DISEASES
OF
HAEMOPOIETIC
SYSTEM
Blood is a unique organ: it is fluid and comes
into contact with almost all other tissues.
The blood cells are non-cohes...
BLOOD CELL
FORMATION
(HAEMOPOIESIS)
SITE OF HAEMOPOIESIS
Fetus 0 – 2 months (yolk sac)
2 – 7 months (liver. Spleen)
5 – 9 months (bone marrow)
Infants Bone ma...
HAEMOPOITIC STEM CELL AND PROGENITOR
CELLS
Haemopoiesis starts with a common, ‘Pluripotent
Haemopoietic Stem Cell’ which ...
DIAGRAMATIC REPRESENTATION OF BONE MARROW PLURIPOTENT
STEM CELL AND CELL LINES THAT ARISE FROM IT
DISEASES OF HAEMOPOIETIC
SYSTEM
1. Anaemias
2. Haematological Malignancies
3. Bleeding Disorders
4. Blood Transfusion
ANAEMIAS
ANAEMIA
Definition: “Reduction in the concentration of
haemoglobin in the blood below the lower limit of
normal for a part...
ADULT REFERENCES OF RED BLOOD CELLS
_________________________________________________
Male Female
________________________...
SYMPTOMS AND SIGNS OF ANAEMIA
Symptoms of Anaemia:
Level of haemoglobin at which patient develops symptoms depends
on the ...
CLASSIFICATION OF ANAEMIA
1. Kinetic Classification of Anaemias
2. Morphologic Classification of Anameias
KINETIC CLASSIFICATION OF
ANAEMIAS
I. ANAEMIAS DUE TO EXCESSIVE BLOOD LOSS
(i) Acute blood loss anaemia
(ii) Chronic blood...
III ANAEMIAS DUE TO EXCESSVIE RED CELLS DESTRUCTION
(HAEMOLYTIC ANAEMIA)
1. HAEMOLYSIS DUE TO RED BLOOD CELLS ABNORMALITY
...
MORPHOLOGIC CLASSIFICATION OF ANAEMIAS
Reticulocyte Count
More than 2.5 % Less than 2.5%
Haemolysis Red Cells Morphology
O...
MORPHOLOGIC CLASSIFICATION OF ANAEMIAS
Causes of Microcytic and Hypochromic Anameias (MCV – Less than 80 fl)
1.Iron Defic...
IRON DEFICIENCY ANAEMIA
Iron deficiency anaemia is the most common cause
of anaemia in every country of world.
It is the...
The causes of a hypochromic microcytic anaemia. These include lack of iron
(iron deficiency), or of iron release from macr...
IRON ABSORPTION AND METABOLISM
Iron Absorption: Iron absorbed from food and iron recovered from senescent red blood cells ...
IRON ABSORPTION AND METABOLISM
Iron Absorption: Iron absorbed from food and iron recovered from senescent red blood cells ...
Iron Absorption
ucosal uptake of heme and nonheme iron is depicted. When the storage sites of the body
e replete with iron...
Iron in
the body
Iron the diet is of two types – Haem Iron and Non Haem Iron.
Much of the dietary iron is non haem iron derived from cerea...
CAUSES OF IRON DEFICIENCY ANAEMIA
I. CHRONIC BLOOD LOSS:
(i) Uterine Bleeding:
-Menorrhagia (excessive menstrual bleeding)...
CLINICAL FEATURES OF IRON DEFICIENCY
ANAEMIA
 The patient develop the general symptoms and signs of anaemia and also
show...
IRON DEFICIENCY: LABORATORY DIAGNOSIS
Peripheral Blood Findings
(i)Haemoglobin is decreased
(ii) Red cells indices (MVC;MC...
Red Blood Cells Morphology: Microcytic and Hypo chromic red cell morphology
with pencil shaped poikilocyttes.
Red cells mo...
TREATMENT OF IRON DEFICIENCY ANAMEIA
It is usual to give 100 to 200 mg of elemental iron daily
to adults and 3 mg/kg body...
Form of
Iron
Preparation Size Iron content Usual oral
dose
FERROUS
SULPHATE
Tablet 300 mg 60 mg 3 tablets
daily
Syrup 40 m...
PARENTRAL IRON
 It is given only in conditions where patient can not
tolerate oral iron or rapid replenishment of stores ...
PARENTRAL IRON: IRON SUCROSE (VENOFER)
The commonly used preparation is Iron Sucrose.
Parental iron preparation contains F...
MEGALOBLASTIC ANAEMIAS
This is a group of anaemias in which, due to
impaired DNA synthesis, the erythroblasts in the
bone...
VITAMIN B 12 AND FOLATE - COMPARISON
VITAMIN B 12 FOLATE
CONTENTS IN
FOOD
Vegetables – Poor
Meat – Rich
Vegetables – Rich
...
ABSORPTION OF VITAMIN B 12
Absorption of B12 requires Intrinsic Factor (IF),
which is secreted by the parietal cells of th...
ABSORPTION OF FOLIC ACID
Folic acid in the diet is in two form
Monoglutamates and Polyglutamates.
 More than 90% is poly...
PATHOGENESIS OF HOW B12 AND FOLATE
DEFICIENCY PRODUCE MEGALOBLASTIC ANAEMIA
Lack of Vitamin B12 or Folate causes slowing o...
CAUSES OF VITAMIN B12 DEFICIENCY
1.Decreased intake of Vitamin B12:
Nutritional deficiency (Vegetarians)
2. Impaired absor...
CAUSES OF FOALTE DEFICIENCY
1. Decreased intake of Folic Acid:
Nutritional Deficiency
2. Impaired Absorption of Folic Acid...
CLINICAL FEATURES OF MEGALOBLASTIC ANAEMIA
Glossitis: tongue is beefy
red and painful
Angular stomatitis
 The onset is us...
CLINCAL MANIFESTATIONS DUE TO VITMAIN B 12 DEFICIENCY
Vitamin B12 is required for two important reactions in the body:
Vit...
Demyelination of the
dorsal and dorsolateral
columns
A baby with neural tube
defect (spina bifida)
VITMAIN B 12 NEUROPATHY...
Lab Diagnosis of Megloblastic Anemia
1.Peripheral blood finidngs
2.Bone Marrow Findings
3.Special Tests
1. Peripheral Bloo...
2.. Bone Marrow Findings in Megaloblastic Anaemia
a. ERYTHROPOIESIS :
- Megaloblasts All the nucleated series of erythroid...
3. Special Tests:
1.Serum Vitamin B12- Decreased
2.Serum Folic Acid-Decreased
Macrocytic Anemia (Meg.):
Megaloblastic Anemia :
Hypersegmented Neutrophils
Oval macrocyte
Marrow smear from a patient
with megaloblastic anaemia:
Megaloblast in various stages
of differentiation.
3. BONE MARROW F...
TREATMENT OF MEGALOBLASTIC ANAEMIA
VITAMIN B12
DEFICIENCY
FOLATE
DEFICIENCY
Compound Hydroxycobalamin Folic Acid
Route Int...
APLASTIC ANAEMIA
Aplastic Anaemia is defined as the presence of pancytopenia
in the peripheral blood and a hypocellular m...
CAUSES OF APLASTIC ANAEMIA
I. CONGENITAL
Fanconi’s Aplastic Anaemia
II. ACQUIRED
1. Idiopathic
2. Chemical and Physical Ag...
PATHOGENESIS OF APLSTIC ANAEMIA
1. DIRECT DAMAGE: Can be caused by direct damage to the
haemopoietic marrow by radiation o...
These cytokines then attach to their
receptor on stem cell and induce
damage to haemopoietic stem cell in
following manne...
CLINICAL FEATURES OF APLASTIC ANAEMIA
The onset is at any age with a peak incidence around 30
years and a slight male pre...
CLINICAL FEATURES OF APLASTIC ANAEMIA…contd
Lymph nodes, liver. Spleen are not enlarged
 Rarely jaundice can be feature ...
LAB DIAGNOSIS OF APALSTIC ANAEMIA:
e peripheral blood film shows
ncytopenia(Anaemia; Leucopenia;Thrombocytopenia)
ere are ...
7. The bone marrow aspirate is usually easily obtained,
typically with many fragments, which appear hypocelluar
(more than...
Vs
Normal bone marrow: From a
section of bone marrow trephine
biopsy. Marrow cells are
interspersed between fat spaces.
Th...
CAUSES OF PANCYTOPENIA
1. Aplastic Anaemia
2. Megloblastic Anaemia
3. Bone marrow infiltration by leukaemias,
lymphomas, m...
HAEMOLYTICHAEMOLYTIC
ANAEMIASANAEMIAS
HAEMOLYTIC ANAEMIAS
The distinguishing feature of all haemolytic anaemias is the
increased rate of red cells destruction
N...
(a) Normal red cell breakdown- Extravascular: This takes place extravascullarly
in the macrophages of the reticuloendothel...
INTRAVASCUALR AND EXTRAVASCULAR
HAAMOLYSIS
There are two main mechanisms by which a red cells are destroyed in
haemolytic ...
INTRODUCTION TO HAEMOLYTIC ANAEMIAS
Haemolytic anaemias are defined as those anaemias
which result from an increase in the...
CLASSIFICATION OF HAEMOLYTIC ANAEMIAS
Hereditary Haemolytic Anaemias are the result of
‘intrinsic’ red cell defects
where...
LABORATORY FINDINGS IN HAEMOLYTIC
ANAEMIAS
The laboratory findings in haemolytic anaemia are
conveniently divided in thre...
2. Features of Increased Red cells Production
Compensatory Erythroid Hyperplasia)
i. Reticulocytosis
ii. Macrocytosis and ...
GENETIC BASIS OF DISEASES
There are three major categories of genetic disorders:
1. MENDELIAN DISORDERS: Disorders relate...
MENDALIAN DISORDERS
All Mendalian disorders are result of expressed
mutations in single or small gene clusters, the
chrom...
HOMOZYGOUS VS HETEROZYGOUS
DOMINANT VS RECESSIVE
Each individual possesses two factors for a
charactersitic.
If these fa...
AUTOSOMAL DOMINANT DISORDERS
Autosomal dominant disorders are manifested in the
heterozygous state. That is, a person wit...
Autosomal dominant disorders tend to be extremely
variable in expression. Some individuals may be so
mildly affected that...
Autosomal recessive inheritance is the single largest
category of mendelian disorders.
These disorders are characterized ...
3. If both the parents are heterozygous (carriers) then in
each pregnancy there are 25% chances that the child can
have ho...
All sex – linked disorders are X- linked, almost all X-
linked recessive. The only gene assigned with certainty to
the Y-...
Since a male transmits an X- chromosome to each of his daughters
and a Y- chromosome to each of his sons, then all the da...
HAEMOGLOBIN DISORDERS
OR
HAEMOGLOBINOPATHIES
Haemoglobin disorders result from:
1. Reduced synthesis of normal Alpha or Be...
THALASSAEMIA
“The Thalassaemias are a heterogeneous group of genetic disorders of
haemoglobin synthesis all of which resul...
MOLECULAR ASPECTS
All the globin genes have three
axons (coding regions) and two
introns (non coding regions whose
DNA is ...
CLINICAL AND GENETIC CLASSIFICATION
OF THALASSAEMIAS
I. BETA (β) THALASSAEMIAS: (Defects in transcription, processing or
t...
II.ALPHA THALASSAEMIAS: Defect is mainly deletion of
genes
(i) Alpha Thalassaemia Silent Carrier( - α/αα)
Asymptomatic; no...
BETA (β ) THALASSAEMIA SYNDROMES
Molecular Pathology of Beta Thalassaemia
Defects in Transcription, Processing or Translat...
Molecular Pathogenesis of Beta Thalassaemia
- Lesions in Beta Thalassameia – Usually point mutations
- Lesions in Alpha Th...
Molecular Pathogenesis of Beta Thalassaemia …contd
3. Splicing Mutations : Mutations that lead to aberrant splicing are th...
COMMON BETA THALASSAEMIA MUTAITONS IN
PAKISTAN
AUTOSOMAL RECESSIVE DISORDER
BETA THALASSAEMIA
•Mediterranean population, Middle East, India, Pakistan, Southeast
Asia, So...
In Beta Thalassaemia Major there is a total lack or a
reduction in the synthesis of structurally normal beta-
globin chain...
Beta Thalassaemia – Clinical Features
CLINICAL FEATURES OF BETA
TAHLASSAEMIA MAJOR
1. Severe anaemia with failure to thrive on 3-7 months of
age after birth
2. ...
•The facial appearance of a child with beta thalassae-
• mia major: Skull is bossed with prominent frontal
•and parietal b...
he patient requires regular blood
nsfusions to sustain an acceptable
emoglobin level. But iron overload
used by repeated t...
LABORATROY DIAGNOSIS OF BETA
THALASSAEMIA MAJOR
1.Peripheral blood film will show severe microcytic and
hypochromic blood ...
Beta Thalassaemia Major
 Microcytic and
hypochromic blood
picture
 Marked anisocytosis &
Poikilocytosis
 Nucleated RBC
...
4. Haemoglobin electrophoresis shows accentuated band
of HbF(Fetal Haemoglobin)
5. Fetal Haemoglibin estimation by alkali ...
Haemoblobin Electrophoresis on Cellulose Acetate
for Haemoglobin Defects
Normal Haemoglobin Electrophoretic Pattern
Beta Thalassaemia Minor (Trait)
Beta Thalassaemia Major
Haemoglobin E trait
Haemoglobin D trait
Haemoglobin C Trait
TREATMENT OF BETA THALASSAEMIA MAJOR
1.Regular blood transfusions are required to maintain the
haemoglobin level over 10 g...
•Thalassaemics receiving blood transfusion
at a thalassaemia center
BETA THALASSAEMIA MINOR (TRAIT)
It is a heterozygous state. The person is carrying abnormal genes
from one parent and nor...
D/D OF MICROCYTIC AND HYPOCHROMIC BLOOD PICTURE
Blood film in Beta Thalasaemia
Major: Microcytic and Hypochromic
with frag...
•Iron Deficiency Vs Beta Thalassaemia Trait
•Iron Deficiency Vs Beta Thalassaemia Trait
Iron Deficiency Vs Beta Thalassaemia Trait
Microcytic hypochromic
morphology of red cells in
proportion to the degree of
a...
“ More than the calf wishes to
suck does the cow wish to suckle”
Teaching Craze !!!
SICKLE CELL ANAEMIA
 Sickle Cell Anaemia or Sickle Cell Disease is an Autosomal
Recessive, Hereditary Haemoglobin disorde...
This substitution confers abnormal physicochemical
properties to red cells. Under unfavourable conditions,
most important...
The change from glutamic acid to valine at
position 6 changes the 3-D structure of the
molecule
This makes hemoglobin st...
PATHOGENESIS OF
SICKLE
CELL DISEASE
On deoxygenation the
HbS molecules under-
go aggregation and
polymerization . This
ch...
 The formation of HbS has two major consequences:
(i) A chronic Haemolytic Anameia
(ii) Occlusion of small blood vessels,...
VARIABILITY IN SICKLING
Sickle cell anaemia runs an extremely variable clinical
course. At one end of the spectrum it is ...
5. Caliber of blood vessels: In high flow areas the shear
stresses can break down the gel structure of HbS, while in
the s...
CLINCAL MANIFESTATIONS OF SICKLE CELL ANAEMIA
CLINICAL PRESENTATION
•High levels of fetal haemoglobin protect against sick...
 During early development of the disease there is often
splenomegaly . In most patients this gradually resolves
due to re...
The complications of Sickle Cell Anaemia can be divided into two groups:
1. Acute and Episodic Crises
2. Chronic Complicat...
CLINICAL CONSEQUENCES
OF SICKLE CELL DISEASE
2. Aplastic Crises: These occur as a result of infection with Parvo
Virus or Folate deficiency. These are characterized by...
5. CNS Crises: Patient presents with a stroke, preceded by bizarre
neurological syndromes similar in nature to transient i...
2. Infarcts following repeated episodes of vascular occlusion:
Result largely from infarcts following repeated episodes of...
COURSE AND PROGNOSIS
The prognosis seems to depend upon the racial
background of the patient, socioeconomic and ill- defi...
LABORATORY DIAGNOSIS OF
SICKLE CELL ANAEMIA
1. Haemoglobin level: The haemoglobin level is usually between
5 to 11 g/dl.
2...
Sickle Cell Disease: Homozygous state.
Numerous Crescent Shaped sickle cells are seen.
Sickle Cell Test
Normal or Alpha
Thalassaemia Trait
Sickle Cell Trait
Sickle Cell Disease
Beta Thalassaemia Trait
Beta Thalassaemia Major
S...
Normal Haemoglobin
Electrophoretic Pattern Sickle Cell Disease Sickle Cell Disease
with high HbF
5. Bilirubin level: Is elevated
6. Serum LDH: Is elevated in crises
7. Blood Gases: should be estimated in crises especial...
POLYMERASE CHAIN REACTION (PCR) FOR SICKLE CELL:
Sickle Cell Anaemia ; Antenatal Diagnosis: Direct DNA analysis. The
DNA h...
SICKLE CELL TRAIT
• This is a benign condition with no anaemia and normal
appearance of red cells on a blood film.
• Scree...
TREATMENT OF SICKLE CELL ANAEMIA
1. Prophylactic: Avoid those factors known to precipitate crises,
especially:
- Dehydrati...
4. Malarial Prophylaxis: Malarial prophylaxis is mandatory for visits to areas
where malaria is endemic
5. Folic Acid: 5 m...
7. Management of Painful Crises: Should be managed in
hospital. Methods employed to manage painful crises are:
- Rest
- Wa...
8. Lung Crises: Once suspected patient should be managed in
hospital in intensive care unit. Oxygen should be administered...
12.Renal problems: Hameaturia and renal failure should be
managed accordingly.
13. Priapism: It occurs quite frequently. I...
15. Splenectomy
Usually upto the end of first decade there is
autosplenectomy. But in patients who have recurrent attacks ...
17. Stroke:
• As the life expectancy of patients with sickle cell disease increases, more
adults presenting with CNS sympt...
18. Bone Marrow OR Stem Cell Transplantation: It can cure
the disease and many patients have now been successfully
treated...
HEREDITARY SPHEROCYTOSIS
Hereditary Spherocytosis is characterized by an
intrinsic defect in the red cell membrane that re...
MOLECULAR
PATHOLOGY
OF HEREDITARY
SPHEROCYTOSIS(HS)
e spheroidal shape of
d blood cells is
aintained by some
egral protein...
As these spherocytes are trapped in the sluggish circulation of
spleen then the deformed red cells are destroyed and engul...
CLINICAL FEATURES OF HEREDITARY
SPHEROCYTOSIS
•The characteristic clinical features of Hereditary
Spherocytosis are :
- An...
LAB DIAGNOSIS OF HEREDITARY
SPHEROCYTOSIS
1. Anaemia is usual but not invariable.
2. Reticulocyte Count: Increased
3. Bloo...
LAB DIAGNOSIS OF HEREDITARY
SPHEROCYTOSIS. contd
4. Serum Bilirubin Increased (indirect
Hyperbrubinemia)
5. Ultrasound abd...
TREATMENT OF HEREDITARY SPHEROCYTOSIS
1 .Splenectomy: It is the principal form of treatment. It should
not be performed un...
GLUCOSE – 6 – PHOSPHATE DEHYDROGENASE
DEFICIENCY
The erythrocyte and its membrane are vulnerable to injury by exogenous
an...
ant G6PD gene
ient or Decreased production
of G6PD Enzyme
ility of glucose 6 phosphate
dize into 6- phospohgluconate
re of...
POPULATION GENETICS
• Sex Linked (x- Linked) : Males are affected
•The inheritance is sex-linked, affecting males, and fem...
AGENTS WHICH MAY CAUSE HAEMOLYTIC ANAMEIA
IN GLUCOSE -6- PHOSPHATE DEHYDROGENASE
DEFICIENCY
I. Infections and other acute ...
CLINCAL FEATURES OF G-6-PD DEFICIENCY
 The haemolysis in G-6-PD is both intravascular and
Extravascular.
 The clinical p...
LABORATORY DIAGNOSIS OF
G- 6 PD DEFICIENCY ANAEMIA
Red cells showing loss of cytoplasm with
separation of remaining haemog...
TREATMENT OF G-6-PD DEFICIENCT ANAEMIA
1. The offending drug is stopped.
2. Any underlying infection is treated.
3. A high...
Malignancies
Of
Haemopoietic
System
Main Type Sub Type
I LEUKAEMIAS 1. Acute Leukaemia
(i) Acute Lymphoblastic Leukaemias(ALL)
L1 ; L2 ; L3
(ii) Acute Myeloid...
LEUKAEMIAS
The leukaemias are group of disorders
characterized by the accumulation of
malignant white cells in the bone m...
CLASSIFICATION OF LEUKAEMIAS
Leukaemias are mainly classified into four types – Acute
and Chronic leukaemias which are fur...
Incidence of Different Types of Leukaemias
According to Age
• ALL is predominantly a childhood disease
• CLL occurs mainly...
Classification OF ACUTE LEUKAEMIAS
 Acute Leukaemia is defined as the presence of over 30%
blast cells in the bone marrow...
Clinical features to some extent are helpful in the
differentiation of ALL and AML, but are not definitive.
The age profi...
MORPHOLOGIC APPROACH TO ACUTE LEUKAEMIAS
The experienced morphologist can classify about 70% of acute
leukaemias as eithe...
DIFFERENCES BETWEEN LYMPHOBLAST AND MYELOBLAST
LYMPHOBLAST MYELOBLAST
Size: Smaller as compared to
myeloblast
Larger than ...
Granules in Cytoplasm: Absent May be present
Auer Rods Absent Present
Accompanying cells: - Maturing cells of myeloid seri...
Lymphoblasts VS Myeloblasts
A: Lymphoblasts have fewer nucleoli than myeloblasts,and the nuclear
chromatin is more condens...
Leukaemia Diagnosis: First Routine Stain
Second: Cytochemistry
Acid Phosphatase Periodic Acid Schiff
Sudan Black
Alpha Nephythyl Acetate Esterase
Third: CD Markers
Flowcytometery
Immunocytochemisrtry
Fourth :Cytogenetics
Acute LeukemiaAcute Leukemia
CYTOCHEMICAL PROFILES IN ACUTE LEUKEMIACYTOCHEMICAL PROFILES IN ACUTE LEUKEMIA
MPO & Sudan Bl...
Immunologic Markers for Classification of ALL and AML
AML
ALL
Marker Precursor T cell* T cell
B Lineage
CD 19 - + -
CD 22 ...
FAB Classification of Acute Lymphoblastic leukaemia
ALL – L1: Small monomorphic blasts with high N:C ratio
ALL – L2: Large...
Acute Lymphoblastic Leukaemia:
FAB Type ALL – L1
Lymphoblasts show monomorphic
appearance, very high N:C ratio;
Nucleoliar...
Acute Leukemia - MorphologyAcute Leukemia - Morphology
ACUTE LYMPHOBLASTIC LEUKEMIAACUTE LYMPHOBLASTIC LEUKEMIA
L1 - small...
Acute Lymphoblastic Leukaemia FAB Type ALL – L2
Large Heterogeneous population of blast cells; Nucleolated;
Low N: C ratio.
Acute Leukemia - MorphologyAcute Leukemia - Morphology
ACUTE LYMPHOBLASTIC LEUKEMIAACUTE LYMPHOBLASTIC LEUKEMIA
L2 - Large...
Acute Lymphoblastic Leukaemia FAB Type ALL – L3:
Lymphoblasts with strongly basophilic cytoplasm and showing
vacuolation.
Acute Leukemia - MorphologyAcute Leukemia - Morphology
ACUTE LYMPHOBLASTIC LEUKEMIAACUTE LYMPHOBLASTIC LEUKEMIA
L3 - Mediu...
Acid Phosphatase staining in ALL: Showing cytoplasmic positivity; Localized
staining in the Golgi zone. Reaction is typica...
Periodic Acid Schiff (PAS) stain in Acute Lymphoblastic Leukaemia
Block Positivity of Cytoplasm. Typical of B- ALL
FRENCH AMERICAN BRITISH(FAB)
CLASSIFICATION OF ACUTE MYELOID
LEUKAEMIA(AML)
AML – M0 : Undifferentiated Myelobalastic leuk...
AML – M0. Undifferentiated blasts which are negative with cytochrmical
reactions for AML(Sudan Black B and ANAE) and posit...
Acute Leukemia - MorphologyAcute Leukemia - Morphology
ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA
AML-M0
• Large...
AML – M1: AML without maturation; 20% of cases ; Very immature
but > 3% are Sudan Black B or myeloperoxidase positive; Few...
Acute Leukemia - MorphologyAcute Leukemia - Morphology
ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA
AML-M1
• A/gra...
AML – M2: AML with Maturation; 30 -40% of cases; Full range of
myeloid maturation through granulocytes. Auer rods present ...
Acute Leukemia - MorphologyAcute Leukemia - Morphology
ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA
AML-M2
• A/gra...
AML – M2 (Sudan Black B): Strong positivity in cytoplasm
AML – M3:Acute Promyelocytic leukaemia; 5 – 10 % of cases;
Majority of cells are hypergranular promyelocytes; often with m...
Acute Leukemia - MorphologyAcute Leukemia - Morphology
ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA
AML-M3
• Major...
AML – M3 variant: Acute Promyelocytic Leukaemia – Variant;
Micro or Hypogranular bilobed promyelocytes
AML – M4: Acute Myelomoncytic Leukaemia with both granulocytic and
monocytic maturation.15 – 20% of cases; Myelocytic and ...
Acute Leukemia - MorphologyAcute Leukemia - Morphology
ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA
• AML-M4
• Mye...
AML – M4: Both Sudan Black B ( left) and Non Specific Esterase -
- ANAE (right) are positive
M5a Acute Monoblastic Leukaemia without maturation in which
more than 80% of blasts are monoblasts
M5b Acute Monoblsatic Leukaemia with Maturation less than
80% of blasts are monoblasts.
AML – M5b: Strong Non Specific Esterase (ANAE) reaction
Acute Leukemia - MorphologyAcute Leukemia - Morphology
ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA
AML-M5
• 80% o...
AML –M5a subtype: A peripheral smear shows one monoblast and five
promonocytes with folder nuclear membranes
AML – M6 Acute Erythroleukaemia; 5% of cases ; Dysplastic ereythroid
precursors (some megaloblatoid, others with giant or ...
Acute Leukemia - MorphologyAcute Leukemia - Morphology
ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA
AML-M6
• >50% ...
AML – M6
AML – M7 : Acute Megakaryocytic Leukaemia; constitute 1% 0f cases; Blasts
of megakaryocytic lineage predominate; Appearanc...
Acute Leukemia - MorphologyAcute Leukemia - Morphology
ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA
AML-M7
• At le...
INCIDENCE OF ACUTE LYMPHOBLASTIC
LEUKAEMIA
Acute Lymphoblastic Leukaemia is
the common form of leukaemia in
children
Its...
CLINICAL FEATURES OF
ACUTE LYMPHOBLASTIC
LEUKAEMIA
Clinical features are secondary to the following:
1. Bone Marrow Failur...
Purpura over the lower limbs in a
Patient with acute leukaemia
Marked cervical lymphadenopathy
in in a patient with Acute Lymphoblastic
Leukaemia
 Haematological investigations reveal normocytic and
normochromic anaemia
 Thrombocytopenia in most cases
 Total Leucoc...
Lab Diagnosis of Acute Myeloid Leukaemia
 Haematological investigations reveal a normochromic
normocytic anaemia
 Thromb...
Prognostic Factors in Acute Lymphoblastic Leukaemia
Determinant Favourable Unfavourable
Age 3-7 yrs <1, >10 yrs
Sex Female...
INCIDENCE OF ACUTE MYELOID
LEUKAEMIA
Acute Myeloid Leukaemia occurs in all age groups. It is
the common form of acute leu...
CLINICAL FEATRUES OF ACUTE MYELOID
LEUKAEMIA
 Clinical features resemble those of Acute Lymphoblastic Leukaemia.
 Anaemi...
Leukemic infiltrates in gums in AML-M5
Mediastinal widening in a 4-year-old boy with ALL
Mediastinal Widening in ALL
TREATMENT OF ACUTE LEUKAEMIAS
General Supportive Treatment:
Blood products support to treat anaemia and
thrombocytopenia....
SPECIFIC TREATMENT OF ACUTE LYMPHOBLASTIC
LEUKAEMIA
I.CHEMOTHERAPY: Induction
Intensification / CNS directed phase
Interim...
SPECIFIC TREATMENT OF ACUTE MYELOID
LEUKAEMIA
Induction
Consolidation
Consolidation
Possible stem cell Further consolidati...
CHRONIC LEUKAEMIAS
The chorinc leukameias are distinguished from acute
leukaemias by their slow progression.
Chronic leuk...
ACUTE LEUKAEMIA CHRONIC
LEUKAEMIA
Leukemic cells do not
differentiate
Leukaemic cells retain
ability to differentiate
Bone...
CHRONIC MYELOID
LEUKAEMIA(CML)
 The disease occurs in either sex (male : female) ratio of
1.4:1), most frequently between...
3. Features of anaemia may include pallor, dyspnoea, and
tachycardia.
4. Bruising, epistaxis, menorrhagia or haemorrhage f...
Massive splenomegaly in chronic myeloid leukaemia. The
palpable margins of the spleen are indicated
LABORATORY FINDINGS IN CHRONIC MYELOID
LEUKAMEIA
White blood cell count : markedly increased. Usually more
than 50 X 10 9
...
Promyelocyte
Myeloblast
Neutrophil
Myelocyte
Metamyelocyte
3. Increased circulating Basophils.
4. Normocytic and normochromic anaemia
5. Platelet count may be increased ( most frequ...
Philadelphia Chromosome
Karyotype showing the t ( 9:22)
The Ph chromosome is arrowed
2 3 4 51
7 8 9 10 11 126
13 14 15 16 17 18
The Philadelphia Chromosome:
a) There is a translocation of the long
arm of chromosome 22 to the long arm
of chromosome 9 ...
Phases of CML
1. Chronic Phase of CML
2. Accelerated phase of CML
3. Blast Crisis in CML
- Myeloid Blast Crisis(80%)
- Lym...
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Haematology
Upcoming SlideShare
Loading in …5
×

Haematology

51,650 views

Published on

Published in: Health & Medicine, Technology

Haematology

  1. 1. DISEASES OF HAEMOPOIETIC SYSTEM
  2. 2. Blood is a unique organ: it is fluid and comes into contact with almost all other tissues. The blood cells are non-cohesive and supported in the fluid medium of blood- the plasma. The blood cells comprise the non- nucleated erythrocytes and platelets, and the nucleated cells or leucocytes. In addition to primary disease of the blood – forming organ – the bone marrow – many disease states produce secondary changes in the blood.  For this reason, the counting and morphological examination of blood cells is routine in the clinical assessment of disease , frequently providing valuable diagnostic information
  3. 3. BLOOD CELL FORMATION (HAEMOPOIESIS)
  4. 4. SITE OF HAEMOPOIESIS Fetus 0 – 2 months (yolk sac) 2 – 7 months (liver. Spleen) 5 – 9 months (bone marrow) Infants Bone marrow (practically all bones) Adults Vertebrae, ribs, sternum, skull, sacrum and pelvis, proximal ends of femur
  5. 5. HAEMOPOITIC STEM CELL AND PROGENITOR CELLS Haemopoiesis starts with a common, ‘Pluripotent Haemopoietic Stem Cell’ which can give rise to erythrocytes, leucocytes (including lymphocytes) and platelets. The pluripotent stem cells possess the ability to renew and differentiation. By a series of cell divisions, cell committed to each cell line are produced and further divisions result in mature cells – erythrocytes, granular leucocytes, megakaryocyte and T- and B- lymphocytes .
  6. 6. DIAGRAMATIC REPRESENTATION OF BONE MARROW PLURIPOTENT STEM CELL AND CELL LINES THAT ARISE FROM IT
  7. 7. DISEASES OF HAEMOPOIETIC SYSTEM 1. Anaemias 2. Haematological Malignancies 3. Bleeding Disorders 4. Blood Transfusion
  8. 8. ANAEMIAS
  9. 9. ANAEMIA Definition: “Reduction in the concentration of haemoglobin in the blood below the lower limit of normal for a particular age and sex of an individual in a particular environment” Value of Haemoglobin less than 13.5 g/dl in males, less than 11.5 g/dl in females and less than 15.0 g/dl in new borns would indicate anaemia
  10. 10. ADULT REFERENCES OF RED BLOOD CELLS _________________________________________________ Male Female _________________________________________________ Haemoglobin (g/dl) 13.5 – 17.5 11.5 – 15.5 Haematocrit ; PCV (%) 40 -52 36 – 48 RBC count(X1012 /l) 4.5 – 6.5 3.9 – 5.6 Mean Cell Volume 80 -95 80 - 95 MCV (fl) Mean Cell HAmeoglobin 27 -34 27 - 34 MCH (pg) Mean Cell Haemoglobin 30 -35 30 -35 Concentration(MCHC) Reticulocytes (%) 1 – 2.5% 1 – 2.5%
  11. 11. SYMPTOMS AND SIGNS OF ANAEMIA Symptoms of Anaemia: Level of haemoglobin at which patient develops symptoms depends on the rate of development of anaemia. If haemoglobin has been falling slowly, symptoms occur late,and if haemoglobin falls rapidly, symptoms occur early. •Patient may present with generalized weakness and easy fatigability. •Palpitation and breathlessness occur if anaemia is severe or patient has underlying heart disease. •Anorexia and indigestion are common Signs of Anaemia: •Pallor is the main sign. Usual sites to look for pallor are the nail bed, hands , skin, lower conjunctiva. •Koilonychia(spoon shaped nails) is seen in iron deficiency anameia •Bone deformities occur in thalassaemia major •Leg ulcers are a feature of sickle cell anaemia •Systolic Murmur may be audible in pulmonary area •Mild jaundice may occur in haemolytic anaemia. •Different other signs and symptoms can be ascribed to a specific cause of anameia
  12. 12. CLASSIFICATION OF ANAEMIA 1. Kinetic Classification of Anaemias 2. Morphologic Classification of Anameias
  13. 13. KINETIC CLASSIFICATION OF ANAEMIAS I. ANAEMIAS DUE TO EXCESSIVE BLOOD LOSS (i) Acute blood loss anaemia (ii) Chronic blood loss anaemia II. ANAEMIAS DUE TO PRODUCTION FAILURE (i) Haematenic deficiency: Iron, Folic Acid and B12 deficiency (ii) Anaemia of chronic disorders: Infection, inflammation, neoplasia; renal failure (iii)Marrow Hypoplasia: Aplastic Anaemia; Pure red cells Aplasia (iv)Marrow Infiltration: Leukaemias; Lymphomas; Myeloproliferative disorders; Myelodysplastic Syndrome
  14. 14. III ANAEMIAS DUE TO EXCESSVIE RED CELLS DESTRUCTION (HAEMOLYTIC ANAEMIA) 1. HAEMOLYSIS DUE TO RED BLOOD CELLS ABNORMALITY (Intracarpuscular Defect) Congenital a. Red blood cells membrane defects: Hereditary Spherocytosis b. Enzyme defects: G.6PD Deficiency c. Haemoglobinopathies: Thalassaemia; Sickle Cell Anaemia Acquired d. Paroxysmal Nocturnal Haemoglobuniria 2. HAEMOLYSIS DUE TO ABNORMALITY OUTSIDE THE RED BLOOD CELL (Extracarpuscular) a. Immune Haemolytic Anaemias: (i) Autiimmune HAemolytic Anaemia (ii)Alloimmune Haemolytic Anaemia b. Non Immune: (i)Malaria (ii)MIcroangiopathic Haemolytic Anaemia
  15. 15. MORPHOLOGIC CLASSIFICATION OF ANAEMIAS Reticulocyte Count More than 2.5 % Less than 2.5% Haemolysis Red Cells Morphology Or Haemorrhage Normocytic Microcytic Macrocytic - Chronic diseases - Iron deficiency - B12 deficiency -Marrow problems - Thalassaemia - Folic Acid deficiency - -Sideroblastic anaemia - Anaemia of Chronic disorders
  16. 16. MORPHOLOGIC CLASSIFICATION OF ANAEMIAS Causes of Microcytic and Hypochromic Anameias (MCV – Less than 80 fl) 1.Iron Deficiency Anaemia 2.Thalassaemias 3.Anaemia of Chronic Disoder 4.Sideroblastic Anaemia  Causes of Normocytic and Normochromic Anaemias (MCV – 80 to 95 fl) 1. Many Haemolytic Anameias 2. Anaemia of Chronic Diseases 3. Acute blood loss 4. Mixed Deficiency Anaemia 5. Aplastic Anaemia 6. Bone marrow infiltration as in Lymphomas, Leukameias,etc Causes of Macrocytic Anaemias (MCV More than 95 fl) Megaloblastic Macrocytic Anameias Megaloblastic Anameia Nonmegaloblastic Anameia 1.Haemolytic Anaemias ( Reticulocytes will appear as Macrocytic cells) 2.Liver Disease 3.3. Alcohol 4.Aplastic Anaemia
  17. 17. IRON DEFICIENCY ANAEMIA Iron deficiency anaemia is the most common cause of anaemia in every country of world. It is the most important cause of a microcytic hypochromic anaemia, in which all the three red blood cell indices (the MCV, MCH and MCHC) are reduced, and the blood film shows small(microcytic) and pale (hypochromic) red cells The important causes of microcytic hypochromic anaemia are: (i) Iron deficiency anaemia (ii) Thalassaemias (iii) Sideroblastic anaemia (iv) Anaemia of chronic disorder
  18. 18. The causes of a hypochromic microcytic anaemia. These include lack of iron (iron deficiency), or of iron release from macrophages to serum (anaemia of chronic inflammation or malignancy). Failure of protoporphyrin synthesis (sideroblastic anaemia) or of globin synthesis (Alpha or Beta Thalassaemia).
  19. 19. IRON ABSORPTION AND METABOLISM Iron Absorption: Iron absorbed from food and iron recovered from senescent red blood cells is transported to the marrow and tissues. The iron laden transferrin binds to transferrin receptors on the surface of erythroid precursors. It is then internalized, at which time the iron is released for use in haemoglobin production. The transferin- transferin receptor complex is then returned to surface of the cell and the transferin is released to complete the cycle
  20. 20. IRON ABSORPTION AND METABOLISM Iron Absorption: Iron absorbed from food and iron recovered from senescent red blood cells is transported to the marrow and tissues. The iron laden transferrin binds to transferrin receptors on the surface of erythroid precursors. It is then internalized, at which time the iron is released for use in haemoglobin production. The transferin- transferin receptor complex is then returned to surface of the cell and the transferin is released to complete the cycle
  21. 21. Iron Absorption ucosal uptake of heme and nonheme iron is depicted. When the storage sites of the body e replete with iron and erytropoietic activity is normal, most of the absorbed iron is lost nto the gut by shedding of the epithelial cells. Conversely, when body iron requirements ncrease or when erytropoieis is stimulated, a greater fraction of the absorbed iron is transferred nto plasma transferrin, with a concomitant decrease in iron loss through mucosal ferritin. MT1,divalet metal transporter 1
  22. 22. Iron in the body
  23. 23. Iron the diet is of two types – Haem Iron and Non Haem Iron. Much of the dietary iron is non haem iron derived from cereals, with a lesser component of haem iron derived from haemoglobin or myoglobin in red or organ meat. Haem Iron is more readily absorbed than non haem iron. The transport and storage of iron is largely mediated by transferrin. Transferrin contain two atoms of iron. It delivers iron to tissues which have transferrin receptors, especially erythroblasts in the bone marrow which incorporate iron in haemoglobin . The transferin is then reutilized . At the end of their life span red cells area broken down into macrophages of the reticuloendothelial system and the iron is released from haemoglibin, enters the plasma and provides most of the iron on transferin. Some of the iron is stored in reticuloendothelial cells as ferritin and haemosidrin.
  24. 24. CAUSES OF IRON DEFICIENCY ANAEMIA I. CHRONIC BLOOD LOSS: (i) Uterine Bleeding: -Menorrhagia (excessive menstrual bleeding). - Postmenopausal bleeding. (ii) Gastrointestinal Bleeding: - Peptic Ulcer - Bleeding Haemorrhoids - Hookworm infestation - Aspirin or other nonsteroidal anti- inflammatory drugs ingestion II. INCREASED DEMAND: Increased iron demand during infancy, adolescence, Pregnancy, lactation and in menstruating women III. MALABSORPTION: Gluten Induced Enteropathy; Gastrectomy IV. DIETRY: Especially vegetarian diet
  25. 25. CLINICAL FEATURES OF IRON DEFICIENCY ANAEMIA  The patient develop the general symptoms and signs of anaemia and also show a painless glossitis, brittle, ridged or spoon shape nails (koilonychia), dysphagia (as a result of pharyngeal webs).  Patients also show unusual dietary craving( Pica; perverted appetite e.g., clay eating)  In children the iron deficiency is particularly significant, as it can cause irritability, poor cognitive function, decline in psychomotor development and learning . Child with iron deficiency anameia can also show behavioral problems Koilonychia: typical ‘spoon’ shaped nails
  26. 26. IRON DEFICIENCY: LABORATORY DIAGNOSIS Peripheral Blood Findings (i)Haemoglobin is decreased (ii) Red cells indices (MVC;MCH;MCHC) are decreased. (iii) Red Blood Cells Morphology: Microcytic and Hypo chromic red cell morphology with pencil shaped poikilocyttes. Red cells morphological changes are proportional to degree of anaemia. (iv) Platelet count is often moderately raised, particularly in cases of localized bleeding site (reactive thrombocytosis) (v) In cases of worm infestation there can be Eosinophilia. Biochemical Findings: (i)Serum iron is decreased (ii) Serum Total Iron Binding Capacity (TIBC) id increased (iii) Serum Ferrritin is decreased Bone Marrow Findings: (i) Erythroid Hyperplasia; Erythroblasts are small (micronormoblasts) and have ragged cytoplamic outlines (ii) Iron Stain: Iron will be absent in stores as well as in erythroid series cells
  27. 27. Red Blood Cells Morphology: Microcytic and Hypo chromic red cell morphology with pencil shaped poikilocyttes. Red cells morphological changes are proportional to degree of anaemia. Pencil Shape Red cell
  28. 28. TREATMENT OF IRON DEFICIENCY ANAMEIA It is usual to give 100 to 200 mg of elemental iron daily to adults and 3 mg/kg body weight as a liquid preparation to children. Treatment response: Rise of haemoglobin at a rate of 2.0g/dl every three weeks . Length of Iron Therapy: In order to replenish iron stores continue iron therapy for 12 months after haemoglobin level becomes normal.
  29. 29. Form of Iron Preparation Size Iron content Usual oral dose FERROUS SULPHATE Tablet 300 mg 60 mg 3 tablets daily Syrup 40 mg/ml 8 mg/ml 25 ml (5 TSP) Drops 125 mg/ml 25 mg/ml FERROUS GLUCON- - ATE Tablet 300 mg 37 mg 5 tablets FERROUS FUMERATE Tablet 300 mg 100 mg 2 tablets
  30. 30. PARENTRAL IRON  It is given only in conditions where patient can not tolerate oral iron or rapid replenishment of stores is indicated. Usually the total amount of iron required is 1.0 g to 2.0 gm
  31. 31. PARENTRAL IRON: IRON SUCROSE (VENOFER) The commonly used preparation is Iron Sucrose. Parental iron preparation contains Ferric Iron. Ferrous iron being freely ionizable is extremely reactive. - Composition: Iron Sucrose - Formulation: 5 ml - Iron Content: 20 mg/ml - Iron content per ampule: 100 mg - Dose in mg to raise Hb by 1 g/dl in adults: 200 - Administration: Intravenous infusion Total calculated dose of venofer is administered as fractional infusions on a daily basis or at convenient intervals. One to two ampules ( 5 – 10 ml) of venofer solution are diluted in 100 ml saline and infused slowly over 1 – 2 hours.
  32. 32. MEGALOBLASTIC ANAEMIAS This is a group of anaemias in which, due to impaired DNA synthesis, the erythroblasts in the bone marrow show a characteristic abnormality – maturation of nucleus being delayed relative to that of cytoplasm ( megaloblast) In megaloblast the nuclear chromatin maintains an open, stippled, lacy appearance despite normal haemoglobin formation in the cytoplasm of the erythroblasts, as they mature Two main deficiencies lead to megaloblasstic anaemia (i) Folic Acid or Folate deficiency (ii) Vitamin B 12 deficiency
  33. 33. VITAMIN B 12 AND FOLATE - COMPARISON VITAMIN B 12 FOLATE CONTENTS IN FOOD Vegetables – Poor Meat – Rich Vegetables – Rich Meat – Moderate EFFECT OF COOKING 10 -30 % loss 60 – 90 % loss SITE OF ABSORPTION Ileum Duodenum & Jejunum NEUROLIGCAL MANIFESTATIONS An Important feature Absent MALNUTRITION Unusual Most common cause of Folate deficiency ONSET Rapid Onset (Takes weeks) Slow (Takes years)
  34. 34. ABSORPTION OF VITAMIN B 12 Absorption of B12 requires Intrinsic Factor (IF), which is secreted by the parietal cells of the fundic mucosa along with hydrochloric acid. Initially the vitamin is released from its protein - bound form by the action of pepsin in the acidic environment of the stomach. The liberated vitamin is then bound to salivary vitamin B12 – binding protein called R- binder. In the duodenum, R- vitamin B12 complexes are broken down by the action of pancreatic enzymes, and the released vitamin B12 then attaches to IF. In this form, IF – B12 complex is then transported to ileum, where it adheres to IF- specific receptors on the ileal cells. Vitamin B12 then transverses the plasma membrane to enter the mucosal cell. It is picked up from the cell by a plasma protein , Transcobalamin II, which is capable of delivering it to liver and other cells of the body, Particularly cells of the bone marrow where it is incorporated in the nuclei for DNA synthesis.
  35. 35. ABSORPTION OF FOLIC ACID Folic acid in the diet is in two form Monoglutamates and Polyglutamates.  More than 90% is polyglutamates . Intestinal conjugases split polyglutamates into monglutamates that are readily absorbed in the proximal jejunum. During intrstinal absorption they are modified so that only 5- methyltetrahydrofolate enters the circulation as the normal transport form of folate.
  36. 36. PATHOGENESIS OF HOW B12 AND FOLATE DEFICIENCY PRODUCE MEGALOBLASTIC ANAEMIA Lack of Vitamin B12 or Folate causes slowing of DNA synthesis in developing erythroblasts with anaccumulation of cells in premitotic phase of cell cycle. The neutropeina and thrombocytopenia also appears to result from ineffective and abnormal precursor cells in the marrow due to impaired DNA synthesis
  37. 37. CAUSES OF VITAMIN B12 DEFICIENCY 1.Decreased intake of Vitamin B12: Nutritional deficiency (Vegetarians) 2. Impaired absorption of Vitamin B12: (i) Pernicious Anaemia ( Lack of Intrinsic Factor; Antibodies against Intrinsic Factor) (ii) Gastrectomy( No release of Intrinsic Factor) 3. Intestinal Causes: (i) Lesions of small intestine (ii) CoeliacDisease (iii) Tropical Sprue (iv) Fish Tapeworm( DIphylobothium Latum) infestation
  38. 38. CAUSES OF FOALTE DEFICIENCY 1. Decreased intake of Folic Acid: Nutritional Deficiency 2. Impaired Absorption of Folic Acid: (i)Coeliac Disease (ii)Tropical Sprue 3. Increased demand: (i) Pregnancy (ii) Haemolytic Anaemias (iii) Myeloproliferative disorders (iv) Carcinoma (v) Inflammatory disorders (vi) Skin diseases 4. Drugs: Dihydrofolate reductase inhibitors
  39. 39. CLINICAL FEATURES OF MEGALOBLASTIC ANAEMIA Glossitis: tongue is beefy red and painful Angular stomatitis  The onset is usually insidious with gradually progressive symptoms and signs of anaemia. The patient may be mildly jaundiced (lemon yellow tint) due to the excess breakdown of haemoglobin resulting from ineffective erythropoiesis in the bone marrow  Glossitis , sore tongue and stomatiitis  Mild symptoms of malabsorption with loss of weight may be present due to epithelial changes  Lethargy, breathlessness and other generalized signs and symptoms of anaemia may be present
  40. 40. CLINCAL MANIFESTATIONS DUE TO VITMAIN B 12 DEFICIENCY Vitamin B12 is required for two important reactions in the body: Vitamin B 12 (i) Homocysteine Methionine Vitamin B 12 (ii) Methylmelonyl CoA Succinyl CoA Absence of B12 then leads to two important consequences: (i) Impaired methylation leads to defects in myelinatioin of neural tissues (ii) Accumulation of Homcysteine has toxic effects on cardiovascular tissue and neural tube
  41. 41. Demyelination of the dorsal and dorsolateral columns A baby with neural tube defect (spina bifida) VITMAIN B 12 NEUROPATHY (SUBACUTE COMBINED DEGENERATION OF SPINAL CORD: B12 deficiency may cause a progressive neurtopathy affecting the peripheral sensory, and posterior columns The neuropathy is symmetrical and affects the lower limbs more than the upper limbs. The patient notices tingling in the feet, difficulty in walking and may fall over in the dark. Rarely optic atrophy or psychiatric symptoms (Megaloblastic Madness) are present NUERAL TUBE DEFECT: The accumulated Homocysteine can act as a toxic substance and can lead to a damage to neural tissue. Supplementation of maternal diet with folic acid during Pregnancy reduces the incidence of neural tube defect By 75% CARDIOVASCULAR DAMAGE: Raised Homocysteine damages cardiac and peripheral and cerebral vascular tissue. So can lead to myocardial Infarction, peripheral and cerebral vascular disease and venous thrombosis
  42. 42. Lab Diagnosis of Megloblastic Anemia 1.Peripheral blood finidngs 2.Bone Marrow Findings 3.Special Tests 1. Peripheral Blood Findings a. Anaemia b. Pancytopenia (Anaemia + Leucopenia+ Thrombocytopnia) c. RBC Morphilogy:Macrocytosis (Oval Macrocytes) d. Hypersegmented Neutrophils: Neutrophils hyper- segmentation is present when more than 5% of the neutrophils have 5 lobes or the film show at least one sixed lobed neutrophils Hyerpsegmentation is early sign of Vitamin B12 or folate deficiency deficiency, and is useful in the diagnosis of megaloblastosis with minimal or no anaemia e. Howel Jolly Bodies f. Basophilic Stippling g. Cabbot’s Rings
  43. 43. 2.. Bone Marrow Findings in Megaloblastic Anaemia a. ERYTHROPOIESIS : - Megaloblasts All the nucleated series of erythroid cells show megaloblastic change - Their abnormal appearance is due to disturbance of cell growth and maturation, resulting from interference with DNA synthesis. - Cells are larger than erythroblasts - Dissociation of Cytoplasmic and Nulcear maturation: Maturation of nucleus lags behind that of cytoplasm . Haemoglobinization of cytoplasm takes place while nucelus is immature - Dyserythropoiesis : Increase in the proportion of more primitive cells b. LEUCOPOIESIS - Shift to left is observed - Giant Metamyelocytes are seen c. MEGAKARYOPOIESIS - Megakaryocytes are norma lor decreased d. IRON STAINING: - Iron increased in fragments with increased siderocytes and sideroblasts - In cases of mixed deficiency (Iron +B12+ Foalate deficeincy) iron will be absent
  44. 44. 3. Special Tests: 1.Serum Vitamin B12- Decreased 2.Serum Folic Acid-Decreased
  45. 45. Macrocytic Anemia (Meg.):
  46. 46. Megaloblastic Anemia : Hypersegmented Neutrophils Oval macrocyte
  47. 47. Marrow smear from a patient with megaloblastic anaemia: Megaloblast in various stages of differentiation. 3. BONE MARROW FINDINGS IN MEGALOBLASTIC ANAEMIA: •Meglablasts:All the nucleated series of erythroid series will show Megaloblastic changes. Cells are larger in size and nuclear chromatin shows open sieve like pattern. •Orthochromatic Normoblasts: Cytoplasm is haemoglobinized but nucleus is not pyknotic (nuclear cytoplasimc dissociation) •Giant Metamyelocytes: Myelocytes are larger in size
  48. 48. TREATMENT OF MEGALOBLASTIC ANAEMIA VITAMIN B12 DEFICIENCY FOLATE DEFICIENCY Compound Hydroxycobalamin Folic Acid Route Intramuscular Oral Daily Dose 100 ug 5 mg Initial Dose 6 X 1000ug over 2-3 weeks Daily for 4 months Maintenance 1000 ug every 3 months Depends on underlying disease ; life long therapy may be needed in chronic haemolytic anaemias , myelofibrosis and renal dialysis Prophylactic -Total Gastrectomy - Ileal Resection Pregnancy; Severe haemolytic anameias; dialysis; prematurity
  49. 49. APLASTIC ANAEMIA Aplastic Anaemia is defined as the presence of pancytopenia in the peripheral blood and a hypocellular marrow in which normal haemopoietic marrow is replaced by fat cells Pancytopenia resulting from aplasia of the bone marrow The diagnosis of aplastic anaemia is made on the basis of (i) Pancytopenia (ii) A hypocellular or aplastic marrow with increased fat spaces (iii) The virtual absence of reticulocytes
  50. 50. CAUSES OF APLASTIC ANAEMIA I. CONGENITAL Fanconi’s Aplastic Anaemia II. ACQUIRED 1. Idiopathic 2. Chemical and Physical Agents a. Agents which regularly produce aplasia if dose is sufficient: - Ionizing radiation - Benzene - Chemotherapeutic agents b. Agents which occasionally produce aplasia: - Antibiotics: Chloramphenicol, Cotrimaxazole -Anti-inflammatory drugs: Phenylbutazone, Indomethacin, Ibuprofen, - Antirheumatic: Gold salts, Penicillamine 3. Viral Infections: Hepatitis (Non A, Non B , Non C) EB virus HIV
  51. 51. PATHOGENESIS OF APLSTIC ANAEMIA 1. DIRECT DAMAGE: Can be caused by direct damage to the haemopoietic marrow by radiation or cytotoxic drugs. 2. CHROMOSOMAL BREAKS: Seen in congenital aplastic anaemia 3. FUNDAMENTAL STEM CELL ABNORMALITY: Some forms of marrow insult presumably cause genetic damage that results in the generation of stem cells with poor proliferative and differentiative capacity 4. IMMUNE MECHANISMS: Different agents activate cytotoxic T cells. These activated cytotoxic T cells then produce different types of cytokines. These cytokines have specific receptors for them on stem cells . The important cytokines in this aspect are : (i) Interferon (IFN) – Gamma (ii) Tumour Necrosis Factor (YNF) (iii) Interleukin – 2 (IL – 2)
  52. 52. These cytokines then attach to their receptor on stem cell and induce damage to haemopoietic stem cell in following manner: (i) Decrease transcription of cellular genes and entery in cell cycle. (ii) Induces the formation of nitric oxide synthase which then produces toxic gas nitric oxide. The nitric oxide then produces further toxicity to other cells (iii) Increased apoptosis (cell death) This scenario is supported by the observation that immuno- suppressive therapy with antithymocyte globulins combined with cyclosporine has a salutary effect in 60% to 70% of patients
  53. 53. CLINICAL FEATURES OF APLASTIC ANAEMIA The onset is at any age with a peak incidence around 30 years and a slight male predominance Onset in insidious or acute with symptoms and signs resulting from anaemia (lethargy, breathlessness, pallor), neutropenia(infections, fever) and thrombocytopenia (bleeding tendencies). Bleeding is often the presenting initial presentation. Bruising, bleeding gums, epistaxis, petechiae and mennorhagia are the most frequent haemorrhagic manifestations Infections, particularly of mouth and the throat are common and generalized infections are frequently life- threatening .
  54. 54. CLINICAL FEATURES OF APLASTIC ANAEMIA…contd Lymph nodes, liver. Spleen are not enlarged  Rarely jaundice can be feature in patients with post hepatitis aplasia. The patients of aplastic anaemia having a history of hepatitis, usually presents at a time when clinically jaundice had already subsided. At presentation it is necessary to take a detailed drug, occupational and symptomatic history to try to establish any etiological agent. Unfortunately it is not always easy.
  55. 55. LAB DIAGNOSIS OF APALSTIC ANAEMIA: e peripheral blood film shows ncytopenia(Anaemia; Leucopenia;Thrombocytopenia) ere are no gross morphological changes in circulating ls, except some macrocytosis of red cells ere is absolute Reticulocytopenia. anulocytes may show increased staining of granules, the called toxic granulation of neutrophils. telets are reduced and are of small and uniform size. nocytes are usually reduced in proportion to nulocytes.
  56. 56. 7. The bone marrow aspirate is usually easily obtained, typically with many fragments, which appear hypocelluar (more than 75% fat). There is relative increase in lymphocytes, plasma cells. The remaining haemopoietic cells are normal. In the early stages Haemophoagocytosis may be prominent 8. Bone marrow trephine is essential to make a diagnosis. It shows fat replacement of marrow, and normal haempoietic tissue is absent or scanty
  57. 57. Vs Normal bone marrow: From a section of bone marrow trephine biopsy. Marrow cells are interspersed between fat spaces. The arrow points to a megakaryocyte Aplastic Anaemia: Bone marrow trephine biopsy showing markedly hypoplastic marrow composed largely of fat cells
  58. 58. CAUSES OF PANCYTOPENIA 1. Aplastic Anaemia 2. Megloblastic Anaemia 3. Bone marrow infiltration by leukaemias, lymphomas, multiple myeloma etc. 4. Myelofibrosis 5. Hypersplenism( peripheral blood pancytopenia with normocellular or hypercellular marrow and splenomegaly)
  59. 59. HAEMOLYTICHAEMOLYTIC ANAEMIASANAEMIAS
  60. 60. HAEMOLYTIC ANAEMIAS The distinguishing feature of all haemolytic anaemias is the increased rate of red cells destruction NORMAL RED CELL DESTRUCTION Red cells destruction usually occurs after a mean lifespan of 120 days when the cells are removed extravasularly by the macrophages of the reticuloendothelial system,(Extrvascular Haemolysis) especially in the marrow but also in liver and spleen. As the red cell have no nucleus, red cells metabolism gradually deteriorates as enzymes are degraded and not replaced and the cells become non- viable. The breakdown of haem from red cells liberates iron for recirculation via plasma transferrin to marrow erythroblasts, and protoporphyrin which is broken down to biilrubin. This circulates to the liver where it is conjugated to glucuronides which are excreted into the gut via bile and converted to stercobilinogen and stercoblin (excreted in faeces). Strecoblinogen and stercolbilin are partly reabsorbed and excreted in urine as urobilinogen and urobilin. Globin chains are broken down to amino acids which are reutilized for general protein synthesis in the body. Intravascular Haemolysis (breakdown of red cells in the blood vessels) play little or no part in normal red cells destruction.
  61. 61. (a) Normal red cell breakdown- Extravascular: This takes place extravascullarly in the macrophages of the reticuloendothelial system (b) Intravascualar Haemolysis: Occurs in some pathological disorders
  62. 62. INTRAVASCUALR AND EXTRAVASCULAR HAAMOLYSIS There are two main mechanisms by which a red cells are destroyed in haemolytic anaemias- Extravascular or Intravascular. In majority of the anaemias the destruction is extravascular. Causes of Extravascular Haemolysis 1.Haemoglobinopathies Thalassaemias Sickle Cell Anaemia 2.Hereditary Spherocytosis 3. Autoimmune Haemolytic Anaemias 4. Malaria 5. G6PD deficiency Causes of Intravascular Haemolysis 1. ABO mismatched blood transfusion 2. Malaria 3. PNH 4. G6PD deficiency
  63. 63. INTRODUCTION TO HAEMOLYTIC ANAEMIAS Haemolytic anaemias are defined as those anaemias which result from an increase in the rate of red cell destruction. Because of erythropoietic hyperplasia and anatomical extension of bone marrow, red cells destruction may be increased several- fold before the patient becomes anaemic- compensated haemolytic disease. The normal adult marrow, after full expansion, is able to produce red cells at six to eight times provided this is ‘effective’. Therefore haemolytic anaemia is not seen unless the red cells life span is less than 30 days.
  64. 64. CLASSIFICATION OF HAEMOLYTIC ANAEMIAS Hereditary Haemolytic Anaemias are the result of ‘intrinsic’ red cell defects whereas Acquired haemolytic anameias are usually the result of an’extracarpuscular’ defect or environmental change. Paroxysmal Nocturnal Haemoglobinuria (PNH) is an exception because it is an acquired disorder but the PNH cells have an intrinsic defect
  65. 65. LABORATORY FINDINGS IN HAEMOLYTIC ANAEMIAS The laboratory findings in haemolytic anaemia are conveniently divided in three groups 1. Features of Increased Haemoglobin Breakdown i. Jaundice and Hyperbilirubenemia ii. Reduced plasma Haptoglobin and Haemopixin iii. Increased serum LDH iv. Haemoglobinemia v. Haemoglobinuria vi. Methhemoglobinemia vii. Haemosidrinuria Evidence of Intravascular Haemolysis
  66. 66. 2. Features of Increased Red cells Production Compensatory Erythroid Hyperplasia) i. Reticulocytosis ii. Macrocytosis and Plychromasia iii. Erythroid hyperplasia of marrow iv. Radiological changes in bones (seen only in congenital anaemia) 3. Feature of Damage to Red Cell: i. Spherocytes ii. Increased red cells fragility iii. Red blood cells fragmentation.
  67. 67. GENETIC BASIS OF DISEASES There are three major categories of genetic disorders: 1. MENDELIAN DISORDERS: Disorders related to mutant genes of large effect. Most of these disorders follow mandalian pattern of inheritance. 2. CHROMOSOMAL DIORDERS: Associated with numerical or structural changes in chromosomes 3. DISEASES WITH MULTIFACTORIAL(POLYGENIC) INHERITENCE: Includes some of the common diseases of humans, such as hypertension and diabetes. These disorders are influenced by both genetic and environmental factors
  68. 68. MENDALIAN DISORDERS All Mendalian disorders are result of expressed mutations in single or small gene clusters, the chromosome number being normal. Mutations involving single – gene disorders typically follow one of three patterns of inheritance: (i) Autosomal Dominant (ii) Autosomal recessive (iii) X- linked.
  69. 69. HOMOZYGOUS VS HETEROZYGOUS DOMINANT VS RECESSIVE Each individual possesses two factors for a charactersitic. If these factors are the same then the indivudual is said to be Homozygous, but if these factors are different then the individual is said to be Heterozygous.  If a character manifests in heterozygous state it is called Dominant If a character manifests in homozygous state it is called Recessive The term coined for these hereditary characters or factors is Gene.
  70. 70. AUTOSOMAL DOMINANT DISORDERS Autosomal dominant disorders are manifested in the heterozygous state. That is, a person with an autosomal dominant trait possesses the abnormal (mutant) gene which causes the disorder as well as normal allele. At least one parent of an index case is usually affected; both males and females can be affected, and both can transmit the disease. When an affected person marries an unaffected one, every child has one in two chance (50%) of having the disease. So, by chance an affected person may well have all normal children. On the other hand, again by chance , he may be unlucky and all his children may be affected.
  71. 71. Autosomal dominant disorders tend to be extremely variable in expression. Some individuals may be so mildly affected that the condition hardly affects their everyday life, but others may be so severely affected that they may become complete invalids. Most affected individuals lie somewhere between these two extremes Common Autodsomal Dominant Haematological Disorders - Hereditary Spherocytosis – Haemolytic Anaemia - von Willebrand Disease – Bleeding Disorder.
  72. 72. Autosomal recessive inheritance is the single largest category of mendelian disorders. These disorders are characterized by following features: 1. Autosomal recessive diseases are only manifest when the gene is present in double dose, i.e, the person is Homozygous for that particular gene and receiving abnormal gene from both parents. 2. Usually Heterozygous are perfectly healthy and all the offspring of an affected person are normal, unless the affected person marries a heterozygote. AUTOSOMAL RECESSIVE DISORDERS
  73. 73. 3. If both the parents are heterozygous (carriers) then in each pregnancy there are 25% chances that the child can have homozygous state (fully expressed Disease or Major state), 25% chances of being normal and 50% chances of having heterozygous state( Carrier or Trait or Minor) 4. Common Autosomal Recessive Haematological Disorders: - Thalasaemias - Sickle Cell Anaemia AUTOSOMAL RECESSIVE DISORDERS….. Contd
  74. 74. All sex – linked disorders are X- linked, almost all X- linked recessive. The only gene assigned with certainty to the Y- chromosome is the determination of testes; males with mutations affecting the Y- linked genes involved in spermatogenesis are usually infertile. And hence there is no Y-linked inheritance. An X- linked recessive trait is one determined by a gene carried on the X- chromosome and manifest in the female only when the allele is in the double dose, that is in the homozygous state. In the male, a mutant allele carried on the single X chromosome is always manifest because there is no normal allele to counteract the effect of the mutant allele as there is in heterozygous female. X- LINKED DISORDERS
  75. 75. Since a male transmits an X- chromosome to each of his daughters and a Y- chromosome to each of his sons, then all the daughters of an affected male will be carriers but none of his sons will be affected. An X- linked disorder is never transmitted from a father to his son. When carrier female marries a normal male then her sons have a 1 in 2 (50%) chance of being affected and her daughters have a 1 in 2 (50%) chance of being carriers like their mother So In X- linked disorders patients are invariably Males, unless a patient of haemophilia marries a female carrier where disease can manifest in a female. The carriers of X- linked disorders are females. COMMON X- LINKED HAEMATOLOGICAL DISORDERS - Haemophilia A and Haemophilia B - Glucose 6 Phosphate Dehydrogenase (G-6PD) deficiency.
  76. 76. HAEMOGLOBIN DISORDERS OR HAEMOGLOBINOPATHIES Haemoglobin disorders result from: 1. Reduced synthesis of normal Alpha or Beta globin chains -Alpha Thalassaemias - Beta Thalassaemias 2. Synthesis of an Abnormal Haemoglobin - Crystaline Haemoglobin (HbS, C, D, E, O etc) (These are produced due to amino acid substitution) - Unstable Haemoglobin
  77. 77. THALASSAEMIA “The Thalassaemias are a heterogeneous group of genetic disorders of haemoglobin synthesis all of which result from reduced rate of production or absence of production of one of the globin chains of haemoglobin” According to globin chain, which is produced in reduced amount, thalassaemias are divided into two important groups: (i) Beta ( β) Thalassaemias: Due to reduced synthesis or absence of synthesis of Beta globin chains. (ii) Alpha ( α ) Thalassaemias: Due to reduced synthesis or absence of synthesis of alpha globin chains. - In Beta thalassaemia the beta chain synthesis is decreased or absent but there will be unimpaired synthesis of Alpha chains -In some thalassaemias no globin chain is synthesized at all and hence are called ……0 or ….. 0 thalassaemias, whereas in others some amount of globin chain is produced but at a reduced rate; these are designated as + +
  78. 78. MOLECULAR ASPECTS All the globin genes have three axons (coding regions) and two introns (non coding regions whose DNA is not represented in the finished protein). The initial RNA is transcribed from both introns and exons, and from this transcript the RNA derived from introns is removed by a process known as splicing. The introns always begin with a G-T dinucleotide and end with an A-G dinucleotide. The splicing machinery recognizes these sequences as well as neighbouring conserved sequences.. The RNA in the nucleus is also ‘capped’ by addition of a structure at the 5/ end which contains a seven methyl- guanosine group. mRNA is polyadenylated at 3/ end.Thalassaemia may arise from mutations or deletions of any of these sequences
  79. 79. CLINICAL AND GENETIC CLASSIFICATION OF THALASSAEMIAS I. BETA (β) THALASSAEMIAS: (Defects in transcription, processing or translation of beta- globin mRNA 1. Beta Thalassaemia Major:) -Homozygous state -Severe anaemia; requires regular blood transfusions 2. Beta Tahlassaemia Minor( Trait) - Heterozygous state - Asymptomatic with mild or no anaemia; red cell abnormalities seen. - Defects in transcription, processing or translation of beta- globin m RNA 3. Beta Thalassaemia Intermedia: -One parent beta thalassaemia minor other parent contributes a gene which lessens the deleterious effects. - Severe, but does not require regular blood transfusions.
  80. 80. II.ALPHA THALASSAEMIAS: Defect is mainly deletion of genes (i) Alpha Thalassaemia Silent Carrier( - α/αα) Asymptomatic; no red cells abnormality (ii) Alpha Thalassaemia trait ( -α/-α) or (--/αα) Asymptomatic like beta thalasseamia trait (iii) HbH Disease --/-α Severe resembles beta thalassaemia intermedia (iv)Hydrops Fetalis (- - / - -) Lethal in utero
  81. 81. BETA (β ) THALASSAEMIA SYNDROMES Molecular Pathology of Beta Thalassaemia Defects in Transcription, Processing or Translation of mRNA: The bulk of eta thalassaemia mutations are single base changes or deletions or insertion f one or two bases at various points in the genes. These mutations occur in oth introns and exons ,and also outside the coding region Deletions: Are rare. A part of mRNA is lost
  82. 82. Molecular Pathogenesis of Beta Thalassaemia - Lesions in Beta Thalassameia – Usually point mutations - Lesions in Alpha Thalassaemia – Mostly Gene Deletions Important mutations in Beta Thalassameia are 2.Promoter Region Mutations: Several point mutations within the promoter sequences reduce binding of RNA ploymerase and therby reduce the transcription rate 75% to 80%. ›Since sone β globin is synthesized, the patients develop β + thalassaemia 2. Chain Terminator Mutations : Two types of mutaitons can cause premature termination of mRNA translation : (a) A point mutation in one of the exons can lead to the formation of a stop codon (b) Single nucleotide substitutions or small deletions alter mRNA reading frames and nitroduce stop codons downstream that terminate protien synthesis ( frameshift mutaitons ) › Premature chain terminaiton by either of these mechanisms geenrates nonfunctional fragments of the β globin gene. leading to β 0 thalassaemia
  83. 83. Molecular Pathogenesis of Beta Thalassaemia …contd 3. Splicing Mutations : Mutations that lead to aberrant splicing are the most common cause of β –thalassaemia .Most of these effect intorn , but som e have been located within exons. If the mutation alters the normal splice junctions, splicing does not occur, and all the mRNA formed is abonrmal. Unspliced mRNA is degraded within nucleus, and β o thalassaemia develops Some mutations affect the intorns at locations away from the normal intron- exon ssplice junction. These mutations create new sites sensitive to the action of splicing enzymes at abnormal locations – within an intron , for example. Because normal splice sites remain unaffected, both normal and abnormal splicing occurs, giving rise to normal as well as abnormal β - globin mRNA. These patients develop β + thalassaemia
  84. 84. COMMON BETA THALASSAEMIA MUTAITONS IN PAKISTAN
  85. 85. AUTOSOMAL RECESSIVE DISORDER BETA THALASSAEMIA •Mediterranean population, Middle East, India, Pakistan, Southeast Asia, Southern Russia, China • Rare in Africa except Liberia and parts of North Africa • Occurs sporadically in all races ALPHA THALASSAEMIA •Widespread in Africa, Mediterranean population. Middle East, Southeast Asia THALASSAEMIA – POPULATION GENETICS
  86. 86. In Beta Thalassaemia Major there is a total lack or a reduction in the synthesis of structurally normal beta- globin chains with unimpaired synthesis of Alpha chains.
  87. 87. Beta Thalassaemia – Clinical Features
  88. 88. CLINICAL FEATURES OF BETA TAHLASSAEMIA MAJOR 1. Severe anaemia with failure to thrive on 3-7 months of age after birth 2. Enlargement of spleen occurs due to excessive red cells destruction, extramedullary haemopoiesis and later because of iron overload. The large spleen increases blood transfusion requirements due to increased pooling of blood. 3. Liver also increased in size. 4. Expansion of bones caused by intense marrow hyperplasia that leads to Thalassaemic Facies and to thinning of the cortex of many bones with a tendency to fractures and bossing of the skull . 5. Infections induced by blood transfusion: - Hepatitis B - Hepatitis C - Human Immunodeficiency Virus (HIV)
  89. 89. •The facial appearance of a child with beta thalassae- • mia major: Skull is bossed with prominent frontal •and parietal bones; the maxilla is enlarged •The skull X- ray in Beta Thalassaemia Major: There is a ‘hair –on-end appearance’ as a result of expansion of the bone marrow into cortical bone
  90. 90. he patient requires regular blood nsfusions to sustain an acceptable emoglobin level. But iron overload used by repeated transfusions is inevit- e unless chelation therapy (removal of n) is given. Each 500 ml of transfused od contains about 250 mg of iron.Iron m the excessive red blood cells break wn and increased gastrointestinal iron sorption also leads to increased iron in body. is iron overload then effects different ans: Liver: Cirrhosis Fibrosis) will take place. Endocrine Organs: Accumulation of iron in different organs will lead to lure of growth, delayed or absent puberty, diabetes mellitus, pothyroidism and hypoparathyroidism. Damage to Myocardium can lead to arrhythmias and cardiac failure. d cardiac damage is the main cause of death
  91. 91. LABORATROY DIAGNOSIS OF BETA THALASSAEMIA MAJOR 1.Peripheral blood film will show severe microcytic and hypochromic blood picture with marked pokilocytosis (fragmented red cells; target cells) 2. Reticulocytes count is increased. 3. Peripheral blood shows normoblasts
  92. 92. Beta Thalassaemia Major  Microcytic and hypochromic blood picture  Marked anisocytosis & Poikilocytosis  Nucleated RBC  Fragmented red ells
  93. 93. 4. Haemoglobin electrophoresis shows accentuated band of HbF(Fetal Haemoglobin) 5. Fetal Haemoglibin estimation by alkali denaturation method (Betke’s method) will show elevated HbF 6. DNA Analysis by Polymerase Chain Reaction (PCR) to look for molecular lesion (mutation or deletion) 7. Prenatal Diagnosis: During pregnancy fetus can be diagnosed by taking Chorionic Villus Sample(CVS) of fetus and then to do PCR to find out that whether fetus is normal or he is having mutations which can lead to beta thalassaemia major or minor. LABORATROY DIAGNOSIS OF BETA THALASSAEMIA MAJOR….. contd
  94. 94. Haemoblobin Electrophoresis on Cellulose Acetate for Haemoglobin Defects
  95. 95. Normal Haemoglobin Electrophoretic Pattern
  96. 96. Beta Thalassaemia Minor (Trait)
  97. 97. Beta Thalassaemia Major
  98. 98. Haemoglobin E trait
  99. 99. Haemoglobin D trait
  100. 100. Haemoglobin C Trait
  101. 101. TREATMENT OF BETA THALASSAEMIA MAJOR 1.Regular blood transfusions are required to maintain the haemoglobin level over 10 g/dl at all times. This usually requires 2-3 units every 4-6 weeks. 2. Regular Folic Acid 5 mg daily. 3. Iron Chelation Therapy is required to treat iron overload. It is accompashied by: (i) Subcutaneous infusion of Desferrioxamine(Desferol) given through infusion pump at a rate of 20 – 40 mg/kg over 8 to 12 hours 5-7 days weekly. (ii) Oral Iron Chelator Deferiprone (L 1) & Asunra 4. Vitamin C 200 mg daily, increases excretion of iron produced by desferrioxamine. 5. Splenectomy, if transfusion requirements are increased. It can not be employed before the age of 5 years 6. Allogeneic Bone Marrow or Stem Cell Transplant: It is curative in selected patients
  102. 102. •Thalassaemics receiving blood transfusion at a thalassaemia center
  103. 103. BETA THALASSAEMIA MINOR (TRAIT) It is a heterozygous state. The person is carrying abnormal genes from one parent and normal from other. It is a common, usually symptom less abnormality characterized by a hypchromic,microcytic blood picture (MCV and MCH very low) with many target cells and minimal anisocytosis. The red cell count (RBC count) is high (More than 5 X1012 /l, and mild anaemia (Haemoglobin 10 – 15 g/dl) The HbA2 level is increased (More than 3.5%; Range 4-6 %) During periods of stress, such as pregnancy or infection the patient can become anaemic. It is important to identify cases of Beta Thalassaemia Minor in a community where Thalassamia is prevalent and where there is increased tendency of cousin marriages, so as to give proper genetic counseling before marriage
  104. 104. D/D OF MICROCYTIC AND HYPOCHROMIC BLOOD PICTURE Blood film in Beta Thalasaemia Major: Microcytic and Hypochromic with fragmented red cells, target cells and nucleated red cells(normoblasts) Blood Film in Iron Deficiency Anaemia: Microcytic and hypochromic blood Picture with pencil- shape cells Blood film in Beta Thalasseamia Minor (Trait): Microcytic and hypochromic blood picture with many target cells and absence of anisocytosis
  105. 105. •Iron Deficiency Vs Beta Thalassaemia Trait
  106. 106. •Iron Deficiency Vs Beta Thalassaemia Trait
  107. 107. Iron Deficiency Vs Beta Thalassaemia Trait Microcytic hypochromic morphology of red cells in proportion to the degree of anaemia, presence of pencil shape red blood cells on peripheral blood, red blood cells count less than 5.0 millions/cmm and decreased MCV usually favours the diagnosis of iron deficiency anaemia. Uniformly microcytic hypochromic blood picture more pronounced as compared to the level of haemoglobin,minimal anisocytosis , presence of target shape cells, red blood cells count more than 5.0 millions/cmm and decreased MCV favours the diagnosis of beta thalassaemia trait
  108. 108. “ More than the calf wishes to suck does the cow wish to suckle” Teaching Craze !!!
  109. 109. SICKLE CELL ANAEMIA  Sickle Cell Anaemia or Sickle Cell Disease is an Autosomal Recessive, Hereditary Haemoglobin disorder.  The homozygous state is produced due to inheritance of an abnormal beta globin gene (HbS gene) from both parents.  The HbS gene is produced by a mutation in beta globin gene, leading to substitution of Valine for Glutamic Acid at the sixth position of Beta Globin Chain of Haemoglobin molecule. Molecular pathology of Sickle Cell Anaemia: There is a single base change in the DNA coding for the amino acid in the sixth position in the β- globin chain ( Adenine is replaced by Thymine) . This leads to an amino acid change from Glutamic Acid to Valine
  110. 110. This substitution confers abnormal physicochemical properties to red cells. Under unfavourable conditions, most importantly hypoxia, the red cells destabilize and undergo repeated cycles of distortion and polymerization labelled as sickling. This ultimately leads to : (i) Increased destruction of red cells manifesting as chronic haemolytic anaemia jaundice (ii) Aggregation of distorted cells in blood circulation leading to ischemic tissue damage
  111. 111. The change from glutamic acid to valine at position 6 changes the 3-D structure of the molecule This makes hemoglobin sticky and changes the shape of the RBC It makes the molecule spiky and rigid rather than round and flexible The RBC can not move through capillaries
  112. 112. PATHOGENESIS OF SICKLE CELL DISEASE On deoxygenation the HbS molecules under- go aggregation and polymerization . This change converts haemoglobin from a freely flowing liquid to a viscous gel, leading ultimately to formation of HbS fibers and resultant distortation of the red cells, which acquire a sickle or holly- leaf shape. Sickling of red cells is initially a reversible phenomenon; with oxygenation, HbS returns to the depolymerazid state; However, with repeated episodes of sickling and unsikling, membrane damage starts and the cell become irreversibly sickled. . These deformed cells retain their abnormal shape even they are fully oxygenated and despite deagregation of HbS .The precipitation of HbS fibers has deleterious effects on red cell membrane.
  113. 113.  The formation of HbS has two major consequences: (i) A chronic Haemolytic Anameia (ii) Occlusion of small blood vessels, resulting in ischemic tissue damage. Substitution of Valine for Glutmaiic Acid at the 6th position of Beta Chain Revresible Sickling Irreversible Sickling Accumulation of sickle aggregates in Loss of red cells membrane microvasculature Viscosity of blood increased Excessive Red Cells Breakdown Blockage of small blood vessels Chronic Haemolytic Anaemia Tissue Infarction Acute Crises Chronic Complications
  114. 114. VARIABILITY IN SICKLING Sickle cell anaemia runs an extremely variable clinical course. At one end of the spectrum it is characterized by a crippling haemolytic anaemia, on the other end it can have a milder course. The reasons are only partly understood: 1. Concentration of HbS: Susceptibility to sickling is proportional to the concentration of HbS. Individuals with sickle cell trait have less than 50% HbS in their cells and are virtually without symptoms. 2. Cellular Dehydration: Cellular dehydration such as in renal papillae will increase sickling 3. Other Haemoglobins: Fetal Haemolgobin inhibit sickling, while HbC and D can enhance sickling 4. Deoxygenation: It is the most important factor in determining sickling. Thus travel to high elevations or in unpressurized aircrafts can precipitate sickling crises.
  115. 115. 5. Caliber of blood vessels: In high flow areas the shear stresses can break down the gel structure of HbS, while in the small or medium sized blood vessels the sickled red cells aggregate. 6. Duration of Hypoxia: Areas of vascular stasis (such as spleen) with lower O2 tension are prone to vascular occlusion 7. Cold Weather: Cold weather may precipitate sickling because of vasoconstriction 8. Acidosis: Acidosis enhances sickling while alkalosis will retard sickling 9. Other precipitating factors: Crises are often precipitated by: (i) Infections (ii) Dehydration due to fever and gastrointestinal loss of fluid (iii) Acidosis due to poor oral intake
  116. 116. CLINCAL MANIFESTATIONS OF SICKLE CELL ANAEMIA CLINICAL PRESENTATION •High levels of fetal haemoglobin protect against sickling for the first 8 to 10 weeks of life. After that the manifestations of sickle cell disease may become apparent. •Typically Sickle cell anaemia presents in infancy with anaemia and jaundice . Most patients go through the rest of their lives with a chronic haemolytic anameia • A common presenting symptom is ‘hand and foot Syndrome’ which occurs early in infancy and is characterized by a painful dactylitis and swelling of the fingers or feet. •Other particular problems in infancy are splenic sequestration and fulminent Pneumonia. Painful swollen fingers (dactylitis) in a child Hand Foot Syndrome: There is marked shortening of the right middle finger because of dactylitis
  117. 117.  During early development of the disease there is often splenomegaly . In most patients this gradually resolves due to repeated infarctions of the spleen (Auto splenectomy) . Indeed it is most unusual to feel the spleen after the end of first decade. If a patient has a palpable spleen after that probably he is suffering from Sickle Cell – Beta Thalassaemia Disease , i.e., receive sickle cell gene from one parent and beta thalassaemia gene from the other parent • Growth and development are usually normal although there may be some skeletal deformities, including frontal bossing of the skull due to expansion of bone marrow.
  118. 118. The complications of Sickle Cell Anaemia can be divided into two groups: 1. Acute and Episodic Crises 2. Chronic Complications I. ACUTE AND EPISODIC CRISES The word ‘crises’ is used to describe an acute and sometimes life- threatening complication of sickle cell anaemia. Different crises are: 1.Painful Vaso- occlusive crisis: These are the most common occurring with a frequency from almost daily to yearly. Tissue hypoxia and infarction can occur any where in the body. It is important to carefully evaluate the patient to differentiate painful crises and pain caused by another process COMPLICATIONS OF SICKLE CELL ANAMEIA
  119. 119. CLINICAL CONSEQUENCES OF SICKLE CELL DISEASE
  120. 120. 2. Aplastic Crises: These occur as a result of infection with Parvo Virus or Folate deficiency. These are characterized by sudden fall in haemoglobin, usually require transfusion. They are characterized by a fall in Reticulocytes and haemoglobin 3. Splenic and Hepatic Sequestration Crises: These are caused by sickling within organs and pooling of blood, often with a severe exacerbation of anaemeia. Hepatic and splenic sequestration crises all may lead to severe illness requiring exchange transfusion . Splenic sequestration is typically seen in infants and presents with an enlarging spleen, falling haemoglobin and abdominal pain. Treatment is with transfusion and patients must be monitored at regular intervals as progression may be rapid. Attacks tend to be recurrent and splenectomy is often advised in recurrent cases. Splenic crises can lead to hypotension and death. 4. Acute Chest Syndrome: Characterized by acute onset of dyspnoea, with chest pain. It is due to sequestration of sickle cells in pulmonary arterial circulaition
  121. 121. 5. CNS Crises: Patient presents with a stroke, preceded by bizarre neurological syndromes similar in nature to transient ischemic attack. It is due to blockage of cerebral vessels by sickle cells. 6. Hyperhemolytic Crises: During painful crises there can be a marked increase in the rate of haemolysis. 7. Megaloblastic Crises: The haemolytic crises can lead to megaloblastic crises. II. CHRONIC COMPLICATIONS 1. Severe Infections: It is the most common cause of death in sickle cell anaemia and it can be associated with one or more of the patterns of crises mentioned above. Babies and children with this disease are particularly prone to develop Pneumococcal Septicemia due to hyposplenism. Salomonella infections of bone leading to Salmonella Osteomyelitis is also seen.
  122. 122. 2. Infarcts following repeated episodes of vascular occlusion: Result largely from infarcts following repeated episodes of vascular occlusions. Almost any organ can be involved Those at particular risk are those which depend on small blood vessels for their blood supply. (i) Bone Infarcts: Bones are particularly prone. Aseptic Necrosis of Femoral or Humeral Head may lead to their destruction and pain. (ii) Renal Infarcts: Damage to vasa rectae system can lead to a loss of ability to concentrate urine and polyuria and nocturnal enuresis. (iii) Pulmonary Infarcts : Occur quite frequently 3. Priapism: Recurrent attacks of painful priapism may lead to permanent deformity of penis 4. Chronic, relapsing leg ulcers: A major problem particularly in adults 5. Occular manifestations: Proliferative retinopathy
  123. 123. COURSE AND PROGNOSIS The prognosis seems to depend upon the racial background of the patient, socioeconomic and ill- defined genetic factors and above all the availability of good paediatric care in the early years. In East Africa USA and Europe it has high mortality in early years In Saudi Arabia and India, a particularly mild form of the condition occurs in which the mortality is extremely low in childhood and a normal survival seems to be the rule. The most common causes of death in the first year or two of life are infections and splenic sequestration. Later in life infection is still the most frequent cause of death. If babies are maintained on prophylactic penicillin from early in life, and if there is good paediatric care, it may be possible to reduce the early mortality of sickle- cell anaemia to a very low level
  124. 124. LABORATORY DIAGNOSIS OF SICKLE CELL ANAEMIA 1. Haemoglobin level: The haemoglobin level is usually between 5 to 11 g/dl. 2. Sickle cells on peripheral blood: Considerable variation on red cells size and shape is noted. Sickled cells and target cells are seen. It is important to calculate the percentage of sickle cells, as the number of sickle cells should remain less than 30% . . Reticulocyte Count: Elevated reticulocyte count ;usually 10 to 20% 4. Screening Test for Sickle Cells: Different substances are used to induce deoxygenation like: - Sodium Metabisulphide - Sodium Dithionite . Haemoglobin Electrophoresis: In Homozygous state (HbSS) all the haemoglobin is HbS, no HbA is detected. The amount of HbF is variable. It is usually between 5 – 15%.
  125. 125. Sickle Cell Disease: Homozygous state. Numerous Crescent Shaped sickle cells are seen.
  126. 126. Sickle Cell Test
  127. 127. Normal or Alpha Thalassaemia Trait Sickle Cell Trait Sickle Cell Disease Beta Thalassaemia Trait Beta Thalassaemia Major Sickle cell / Beta Thalassaemia Sickle cell/ HbC disease Haemoglobin H Disease Haemoglobin electrophoretic pattern in normal adult human blood and in subjects with sickle cell (HbS) trait or disease, beta thalassaemia trait, beta thalassaemia minor, HbS/beta thalassaemia or HbS/HbC disease
  128. 128. Normal Haemoglobin Electrophoretic Pattern Sickle Cell Disease Sickle Cell Disease with high HbF
  129. 129. 5. Bilirubin level: Is elevated 6. Serum LDH: Is elevated in crises 7. Blood Gases: should be estimated in crises especially to estimate pCO2 8. Renal Function Tests: To assess for any degree of renal impairment 9. Prenatal Diagnosis: Prenatal diagnosis is possible by obtaining fetal chorionic Villous sample (CVS) and the by performing Polymerase Chain Reaction (PCR) analysis.
  130. 130. POLYMERASE CHAIN REACTION (PCR) FOR SICKLE CELL: Sickle Cell Anaemia ; Antenatal Diagnosis: Direct DNA analysis. The DNA has been digested by the restriction enzyme Mst II. The replacement of an adenine base in the normal beta- globin gene by thymine in the sickle cell gene removes a normal restriction site for Mst II, producing a larger 1.3 – kb fragment that the normal 1.1 – kb fragment to hybridize with the β - globin gene probe. In this case the trophoblast DNA (T) shows both normal (A) and sickle (S) restriction fragments and so is AS (sickle trait) , while the case of sickle cell anaemia (SS) shows only band of HbS
  131. 131. SICKLE CELL TRAIT • This is a benign condition with no anaemia and normal appearance of red cells on a blood film. • Screening Tests for Sickle cell are positive • Haemoglobin electrophoresis will show 25 – 45% HbS, rest is HbA. • Haematuria is the most common symptom and is thought to be caused by infarcts of renal papillae. • Care must be taken with anaesthesia, pregnancy, at high altitudes and during strenuous exercises.
  132. 132. TREATMENT OF SICKLE CELL ANAEMIA 1. Prophylactic: Avoid those factors known to precipitate crises, especially: - Dehydration - Anoxia - Infections - Stasis of circulation - Cooling of skin surface 2. Penicillin: Early deaths due to infections and crises can be reduced by the administration of prophylactic oral penicillin. Oral penicillin (62.5 mg t.d.s up to 1 year of age; 125 mg b.d at 1-3 years of age; 250 mg b.d thereafter) should be started as early as possible and maintained throughout childhood and early adolescence. 3. Vaccines: Pneumococcal , Meningococcal and Haemophilus influenzae vaccines should also be given. As due to loss of splenic functions body will not be in a position to get rid of capsulated organisms.
  133. 133. 4. Malarial Prophylaxis: Malarial prophylaxis is mandatory for visits to areas where malaria is endemic 5. Folic Acid: 5 mg daily 6. Blood Transfusions: •Adaptation to anaemia is particularly successful because of the low oxygen affinity of HbS. •Patients with sickle cell anaemia manage well with relatively low haemoglobin levels. Regular blood transfusion is not required. •The blood transfusions are sometimes given repeatedly or prophylactically, to patients: -who have frequent crises; -who have had major organ damage, for example of the brain. - who have aplastic crisis. •The aim is to suppress HbS production over a period of several months or even years. • To take care of the complications of chronic blood administration: - Iron Overload - Viral Infections like Hepatits B , C and HIV - Alloimmunization to minor blood group antigens
  134. 134. 7. Management of Painful Crises: Should be managed in hospital. Methods employed to manage painful crises are: - Rest - Warmth - Rehydration (i) Oral fluids (ii) Intravenous normal saline (3 liters in 24 hours) - Antibiotics, if infection is present - Analgesics: Suitable drugs are paracetamol, a non- steroidal anti-inflammatory agent and opiates, e.g. continuous infusion of Dimorphine. - Blood transfusion if severe anaemia - Exchange blood transfusion – if there is repeated occurrence of painful crises.
  135. 135. 8. Lung Crises: Once suspected patient should be managed in hospital in intensive care unit. Oxygen should be administered and blood gase should be monitored. 9. Exchange Transfusion: Exchange transfusion is usually required under following circumstances in sickle cell anaemia: - For major surgical emergencies - For repeated painful crises - In patients with neurological symptoms - Visceral damage - If priapism failed to treat by conservative management. The aim of exchange transfusion is to decrease the number of sickle cells less than 30 -40%. 10. Total Hip Replacement: Chronic hip pain and difficulty with walking due to aseptic necrosis of the femoral heads may require total hip replacement. 11. Eye changes: Laser therapy
  136. 136. 12.Renal problems: Hameaturia and renal failure should be managed accordingly. 13. Priapism: It occurs quite frequently. It has been found that nearly two third of major episodes are preceded by ‘stuttering’ attacks and effective therapy at this stage is necessary. Measures for treating priapism are: - Stilbestrol – 5 mg daily may be effective in preventing a major episode of priapism in patients who have minor episodes. - Exchange transfusion - Corporal aspiration with irrigation of normal saline -Cavernous spongiosum shunt 14. Leg Ulcers: Bed rest; elevation; zinc sulphate dressings. A transfusion program or skin grafting can enhance healing.
  137. 137. 15. Splenectomy Usually upto the end of first decade there is autosplenectomy. But in patients who have recurrent attacks of splenic sequestration syndrome, splenectomy is indicated. 16. Antisickling Agents: Hydroxyurea: (15.0 to 20.0 mg/kg per day) can increase HbF level and has been shown to improve the clinical course of patients who are having three or more painful crises each year. It should not be used during pregnancy.
  138. 138. 17. Stroke: • As the life expectancy of patients with sickle cell disease increases, more adults presenting with CNS symptoms can be anticipated. • Adults are at greater risk for intracranial haemorrhage and children at risk for ischemic stroke. • In adults patients with sickle cell anaemia it is important to identify and address other risk factors for stroke, such as hypertension, abnormal lipids, diabetes and smoking. • The clinical presentation of intracranial haemorrhage is usually more dramatic than that of ischemia and may include severe headache, vomiting , stupor, coma and haemipresis. • Blood transfusions and exchange are the main stay of treatment. • Hydroxyurea has been used for secondary prevention by different groups and appears promising as an alternative to transfusion in older children and young adults. • For transient ischemic attacks Aspirin, Colopidegral, Dupyridamol, along with Warfarin. • For ischemic stroke: (i) Tissue Plasminogen (tPA) within 3 hours . Risk of brain haemorrhage is 6.4%, and out of these half are fatal. (ii) Aspirin – 325 mg one – time dose within first 48 hours.
  139. 139. 18. Bone Marrow OR Stem Cell Transplantation: It can cure the disease and many patients have now been successfully treated. The mortality rate is less than 10%. Transplantation is only indicated in the severest of cases whose quality of life or life expectancy are substantially impaired 19. Gene Therapy: Hameoglobinopathies like sickle cell anaemia and beta thalassaemia major are also a target for gene therapy. Gene therapy with lentivirus vectors has been successful in sickle transgenic mice. An anti-sickling beta globin gene was introduced into the sickle transgenic mice. Long term expression (upto 10 months) was achieved. In addition haematological parameters correction, inhibition of cellular dehydration and loss of sickling tendency were also demonstrated.
  140. 140. HEREDITARY SPHEROCYTOSIS Hereditary Spherocytosis is characterized by an intrinsic defect in the red cell membrane that renders erythrocytes spheroidal, less deformable, and vulnerable to splenic sequestration and destruction. POPLUATION GENETICS Its prevalence is high in North Europe. In approximately 75% of cases, the inheritance is Autosomal Dominant . The remaining have an autosomal recessive form of the disease that is much more severe than the autosomal dominant form
  141. 141. MOLECULAR PATHOLOGY OF HEREDITARY SPHEROCYTOSIS(HS) e spheroidal shape of d blood cells is aintained by some egral proteins in its embrane. In HS netically there are utations in any of ese proteins. The portant membrane otein abnormalities in association with Hereditary Spherocytosis are: •Spectrin deficiency or mutations • Ankyrin deficiency or mutations •Pallidin (Protein 4.2) abnormalities A deficiency of Spectrin seems to be most common abnormality in all tients with Hereditary Spherocytosis. Spectrin deficiency is associated with duced membrane stability and loss of membrane fragments as the cells are posed to shear stresses in the circulation. The resulting reduction in cell rface to volume ratio “forces” the cells to assume the smallest possible ameter for a given volume namely ,a sphere
  142. 142. As these spherocytes are trapped in the sluggish circulation of spleen then the deformed red cells are destroyed and engulfed by phaogocytic cells (macrophages). The cardinal role of spleen in the premature demise of the spherocytes is proved by the invariably beneficial effect of splenectomy . Red cells membrane proteins defects Destabilization of red cell membrane Loss of red cells membrane Formation of Spherocytes Entrapment of Spherocytes in sluggish splenic circulation Phagocytosis of spherocytes by macrophages
  143. 143. CLINICAL FEATURES OF HEREDITARY SPHEROCYTOSIS •The characteristic clinical features of Hereditary Spherocytosis are : - Anaemia - Splenomegaly - Jaundice • The severity of the disease varies from patient to patient. The anaemia may present at any age from neonatal ( rarely as a case of neonatal jaundice), to infancy to adult hood to old age. • Jaundice is typically fluctuating. • In 20% to 30% the disease is largely asymptomatic. • Splenomegaly occurs in most patients • Pigment gall stones, derived from bilirubin released after red cells breakdown, are frequent • Aplastic crises, usually precipitated by parvovirus infection, may cause a sudden increase in severity of anaemia
  144. 144. LAB DIAGNOSIS OF HEREDITARY SPHEROCYTOSIS 1. Anaemia is usual but not invariable. 2. Reticulocyte Count: Increased 3. Blood Film: Shows Microspherocytes which are densely staining red cells with smaller diameter then normal red cells. HS: Note the anisocytosis and several dark- appearing spherocytes with no area of central pallor Deeply staining spherocytes with large polychromatic cells (reticulocytes)
  145. 145. LAB DIAGNOSIS OF HEREDITARY SPHEROCYTOSIS. contd 4. Serum Bilirubin Increased (indirect Hyperbrubinemia) 5. Ultrasound abdomen: To rule out gall stones 6. Osmotic Fragility Test: To demonstrate the increased fragility of spherocytes. The red cells lyse in hypotonic saline solutions.
  146. 146. TREATMENT OF HEREDITARY SPHEROCYTOSIS 1 .Splenectomy: It is the principal form of treatment. It should not be performed unless clinically indicated because of severe anaemia or gall stones. It is preferable to perform Splenectomy after the age Of 5 years. It will produce a rise in haemoglobin level to normal. Spherocytes will remain. 2. Folic Acid: In severe cases to prevent folate deficiency.
  147. 147. GLUCOSE – 6 – PHOSPHATE DEHYDROGENASE DEFICIENCY The erythrocyte and its membrane are vulnerable to injury by exogenous and endogenous oxidants. Abnormalities in the Hexose monophosphate shunt or in Glutathione metabolism resulting from deficient or impaired enzyme function reduce the ability of red cells to protect themselves against oxidative injuries and lead to Hemolytic Anaemia. The most important of these enzyme derangements is Deficiency of Glucose – 6 – Phosphate Dehydrogenase(G6PD) activity.G6PD reduces NADP to NADPH while oxidizing glucose -6- phosphate. NADPH then provides the reducing power that converts oxidized glutathione to reduced glutathione. The reduced glutathione so generated protects against oxidant injury by catalyzing the breakdown of oxidant compounds like H2 O2
  148. 148. ant G6PD gene ient or Decreased production of G6PD Enzyme ility of glucose 6 phosphate dize into 6- phospohgluconate re of reduction of NADP to NADPH re of NADPH to reduce oxidized Glutathione e to breakdown oxidants like HH22 OO22 ased Red cell Susceptibility to get maged by different Oxidants ions and other oxidants causes oxidation of sufhydryl groups of the globin chains uration of hemoglobin and formation of precipitates (HEINZ BODIES) Bodies get attached with red cells m membrane d Cells break down (Haemoyltic Anaemia)
  149. 149. POPULATION GENETICS • Sex Linked (x- Linked) : Males are affected •The inheritance is sex-linked, affecting males, and females are carriers. The female carriers show approximately half normal red cell G6PD values. • The main races affected are in West Africa, the Mediterranean, the Middle East and South East Asia. The degree of deficiency varies, often being mild (10 – 15% of normal activity) in Black Africans, more severe in Orientals and most severe in Mediterranean's. Severe deficiency occurs occasionally in white people • G-6-PD DEFICIENT VARIANTS: More than 400 deficient variants due to pint mutation or gene deletion are identified. G6PD type B is the most common. The others are G-6PD A- and G6PD Mediterranean
  150. 150. AGENTS WHICH MAY CAUSE HAEMOLYTIC ANAMEIA IN GLUCOSE -6- PHOSPHATE DEHYDROGENASE DEFICIENCY I. Infections and other acute illnesses: Diabetic ketoacidosis II. Drugs : -Antimalarials: Primaquine, Pamaquine, Chloroquine, Fansidar, Maloprim -Sulphonamides and Sulphones, e.g., Cotrimaxazole - Other Antibacterials – Nitrofurantoin, Chloramphenicol,. -Analgesics – Aspirin, Phenecitin - Miscellaneous – Naphthol, Naphthalene (moth balls), Vitamin K analogues. III. Fava beans (possibly other vegetables) (Usually the patient having a deficiency of G6PD is asymptomatic, but when ever he is exposed to an oxidant stress (drugs,fava beans or infections) haemolytic anaemia will take place)
  151. 151. CLINCAL FEATURES OF G-6-PD DEFICIENCY  The haemolysis in G-6-PD is both intravascular and Extravascular.  The clinical presentation can be of three types: 1. Acute Haemolytic Anameia: It is the most common presentation. There is rapidly developing intravascular haemolysis with haemoglobinuria, hemoglbinemia and sudden fall in haemoglobin level, which is precipitated by infection and other acute illnesses, drugs or the ingestion of fava beans. The anaemia may be self limiting as new red cells are made with near normal enzyme levels 2. Neonatal Jaundice: 3. Continuous congenital haemolytic anaemia: Very rare.
  152. 152. LABORATORY DIAGNOSIS OF G- 6 PD DEFICIENCY ANAEMIA Red cells showing loss of cytoplasm with separation of remaining haemoglobin from cell membrane Blister cells or Bite Cells) “Bite cell” in the center and supravital stain showing Heinz Bodies 1. Between crises blood count is normal. 2. Screening Tests: Different screening tests can demonstrate the ability of G6PD to reduce NADP 3.G6PD Assay: Quantitative assessment of G6PD levels in red cells. 4. “Blister Cells” or “Bite cells”: These are the cells in which Heinz bodies are removed by spleen. 5. Demonstration of Heinz Bodies: Heinz bodies are formed by denatured haemoglobin. Can be seen in reticulocyte stain. 6.Features of Intravascular Haemolysis: Haemoglobinuria. 7 Hyperbilirubinemia: Indirect Bilirubin is increased
  153. 153. TREATMENT OF G-6-PD DEFICIENCT ANAEMIA 1. The offending drug is stopped. 2. Any underlying infection is treated. 3. A high urine output is maintained. 4. Blood transfusion undertaken where necessary for severe anaemia 5. G-6PD deficient babies are prone to neonatal jaundice and in severe cases phototherapy and exchange transfusion may be needed.
  154. 154. Malignancies Of Haemopoietic System
  155. 155. Main Type Sub Type I LEUKAEMIAS 1. Acute Leukaemia (i) Acute Lymphoblastic Leukaemias(ALL) L1 ; L2 ; L3 (ii) Acute Myeloid Leukaemias(AML) M0; M1; M2; M3; M4; M5; M6; M7 2. Chronic Leukaemia (i) Chronic Myeloid Leukaaemia(CML) (ii) Chronic Lymphocytic Leukaemia(CLL) II PLASMA CELL DYSCRASIAS Multiple Myeloma and other plasma cell dyscrasia III MYELOPROLIFRATIV E DISORDERS 1. Chronic Myeloid Leukaemia 2. Polycythemia Rubra Vera 3. Essential Thrombocythemia 4. Myelofibrosis IV LYMPHOMAS 1. Non – Hodgkin's Lymphomas (i) B – Cell (ii) T- Cell 2. Hodgkin’s Lymphoma (i) Lymphocyte Predominant (ii) Lymphocyte Rich(iii) Lymphocyte Depleted (iv) Mixed Cellularity (v) Nodular Sclerosis
  156. 156. LEUKAEMIAS The leukaemias are group of disorders characterized by the accumulation of malignant white cells in the bone marrow and blood Neoplastic proliferation of cells of haemopoetic origin which arises after somatic mutation in a single haemopoetic stem cell, the progeny of which forms a clone of leukaemic cells.
  157. 157. CLASSIFICATION OF LEUKAEMIAS Leukaemias are mainly classified into four types – Acute and Chronic leukaemias which are further subdivided into Lymphoid and Myeloid. Leukaemia Acute Chronic Lymphoid Myeloid Lymphoid Myeloid ACUTE LEUKAEMIA: Acute Myeloid Leukaemia : M 0 to M7 Acute Lymphoblastic Leukaemia: L1 to L3 CHRONIC LEUKAEMIAS: Chronic Myeloid Leukaemia Chronic Lymphocytic Leukaemia
  158. 158. Incidence of Different Types of Leukaemias According to Age • ALL is predominantly a childhood disease • CLL occurs mainly in the elderly • AML and CML have a wider age distribution.
  159. 159. Classification OF ACUTE LEUKAEMIAS  Acute Leukaemia is defined as the presence of over 30% blast cells in the bone marrow at the time of presentation  It is further subdivided into Acute Myeloid Leukaemia (AML) and Acute Lymphoblastic Leukaemia (ALL) on the basis of whether the blasts are shown to be myeloblasts or lymphoblasts. Acute Leukaemias are usually aggressive disease in which the malignant transformation causes accumulation of early bone marrow haemopoietic progenitors , called blast cells. The dominant clinical feature of these diseases is usually bone marrow failure caused by accumulation of blast cells. If untreated these diseases are usually fairly rapidly fatal.
  160. 160. Clinical features to some extent are helpful in the differentiation of ALL and AML, but are not definitive. The age profiles of acute luekaemias are distinctive. ALL has a peak in childhood and AML in adult age. The overlap is such, that age is not a useful criterion in classifying leukaemias. Significant lymph node enlargement (diameter exceeding 2 cm) is more common in ALL than AML. Solid masses of leukaemic cells (Chloromas or Granulocytic Sarcomas) are indicative of AML. Extensive involvement of gums is seen characteristically in Acute Moncytic Leukaemia (M4 or M5)
  161. 161. MORPHOLOGIC APPROACH TO ACUTE LEUKAEMIAS The experienced morphologist can classify about 70% of acute leukaemias as either ALL or AML by the blast appearance on routine haematology stains (Romanosky stain) , on the basis of nuclear or cytoplasmic features. For the rest of the cases different diagnostic modalities are employed to make a diagnosis. The modalities which can be used to diagnose haematological malignancies are: 1. Routine haematological stains: On the basis of that the lineage of abnormal cells can be determined in 70% of cases 2. Cytochrmistry or Special stains: There are different special stains for every specific cell type 3. Monoclonal antibodies or CD (Cluster Differentiating) markers: Specific antibodies for every lineage are used. They are employed by two ways: (i) Immunocytochrmistry (ii) Flowcytometery
  162. 162. DIFFERENCES BETWEEN LYMPHOBLAST AND MYELOBLAST LYMPHOBLAST MYELOBLAST Size: Smaller as compared to myeloblast Larger than lymphoblast Nuclear Chromatin: More clumped and irregularly distributed Chromatin is fine, delicately stippled or lacy, and evenly distributed Nucleoli: May be indistinct or number varies from 1 to 2 . There is a condensation of chromatin along with the nuclear and nucleolar membrane Nucleoli are single or multiple and are usually prominent. Nuclear and molecular membranes are indistinct. Nuclear outlines: May be folded or convoluted. - Amount of Cytoplasm: Scant Abundant cytoplasm, as compared to lymphoblast N:C Ratio: Very high High
  163. 163. Granules in Cytoplasm: Absent May be present Auer Rods Absent Present Accompanying cells: - Maturing cells of myeloid series may be seen Cytochrmistry or Special Stains: • Acid Phosphatase (ACP) stain: Positive in T-ALL • Periodic Acid Schiff (PAS) stain: Positive in B - ALL • •Sudan Black B stain: Positive in M1, M2, M3,M4 • Non Specific Esterase (ANAE): Positive in M4 and M5 Flowcytometery and Immunocytochemistry: Will show T- Lymphoid or B- Lymphoid markers T – ALL: CD 3 ; CD7 B- ALL: CD 19; CD22; CD10 Will show Myeloid or Moncytic or Megakaryocytic markers CD Marker for Myeloid series: CD 13; CD33
  164. 164. Lymphoblasts VS Myeloblasts A: Lymphoblasts have fewer nucleoli than myeloblasts,and the nuclear chromatin is more condensed. Cytoplasmic granules are absent B: Myeloblasts have a delicate nuclear chromatin, prominent nucleoli, and fine azurophilc granules in the cytoplasm
  165. 165. Leukaemia Diagnosis: First Routine Stain
  166. 166. Second: Cytochemistry Acid Phosphatase Periodic Acid Schiff Sudan Black Alpha Nephythyl Acetate Esterase
  167. 167. Third: CD Markers Flowcytometery Immunocytochemisrtry
  168. 168. Fourth :Cytogenetics
  169. 169. Acute LeukemiaAcute Leukemia CYTOCHEMICAL PROFILES IN ACUTE LEUKEMIACYTOCHEMICAL PROFILES IN ACUTE LEUKEMIA MPO & Sudan Black B NSE PAS AML + + ± (Monocytic, diffuse) ALL - ± + (Focal) (75%) +, positive; -, negative; ±, not definitive. NSE: Non Specific Esterase PAS: Periodic Acid Schiff
  170. 170. Immunologic Markers for Classification of ALL and AML AML ALL Marker Precursor T cell* T cell B Lineage CD 19 - + - CD 22 - + - CD 10 - +or - - T Lineage CD 7 - - + CD 3 - - + tdT* - + + Myeloid CD 13 + - - CD 33 + - - Glycophorin +(M6) - - CD 41 +(M7) - - Myeloperoxidase +(M0) *B-ALL resembles precursor B-ALL immunologically,but has surface Ig and is tdT negative
  171. 171. FAB Classification of Acute Lymphoblastic leukaemia ALL – L1: Small monomorphic blasts with high N:C ratio ALL – L2: Large. hetrogenous,nucleolated , low N:C ratio ALL - L3: Burkitt type , basophilic and vacuolated cytoplasm. The L1 blast show uniform, small uniform, small blast cells with scanty cytoplasm. The L2 type comprise mixed population of small and large blasts with more prominent nucleoli and cytoplasm. The L3 blasts are large with prominent nucleoli. strongly basophilic cytoplasm and cytoplasmic vacuoles. FRENCH AMERICAN AND BRITISH (FAB) CLASSIFICATION OF ACUTE LYMPHOBLASTIC LEUKAAEMIAS
  172. 172. Acute Lymphoblastic Leukaemia: FAB Type ALL – L1 Lymphoblasts show monomorphic appearance, very high N:C ratio; Nucleoliare indistinct; Chromatin is course and there is condensation of chromatin along nuclear membrane
  173. 173. Acute Leukemia - MorphologyAcute Leukemia - Morphology ACUTE LYMPHOBLASTIC LEUKEMIAACUTE LYMPHOBLASTIC LEUKEMIA L1 - small cells with scant cytoplasm, regular nuclear contours with occasional clefting, and scant cytoplasm.
  174. 174. Acute Lymphoblastic Leukaemia FAB Type ALL – L2 Large Heterogeneous population of blast cells; Nucleolated; Low N: C ratio.
  175. 175. Acute Leukemia - MorphologyAcute Leukemia - Morphology ACUTE LYMPHOBLASTIC LEUKEMIAACUTE LYMPHOBLASTIC LEUKEMIA L2 - Large cells with variable cytoplasm, irregular nuclear contours with clefting, and prominent one or more nucleoli.
  176. 176. Acute Lymphoblastic Leukaemia FAB Type ALL – L3: Lymphoblasts with strongly basophilic cytoplasm and showing vacuolation.
  177. 177. Acute Leukemia - MorphologyAcute Leukemia - Morphology ACUTE LYMPHOBLASTIC LEUKEMIAACUTE LYMPHOBLASTIC LEUKEMIA L3 - Medium to large cells with moderately abundant intensely basophilic cytoplasm and prominent cytoplasmic vacuolation, regular nuclear contours, prominent nucleoli.
  178. 178. Acid Phosphatase staining in ALL: Showing cytoplasmic positivity; Localized staining in the Golgi zone. Reaction is typical of T- ALL
  179. 179. Periodic Acid Schiff (PAS) stain in Acute Lymphoblastic Leukaemia Block Positivity of Cytoplasm. Typical of B- ALL
  180. 180. FRENCH AMERICAN BRITISH(FAB) CLASSIFICATION OF ACUTE MYELOID LEUKAEMIA(AML) AML – M0 : Undifferentiated Myelobalastic leukaemia ; require cell markers. AML – M1: Myeloblastic without maturation; requires peroxidase stain or Sudan Black B stain AML – M2: Myeloblastic with maturation AML – M3: Acute Promyelocytic Leukaemia; Hypergranular promyelocytes AML – M3 VARIENT: Micro or Hypogranular promyelocytes AML – M4: Acute Myelomoncytic leukaemia; with both granulocytic and monocytic maturation AML – M5: Acute Monoblastic Leukaemia AML – M6: Erythroleukaemia AML – M7: Acute megakaryoblastic Leukaemia
  181. 181. AML – M0. Undifferentiated blasts which are negative with cytochrmical reactions for AML(Sudan Black B and ANAE) and positive with antibodies against myeloid antigens :CD13 and CD33. Morphologically these blasts resemble L2 type but this case is not ALL because all lymphoid markers (B and T ) were negative .
  182. 182. Acute Leukemia - MorphologyAcute Leukemia - Morphology ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA AML-M0 • Large agranular blasts. • Myeloperoxidase negative or <3% +. • CD13 and/or CD33 +. • B and T-lineage markers are negative.
  183. 183. AML – M1: AML without maturation; 20% of cases ; Very immature but > 3% are Sudan Black B or myeloperoxidase positive; Few granules or Auer rods and little maturation beyond the myeloblast stage AML – M1: Auer rod highlighted by the peroxidase cytochemical reaction (peripheral blood)
  184. 184. Acute Leukemia - MorphologyAcute Leukemia - Morphology ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA AML-M1 • A/granular blasts, >90% of non-erythroid cells. • At least 3% are myeloperoxidase or Sudan black +. • Remaining 10% or less cells are maturing granulocytes. Auer Rod
  185. 185. AML – M2: AML with Maturation; 30 -40% of cases; Full range of myeloid maturation through granulocytes. Auer rods present in most cases. Presence of t (8 ; 21) defines a prognostically favourable subgroup.
  186. 186. Acute Leukemia - MorphologyAcute Leukemia - Morphology ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA AML-M2 • A/granular blasts, from 39-80% of non- erythroid cells. • Monocytic cells <20%. • Granulocytes (promyelocytes to neutrophils >10%).
  187. 187. AML – M2 (Sudan Black B): Strong positivity in cytoplasm
  188. 188. AML – M3:Acute Promyelocytic leukaemia; 5 – 10 % of cases; Majority of cells are hypergranular promyelocytes; often with many Auer rods per cells; Aggregates of Auer rods (Faggots) can also be seen; Patients are younger in age (median age 35 – 40 years) and often develop Disseminated Intravascular Coagulation (DIC). The t (15 ; 17 ) is characteristic. Aggregates of Auer Rods
  189. 189. Acute Leukemia - MorphologyAcute Leukemia - Morphology ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA AML-M3 • Majority of cells are abnormal promyelocytes. • Faggot cells (cell with bundles of Auer rods). • Microgranular variant also occurs.
  190. 190. AML – M3 variant: Acute Promyelocytic Leukaemia – Variant; Micro or Hypogranular bilobed promyelocytes
  191. 191. AML – M4: Acute Myelomoncytic Leukaemia with both granulocytic and monocytic maturation.15 – 20% of cases; Myelocytic and monocytic differentiation evident; Myeloid elements resemble M2 AML. Monoblasts are positive for nonspecific esterases.
  192. 192. Acute Leukemia - MorphologyAcute Leukemia - Morphology ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA • AML-M4 • Myeloblasts and monoblasts are >30%. • Myeloblasts and granulocytes are 20-80%. • Monocytic cells >20%. • PBM >5.0 x 109/L. • AML-M4Eo
  193. 193. AML – M4: Both Sudan Black B ( left) and Non Specific Esterase - - ANAE (right) are positive
  194. 194. M5a Acute Monoblastic Leukaemia without maturation in which more than 80% of blasts are monoblasts
  195. 195. M5b Acute Monoblsatic Leukaemia with Maturation less than 80% of blasts are monoblasts.
  196. 196. AML – M5b: Strong Non Specific Esterase (ANAE) reaction
  197. 197. Acute Leukemia - MorphologyAcute Leukemia - Morphology ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA AML-M5 • 80% of non-erythroid cells are of monocytic lineage. • M5a (80% monoblasts). • M5b (<80% monoblasts).
  198. 198. AML –M5a subtype: A peripheral smear shows one monoblast and five promonocytes with folder nuclear membranes
  199. 199. AML – M6 Acute Erythroleukaemia; 5% of cases ; Dysplastic ereythroid precursors (some megaloblatoid, others with giant or multiple nuclei) predominate , and within the nonerythroid cells, >30% are myeloblasts; seen in advanced age.
  200. 200. Acute Leukemia - MorphologyAcute Leukemia - Morphology ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA AML-M6 • >50% of all nucleated marrow cells are of erythroid lineage. • 30% of the non-erythroid cells are blasts.
  201. 201. AML – M6
  202. 202. AML – M7 : Acute Megakaryocytic Leukaemia; constitute 1% 0f cases; Blasts of megakaryocytic lineage predominate; Appearance of blasts is like lymphoblasts with budding cytoplasm; Platelets appear to shed out from the cytoplasm; Cytoplasmic blebs are PAS and ANAE positive; Blasts react with platelet specific antibodies GP IIb/ IIIa;. Myelofiborsis or increased marrow reticulin seen in most cases;
  203. 203. Acute Leukemia - MorphologyAcute Leukemia - Morphology ACUTE MYELOGENOUS LEUKEMIAACUTE MYELOGENOUS LEUKEMIA AML-M7 • At least 30% of nucleated cells are blasts. • The blasts demonstrate platelet peroxidase by EM or IHC.
  204. 204. INCIDENCE OF ACUTE LYMPHOBLASTIC LEUKAEMIA Acute Lymphoblastic Leukaemia is the common form of leukaemia in children Its incidence is highest at 3 – 7 years, falling off by 10 years. There is a lower frequency of ALL after 10 years of age with a secondary rise after the age of 40.
  205. 205. CLINICAL FEATURES OF ACUTE LYMPHOBLASTIC LEUKAEMIA Clinical features are secondary to the following: 1. Bone Marrow Failure leading to: - Anaemia (pallor; lethargy; dyspnoea) - Neutropenia (fever; malaise; features of mouth , throat, skin, respiratory, perianal or other infections) - Thrombocytopenia (spontaneous bruises; purpura; bleeding gums and menorrhagia) 2. Organ infiltration – tender bones, lymphadenopathy, moderate splenomegaly, hepatomegaly If CNS involvement then headache, nausea, vomiting blurring of vision and diplopia
  206. 206. Purpura over the lower limbs in a Patient with acute leukaemia
  207. 207. Marked cervical lymphadenopathy in in a patient with Acute Lymphoblastic Leukaemia
  208. 208.  Haematological investigations reveal normocytic and normochromic anaemia  Thrombocytopenia in most cases  Total Leucocytes Count (TLC) may be decreased, normal or increased to 200 X 10 9 /l or more  The blood film typically shows a variable numbers of blast cells  The bone marrow is hypercellular with >30% leukaemic blasts.  Blast cells are characterized by morphology, cytochemistry, immunological markers and cytogenetic analysis Lab Diagnosis of Acute Lymphoblastic Leukaemia
  209. 209. Lab Diagnosis of Acute Myeloid Leukaemia  Haematological investigations reveal a normochromic normocytic anaemia  Thrombocytopenia in most cases  Total Leucocytes count (TLC) is usually increased and blood film examination typically shows a variable numbers of blast cells  The bone marrow is hypercellular and typically contains many leukaemic blasts(> 30%)  Blast cells are characterized by morphology, cytochemistry, immunological markers and cytogenetic analysis  Tests for DIC are often positive in patients the promyelocytic variant of AML
  210. 210. Prognostic Factors in Acute Lymphoblastic Leukaemia Determinant Favourable Unfavourable Age 3-7 yrs <1, >10 yrs Sex Female Male Race White Black WBC Count <10 X 10 9 /l >50 X10 9 /l Time of Remission <14 days >28 days Organomegaly Absent Massive Mediastinal mass Absent Present CNS Leukaemia Absent Present FAB Morphology L1 L2,L3 Immunophenotype Early Pre B cell T-cell,B-cell Cytogenetics Hyperdiploidy Pseudodiploidy t(9;22) t(8;14) t(4;11) t(14q+ )
  211. 211. INCIDENCE OF ACUTE MYELOID LEUKAEMIA Acute Myeloid Leukaemia occurs in all age groups. It is the common form of acute leukaemia in adults and is increasingly common with age. AML forms only a minor fraction (10 – 15%) of the leukaemia in childhood.
  212. 212. CLINICAL FEATRUES OF ACUTE MYELOID LEUKAEMIA  Clinical features resemble those of Acute Lymphoblastic Leukaemia.  Anaemia and Thrombocytopenia are often profound.  A bleeding tendency and disseminated intravascular coagulation (DIC) is characteristic of the M3 variant of AML.  Tumour cells can infiltrate a variety of tissues . Gum hypertrophy and infiltration , skin involvement and CNS disease are characteristic of Myelomoncytic  (M4) and monocytic (M5) types. An isolated mass of leukaemic blasts is usually referred to as a granulocytic sarcoma.
  213. 213. Leukemic infiltrates in gums in AML-M5
  214. 214. Mediastinal widening in a 4-year-old boy with ALL Mediastinal Widening in ALL
  215. 215. TREATMENT OF ACUTE LEUKAEMIAS General Supportive Treatment: Blood products support to treat anaemia and thrombocytopenia.  Antibiotic coverage to treat infections. Insertion of central venous catheter (Hickman) via a skin tunnel from the chest into the superior vena cava to give access for giving chemotherapy , blood products, antibiotics, intravenous feeding, etc.  Prevention of vomiting.
  216. 216. SPECIFIC TREATMENT OF ACUTE LYMPHOBLASTIC LEUKAEMIA I.CHEMOTHERAPY: Induction Intensification / CNS directed phase Interim Maintenance I Delayed Intensification I Interim Maintenance II Delayed Intensification II Maintenance Chemotherapy II.BONE MARROW OR PERIPHERAL BLOOD STEM CELL TRANSPLANT: In most children it is usual to attempt to cure without transplant. It is reserved for patients with poor prognostic factors
  217. 217. SPECIFIC TREATMENT OF ACUTE MYELOID LEUKAEMIA Induction Consolidation Consolidation Possible stem cell Further consolidation transplantation
  218. 218. CHRONIC LEUKAEMIAS The chorinc leukameias are distinguished from acute leukaemias by their slow progression. Chronic leukaemias can be broadly subdivided into : (i) Chronic Myeloid Leukaemia (ii) Chronic Lymphocytic leukaemia
  219. 219. ACUTE LEUKAEMIA CHRONIC LEUKAEMIA Leukemic cells do not differentiate Leukaemic cells retain ability to differentiate Bone marrow failure Proliferation without bone marrow failure Rapidly fatal if untrateated Survival for a few years Potentially curable Not presently curable without bone marrow transplant Acute leukaemia Vs Chronic Leukaemia
  220. 220. CHRONIC MYELOID LEUKAEMIA(CML)  The disease occurs in either sex (male : female) ratio of 1.4:1), most frequently between the ages of 40 and 60 years. However it may occur in children and neonates, and in very old.  In most cases there are no predisposing factors but the incidence was increased in survivors of the atom bomb exposures in Japan. CLINICAL FEATURES The clinical features of Chronic Myeloid Leukaemia include: 1.Symptoms related to hypermetabolism, eg., weight loss, lassitude, anorexia or night sweats. 2. Splenomegaly is nearly always present and is frequently massive.
  221. 221. 3. Features of anaemia may include pallor, dyspnoea, and tachycardia. 4. Bruising, epistaxis, menorrhagia or haemorrhage from other sites because of abnormal platelet function. 5. Gout or renal impairment caused by hyperuricaemia from excessive purine breakdown may be a problem. 6. In many patients diagnosis is made incidentally from a routine blood count
  222. 222. Massive splenomegaly in chronic myeloid leukaemia. The palpable margins of the spleen are indicated
  223. 223. LABORATORY FINDINGS IN CHRONIC MYELOID LEUKAMEIA White blood cell count : markedly increased. Usually more than 50 X 10 9 /l. On differential cell count a complete spectrum of myeloid cell is seen in the peripheral blood. The levels of Neutrophils and Myelocytes exceed those of blast cells and promyelocytes Bimodal peak of neutrophils and myelocytes) Peripheral blood film in CML showing leucocytosis alongwith immature forms of Myeloid series
  224. 224. Promyelocyte Myeloblast Neutrophil Myelocyte Metamyelocyte
  225. 225. 3. Increased circulating Basophils. 4. Normocytic and normochromic anaemia 5. Platelet count may be increased ( most frequently), normal or decreased. 6. Neutrophil alkaline Phosphatase score (NAP ) is invariably low. 7. Bone marrow is hypercullar with granulopoieitc predominance. 8. Demonstration of Philadelphia Chromosome on cytogenetic analysis of blood or bone marrow. It is a reciprocal translocation between chromosome between 9 and 22 – t(9;22)
  226. 226. Philadelphia Chromosome Karyotype showing the t ( 9:22) The Ph chromosome is arrowed
  227. 227. 2 3 4 51 7 8 9 10 11 126 13 14 15 16 17 18
  228. 228. The Philadelphia Chromosome: a) There is a translocation of the long arm of chromosome 22 to the long arm of chromosome 9 and reciprocal translocation of the long arm of chromosome 9 to chromosome 22. This reciprocal translocation brings most of the ABL gene into the BCR region of chromosome 22(and part of the BCR gene into juxtaposition with the remaining portion of ABL on chromosome number 9. b) The breakpoint in ABL is between exons 1 an 2. The breakpoint in BCR is at one of the points in the major breakpoint cluster regions m- BCR. c)This results in a 210 – kDa fusion protein product derived from the BCR- ABL fusion gene.
  229. 229. Phases of CML 1. Chronic Phase of CML 2. Accelerated phase of CML 3. Blast Crisis in CML - Myeloid Blast Crisis(80%) - Lymphoid Blast Crisis(20%)

×