the following are poor prognostic
indicators in AML except:
a. Inv 16
b. Complex karyotype
c. AMLM7
d. Deletion 7q
2. AML with gum infiltration, hepato-
splenomegaly is most likely to be:
a. ALL
b. M3
c. M2
d. M4
2. AML with gum infiltration, hepato-
splenomegaly is most likely to be:
a. ALL
b. M3
c. M2
d. M4
3. AML with worst prognosis:
a. 8/21 translocation
b. Inversion 16
c. Normal cytogenetics
d. Monosomy 7
3. AML with worst prognosis:
a. 8/21 translocation
b. Inversion 16
c. Normal cytogenetics
d. Monosomy 7
4. Chromosomal translocation characteristic in
acute promyelocyte leukemia is:
a. t ( 15: 17)
b. t ( 22: 9)
c. t (21: 17)
d. t ( 8: 21)
4. Chromosomal translocation characteristic in
acute promyelocyte leukemia is:
a. t ( 15: 17)
b. t ( 22: 9)
c. t (21: 17)
d. t ( 8: 21)
5. Auer rods are seen in:
a. Lymphoblast
b. Myeloblast
c. Erythroblast
d. Megakaryoblast
5. Auer rods are seen in:
a. Lymphoblast
b. Myeloblast
c. Erythroblast
d. Megakaryoblast
6. D.I.C. is seen in-
a. Acute promyelocytic leukemia
b. Acute myelomonocytic leukemia
c. CMC
d. Autoimmune hemolytic anemia
6. D.I.C. is seen in-
a. Acute promyelocytic leukemia
b. Acute myelomonocytic leukemia
c. CMC
d. Autoimmune hemolytic anemia
7. Favourable prognosis for AMI. with-
a. t(8, 21)
b. Deletion 5q
c. Preceeding MDS
d. Age < 2years
7. Favourable prognosis for AMI. with-
a. t(8, 21)
b. Deletion 5q
c. Preceeding MDS
d. Age < 2years
Acute lymphoblastic leukemia /
lymphoma

Dr. PRIYANKA SACHDEV , MD

 CML AML ALL CLL
Introduction
Age
Pathogenesis
Classification
Clinical features

Lab diagnosis
Treatment

Prognosis
 Acute lymphoblastic leukemia/lymphoma

• Acute lymphoblastic leukemia (ALL) encompasses a group

of neoplasms composed of immature, precursor B (Pre-
B) or T (Pre-T) lymphocytes referred to as lymphoblast.

TYPES →
1. Pre B-cell ALL
2. Pre T-cell ALL
 Age

• Pre B-cell ALL → 3-5 years

• Pre T-cell ALL → Adolescent male

 Pathogenesis
Pre B cell ALL
 Pathogenesis
Pre B cell ALL

▪Hyperdiploidy (i.e. more than 50 chromosomes) is most

common cytogenetic change in ALL
▪Hypodiploidy (chromosome number less than 50).
▪Loss-of function mutations in PAX5, E2A, or EBF genes.
▪t(12;21) translocations involving genes ETV6 and
RUNX1.
▪t(9;22) translocations involving genes BCR and ABL
 Don’t forget
t(9;22); (BCR-ABL protein):

1. B-ALL
▪BCR-ABL protein is 190 kDa in size.
▪They have stronger tyrosine kinase activity.

2. CML
• BCR-ABL protein of 210 kDa in size.
▪Weaker tyrosine kinase activity.
Pre B cell ALL
• Gain-of function mutations in NOTCH 1 gene
FAB Classification
 FAB Classification
L1 ALL L2 ALL L3 ALL
Commonest type Next common type Rarest type
Best prognosis Worse prognosis. Worst prognosis.

Small homogenous blast, Large, heterogenous blast, Large homogenous blast,

scanty cytoplasm, indistinct indented nuclei, one or more abundant basophilic cytoplasm
nucleoli nucleoli, moderately with prominent cytoplasmic
abundant cytoplasm, minimal vacoulation staining positive
cytoplasmic vacoulation with Oil Red '0'.
 Clinical Features
I. Due To Bone Marrow Failure→

a) Anaemia producing pallor, lethargy, dyspnoea.

b) Bleeding manifestations causing spontaneous bruises,
petechiae, bleeding from gums and other bleeding
tendencies.
c) Infections
II. Due to organ infiltration by leukaemic cells →

1. Bone pain and tenderness → Resulting from infiltration of the

subperiosteum.

2. Generalized lymphadenopathy, splenomegaly and

hepatomegaly→ neoplastic infiltration of these tissues.

3. Compression of large mediastinal vessels or airway → In Pre-T ALL of

thymus.

4.CNS manifestation (Headache, vomiting, nerve palsies)→ Due to

meningeal involvement.

5. Testicular involvement
 Lab diagnosis

Blood Bone Marrow Cytogenetics Cytochemistry

a) Hb a) Cellularity a) Hyperdiploidy a) MPO

b) Platelets b) Leukaemic cells b) Hypodiploidy b) Sudan black
c) WBC c) Erythropoiesis c) t (9;22) c) NSE
d) Megakaryopoiesis d) PAS
e) Acid phosphatase
 I. Blood Picture
a) Anaemia

b) Thrombocytopenia

c) White blood cells →

• Leucopenia to- normal TLC to leucocytosis
• Large number of circulating lymphoblasts
 II. Bone Marrow Examination

a) Cellularity → Hypercellular

b) Leukaemic cells
• FAB diagnostic criteria → > 30 % blasts in bone marrow
• WHO criteria → > 20% blast in bone marrow

c) Erythropoiesis → Reduced

d) Megakaryocytes → Reduced
 III. Cytogenetics

• Hyperdiploidy
• Hypodiploidy
• Trisomy 4, 7,10
• t (9;12)
• t (12;21)
• t (9;22)
• t (4;11)
 IV. Cytochemistry

a) Periodic acid-Schiff (PAS): Positive

b) Acid phosphatase: Focal positivity in leukaemic blasts in
ALL.
c) Myeloperoxidase: Negative
d) Sudan Black: Negative
e) Non-specific esterase (NSE): Negative
 Lab diagnosis

Blood Bone Marrow Cytogenetics Cytochemistry

a) Hb a) Cellularity a) Hyperdiploidy a) MPO

b) Platelets b) Leukaemic cells b) Hypodiploidy b) Sudan black
c) WBC c) Erythropoiesis c) t (9;22) c) NSE
d) Megakaryopoiesis d) PAS
e) Acid phosphatase
 Treatment
1. Treatment Of Anaemia And Haemorrhage
a) Anaemia and haemorrhage → fresh blood transfusions and
platelet concentrates.
b) Severe thrombocytopenia → regular platelet transfusions

2. Chemotherapy
1. Vincristine
2. Prednisolone
3. Anthracyclines (daunorubicin, adriamycin)
4. L-asparaginase

3. Allogenic bone marrow (stem cell) transplantation

 Prognostic factors
Criteria Good Bad
1. Age 2-10 years <2 years; > 10 years
2. Gender Female Male
3. Race White Black
4. CNS, mediastinum, Absent Present
testicular involvement
5. Peripheral blood blast <100000 >100000
count
6. Cytogenetics a. Hyperdiploidy (>50 a. Hypodiploidy (<50
chromosomes) chromosomes)
b. Trisomy 4, 7, 10 b. t(8:14)
c. t (9:12) and t(12:21) c. t(1:19)
d. t(9:22)
e. t(4:11)
MCQs
1. A 17-year-old boy presented with TL.C of
138 x 109 /L with 80% blasts on the
peripheral smear. Chest X-ray demosnstrated a
large mediastinal mass. Immunophenotyping of
this patient's blasts would most likely
demonstrate -
a. No surface antigens (null phenotype)
b. An immature T cell phenotype (Tdt/D34/CD7 positive)
c. Myeloid markers, such as CD13, CD33 and CD 1 5
d. B cell markers, such as CD 1 9, CD20 and CD22
1. A 17-year-old boy presented with TL.C of
138 x 109 /L with 80% blasts on the
peripheral smear. Chest X-ray demosnstrated a
large mediastinal mass. Immunophenotyping of
this patient's blasts would most likely
demonstrate -
a. No surface antigens (null phenotype)
b. An immature T cell phenotype (Tdt/D34/CD7 positive)
c. Myeloid markers, such as CD13, CD33 and CD 1 5
d. B cell markers, such as CD 1 9, CD20 and CD22
2. Poor prognostic indicator of ALL is
a. Female sex
b. Leukocyte count < 50,000
c. Age greater than 1 year
d. Hypodiploidy
2. Poor prognostic indicator of ALL is
a. Female sex
b. Leukocyte count < 50,000
c. Age greater than 1 year
d. Hypodiploidy
3. Good prognosis of A IX
a. Hyperdiploidy
b. Hypodiploidy
c. T cell line
d. Philadelphia chromosome
3. Good prognosis of A IX
a. Hyperdiploidy
b. Hypodiploidy
c. T cell line
d. Philadelphia chromosome
4. ALL L3 morphology is a malignancy' arising
from which cell lineage -
a. Mature B cell
b. Precursor B cell
c. Immature T cell
d. Mixed B cell & T cell
4. ALL L3 morphology is a malignancy' arising
from which cell lineage -
a. Mature B cell
b. Precursor B cell
c. Immature T cell
d. Mixed B cell & T cell
5. Periodic acid schiff stain shows block
positivity in -
a. Myeloblasts
b. Lymphoblasts
c. Monoblasts
d. Megakaryoblasts
5. Periodic acid schiff stain shows block
positivity in -
a. Myeloblasts
b. Lymphoblasts
c. Monoblasts
d. Megakaryoblasts
Chronic Lymphocytic Leukaemia (CLL) /
Small Lymphocytic Lymphoma (SLL)

Dr. PRIYANKA SACHDEV , MD

Long questions

1. Define and classify leukemias. Discus peripheral blood

smear, bone marrow biopsy and Immunophenotyping
associated with Chronic Lymphocytic Leukaemia (CLL)
2. Define and classify leukemias. Describe the clinical
features and laboratory diagnosis of Chronic Lymphocytic
Leukaemia (CLL)
Short questions
1. Peripheral blood smear of CLL
2. Bone marrow biopsy of CLL
3. Immunophenotyping of CLL
4.Laboratory diagnosis of Chronic Lymphocytic Leukaemia
5.Poor Prognostic factors of CLL
6.Small Lymphocytic Lymphoma (SLL)

Very Short questions

1. Smudge cells
 CML AML ALL CLL
Introduction
Age
Pathogenesis
Classification
Clinical features

Lab diagnosis
Treatment

Prognosis
 Chronic Lymphocytic Leukaemia (CLL) /
Small Lymphocytic Lymphoma (SLL)

• CLL and SLL are identical neoplasms arise due to an abnormal

neoplastic proliferation of B cells.

• CLL is defined as an absolute lymphocyte count more than 5000

per mm3.
• Some patient may not have lymphocytosis and are classified as
SLL

❖CLL involves primarily bone marrow and blood

❖SLL involves lymph nodes.
 Age

Median age of 60 years

 Pathogenesis

• Deletions of 13q, 11q, and 17p

• Trisomy 12q

• Most common chromosomal change in CLL is

deletions 13q (good prognosis).
 Clinical Features
1. Due To Bone Marrow Failure→

a) Anaemia producing pallor, lethargy, dyspnoea.

b) Bleeding manifestations causing spontaneous bruises,
petechiae, bleeding from gums and other bleeding tendencies.
c) Infections

2. Enlargement of superficial lymph nodes is a very common

finding. The lymph nodes are usually symmetrically enlarged,
discrete and non-tender.

3. Splenomegaly and hepatomegaly

 Lab diagnosis

Blood Immunophenotyping Lymph Node Biopsy

a) Hb CD19 Diffuse proliferation

b) Platelets CD20 Pseudofollicles
c) WBC CD 23
CD5
Surface IgM and IgD
 I. Blood Picture
a) Anaemia → Normocytic normochromic

b) Thrombocytopenia

c) White blood cells →

• Marked leukocytosis (50,000-200,000/ μl).
• More than 90% of leucocytes are mature small lymphocytes.
• Smudge or basket cells (degenerated forms) are present due
to damaged nuclei of fragile malignant lymphocytes.
 Smudge or basket cells

• Slide artefact formed by deficiency of cytoskeletal

protein Vimentin, which is required for rigidity and
integrity of cells
 II. Immunophenotyping

• CLL is a tumor of mature B-ceIls

• Therefore it expresses the B-cell markers such as CD19
,CD20 and surface IgM and IgD
• In addition CD 23 and CD5 are also present
III. Lymph Node Biopsy
 III. Lymph Node Biopsy

▪Lymph nodes involved by SLL show an effacement of

normal nodal architecture by a diffuse proliferation of
small, round lymphoid cells with coarse chromatin,
indistinct nucleoli, and scant cytoplasm
▪In addition, scattered nodules (so-called pseudofollicles,
growth centers, or proliferation centers) composed of
medium sized and large lymphoid cells with dispersed
chromatin and distinct nucleoli are observed.
▪The diffuse proliferation of small lymphoid cells with
pseudofollicles is pathognomonic for SLL.
 III. Lymph Node Biopsy

▪Effacement of normal nodal architecture by a diffuse

proliferation of small, round lymphoid cells with coarse
chromatin, indistinct nucleoli, and scant cytoplasm
▪Scattered nodules (pseudofollicles) composed of medium
sized and large lymphoid cells with dispersed chromatin and
distinct nucleoli are observed.

▪The diffuse proliferation of small lymphoid cells with

pseudofollicles is pathognomonic for SLL.
 Lab diagnosis

Blood Immunophenotyping Lymph Node Biopsy

a) Hb CD19 Diffuse proliferation

b) Platelets CD20 Pseudofollicles
c) WBC CD 23
CD5
Surface IgM and IgD
 Treatment

• Unlike other leukaemias, none of the available drugs

and radiation therapy are capable of eradicating CLL
and induce true complete remission.

• Treatment is palliative and symptomatic

 Prognosis
Poor prognosis →

1) Deletions of 11q or 17p and trisomy 12.

2) Expression of ZAP-70 protein that augments
immunoglobulin receptor signalling activity.
3) Presence of notch1 mutations.
MCQs
1. A 80 year-old man presents with painless
cervical lymphadenopathy .Peripheral smear
shows presence of
a. ALL
b. AML
c. CLL
d. CML
1. A 80 year-old man presents with painless
cervical lymphadenopathy .Peripheral smear
shows presence of
a. ALL
b. AML
c. CLL
d. CML
Hodgkins lymphoma

Dr. PRIYANKA SACHDEV , MD

 Overview
• Introduction
• Types of RS cells
• WHO Classification of Hodgkins lymphoma →
❖Nodular sclerosis
❖Mixed Cellularity
❖Lymphocyte Rich
❖Lymphocyte depleted
❖Lymphocyte predominant
• Clinical Features
• Ann Arbor Staging
 Hodgkins lymphoma
It has following characteristics→

• It arises in a single lymph node or chain and follow

orderly spread to anatomically contiguous lymphoid
tissue

• Presence of distinctive neoplastic giant cells called

Reed-Sternberg (RS) cells derived from germinal center
or post-germinal center B-cells
 TYPES OF RS Cells

4 Types →

• A) Classical Reed-Sternberg cells

• B) Lacunar cells
• C) Lympho-histocytic (L&H cells) or popcorn cells
• D) Pleomorphic variant
 A) Classical Reed-Sternberg cells
• Large cell with bilobed nucleus appearing as mirror image of
each other
• Each lobe of the nucleus contains a prominent, eosinophilic,
inclusion-like nucleolus with a clear halo around it, giving an
owl-eye appearance.
 B) Lacunar cells

• It is smaller
• In addition to Classical RS features has a pericellular
space or lacuna in which it lies, which is due to
artefactual shrinkage of the cell cytoplasm.
C) Lympho-histocytic (L&H cells) or popcorn cells

• Large with lobulated nucleus in the shape of popcorn

 D) Pleomorphic variant

• These cells have pleomorphic and atypical nuclei.

Types of RS cells
 WHO Classification of Hodgkins lymphoma

The classification is based on Immunophenotyping of RS cells

1. Classical HD → RS cells positive for CD 15 and CD 30

2. Non-classical /Nodular lymphocytic predominance type → RS

cells positive for CD 20 and BCL-6 but are negative for CD 15 and
CD 30.
Classic subtypes Non classical

• Nodular sclerosis • Lymphocyte predominant

• Mixed cellularity
• Lymphocyte-rich
• Lymphocyte depletion
 Lymphocyte
Lymphocyte
Nodular sclerosis Mixed cellularity Lymphocyte rich predominant
depleted
(non classical HL)
‒ MC type ‒ MC type in India ‒ Associated with
HIV
‒ M=F ‒ M>F ‒ M>F ‒ M>F ‒ M>F

‒ Lacunar cell RS ‒ Classical RS cells ‒ Classical RS cells ‒ Pleomorphic RS ‒ LH (popcorn cells)

cell cell RS cell
‒ CD15 + CD30+ ‒ CD15 + CD30+ ‒ CD15 + CD30 + ‒ CD15 + CD30+ ‒ CD20 + BCL6 +,
and CD20- EMA +
‒ CD15—, CD30-

‒ No association ‒ Associated with ‒ Associated with ‒ Associated with ‒ Not associated

with EBV EBV EBV EBV with EBV
‒ Excellent ‒ Prognosis very ‒ Good to excellent ‒ Poor prognosis ‒ Excellent
prognosis good prognosis prognosis
‒ Adolescent ‒ Biphasic incidence ‒ Old age group ‒ Old age group ‒ Young males
(young and > 55
years)
1. The subtype of Hodgkin's disease, which is
histogentically distinct from all the other
subtypes, is-
a. Lymphocyte predominant
b. Nodular sclerosis
c. Mixed cellularity
d. Lymphocyte depleted
1. The subtype of Hodgkin's disease, which is
histogentically distinct from all the other
subtypes, is-
a. Lymphocyte predominant
b. Nodular sclerosis
c. Mixed cellularity
d. Lymphocyte depleted
2. Reed Sternberg cells are found in –
a. Hodkin's disease
b. Sickle cell anaemia
c. Thalassemia
d. CML
2. Reed Sternberg cells are found in –
a. Hodkin's disease
b. Sickle cell anaemia
c. Thalassemia
d. CML
3. The lymphocytic and histiocytic variant of
Reed-Sternberg cell is seen in –
a. Follicular center lymphoma
b. Lymphocyte depleted Hodgkin's disease
c. Nodular sclerosis Hodgkin's disease
d. Lymphocyte predominant Hodgkin's disease
3. The lymphocytic and histiocytic variant of
Reed-Sternberg cell is seen in –
a. Follicular center lymphoma
b. Lymphocyte depleted Hodgkin's disease
c. Nodular sclerosis Hodgkin's disease
d. Lymphocyte predominant Hodgkin's disease
4. Best prognostic type of Hodgkin's
lymphoma is –
a. Lymphocytic predominant
b. Lymphocytic depletion
c. Mixed cellularity
d. Nodular sclerosis
4. Best prognostic type of Hodgkin's
lymphoma is –
a. Lymphocytic predominant
b. Lymphocytic depletion
c. Mixed cellularity
d. Nodular sclerosis
5. Most common type of hodgkins lymphoma
is
a. Lymphocyte predominant
b. Lymphocyte depletion
c. Nodular sclerosis
d. Mixed cellularity
5. Most common type of hodgkins lymphoma
is
a. Lymphocyte predominant
b. Lymphocyte depletion
c. Nodular sclerosis
d. Mixed cellularity
6. Most common type of hodgkin's lymphoma
in India is –
a. Nodular sclerosing
b. Lymphocyte predominance
c. Mixed cellularity
d. Lymphocyte depletion
6. Most common type of hodgkin's lymphoma
in India is –
a. Nodular sclerosing
b. Lymphocyte predominance
c. Mixed cellularity
d. Lymphocyte depletion
7. Classical markers for Hodgkin's disease
a. CD 15 and CD 30
b. CD 15 and CD 22
c. CD 15 and CD 20
d. CD 20 and CD 30
7. Classical markers for Hodgkin's disease
a. CD 15 and CD 30
b. CD 15 and CD 22
c. CD 15 and CD 20
d. CD 20 and CD 30
8. 'Popcorn-cells' are seen in which variety of
hodgkin's disease –
a. Nodular sclerosis
b. Mixed cellularity
c. Lymphocyte predominance
d. Lymphocyte depletion
8. 'Popcorn-cells' are seen in which variety of
hodgkin's disease –
a. Nodular sclerosis
b. Mixed cellularity
c. Lymphocyte predominance
d. Lymphocyte depletion
9. Lacunar cells are seen in which type of
Hodgkin’s lymphoma –
a. Lymphocyte predominance
b. Lyphocyte depletion
c. Nodular sclerosing
d. Mixed cellularity
9. Lacunar cells are seen in which type of
Hodgkin’s lymphoma –
a. Lymphocyte predominance
b. Lyphocyte depletion
c. Nodular sclerosing
d. Mixed cellularity